Revision as of 02:22, 2 November 2017

Design

We designed a novel toolbox to gain the complete control over all major functions of the peroxisome. The toolbox is our solution for improving the engineering workflow and predictability of synthetic constructs. Interested? Find out how.

Scientific background

The root problem

Synthetic biology is an engineering discipline. And while we are able to plan our constructs with tools like biobricks, a major difference to e.g. electrical engineering is that they are not nicely isolated on a chip, but surrounded by all types of interfering agents. One of the major issues regarding protein expression in a novel chassis is unwanted and unexpected crosstalk between engineered pathways and the native cellular processes of the production host. A further issue is toxicity of the products or intermediates of the pathway. Both can greatly change our system’s behaviour which in some cases leads us to having to trial-and-error find a solution, making our previously modeled optimization useless.

Our approach

The natural approach of organisms to deal with metabolic interference and toxic byproducts is subcellular compartmentalization. This has proven to be a functional solution in naturally occurring pathways in eukaryotes as well as new synthetic pathways for biotechnological application. Thus, the creation of a synthetic organelle presents a suitable strategy to increase the efficiency and yield of non-native pathways. A common approach is to build up artificial compartments from scratch. Many breakthroughs have been achieved in the last decade, however the creation of a fully synthetic compartment is yet a milestone to reach for. We on the contrary want to start by engineering artificial compartments through orthogonalization.

The peroxisome is the ideal starting candidate as it has many advantages over other compartments, including it being able to import fully folded proteins and not being essential in yeast under optimal growth conditions. Our project’s aim is to create a toolbox for manipulating and creating customizable peroxisomes as a first step towards synthetic organelles.

By modifying the import machinery of yeast peroxisomes only selected proteins will be imported into the peroxisomes leaving the researcher in full control over the content of the peroxisomal lumen. Furthermore our toolbox will include a secretion mechanism for the synthesized products, various intra-compartmental sensors, modules for the integration of proteins into the peroxisome membrane, as well as optogenetic control for some of these parts for a more precise spatiotemporal control.

As a proof of concept for the functionality of the toolbox and the customizable compartment two metabolic pathways will be integrated into the altered peroxisome: (i) Violacein biosynthesis and (ii) nootkatone biosynthesis. Violacein, a bisindole formed by condensation of two tryptophan molecules, is a violet pigment and thus easy to quantify in the cell. Nootkatone on the other side is a natural compound found inside the peel of the grapefruit, which gives it its characteristic taste and smell. In addition, nootkatone is a natural mosquito and tick repellent that is already being commercially used and industrially manufactured. Unfortunately, the production costs are extremely high. Furthermore, the production of nootkatone inside yeast is challenging as it is toxic for yeast and thus, the production efficiency is rather low. A successful implementation of the synthetic compartment will show increased yields in the production of these compounds and showcase the potential of this approach for similar future applications.

Cloning strategies and the Yeast Toolbox for Multipart-Assembly

While describing our cloning strategies we mentioned several levels, which stand for different stages of our plasmids. They are further described in the work of J.M. Dueber and colleagues, who designed the well established yeast toolkit we used in this project (Dueber). The toolkit offers the possibility to design plasmids with desired antibiotic resistances, promoters as well as terminators from standardized parts. It also provides fluorescence proteins, protein-tags and many more useful components as part plasmids. These part plasmids are distinguished in different part types due to their specific overhangs to ensure their combination in the correct order (e.g. promoter - gene of interest - terminator) all in a versatile one-pot Golden Gate reaction without time-consuming conventional cloning steps.

**Figure 1: The yeast toolkit starter set comprises of 96 parts and vectors.** The eight primary part types can be further divided into subtypes. (Dueber)

The cloning steps regarding the plasmid levels are implemented in E.coli in order to reduce the required time to generate the final plasmids. The different levels are therefore defined by their part content and their antibiotic resistances.

To generate a level 0 plasmid, the gene of interest is ligated into the provided level 0 backbone via Golden Gate assembly using the enzyme BsmBI. The backbone contains a resistance to Chloramphenicol, as well as an origin of replication, creating a very basic yet functional plasmid.

The level 1 plasmid contains a promoter and terminator suited for S. cerevisiae. There is the possibility of including a polyhistidine-tag if there is a need for Western blot analysis. The antibiotic resistance contained in the level 1 plasmid changes from chloramphenicol to ampicillin which enables filtering out residual level 0 plasmids contained in the Golden Gate product. Furthermore, the Dueber toolbox includes the possibility of designing GFP-Dropout cassettes. These are custom-built level 1 backbones whose inserts are sfGFP as well as promoter and terminator suited for E. coli. Upon a successful cloning step the GFP is replaced by the part(s) of interest, and correct colony shows a white colour. In case of a wrong ligation event colonies show a green fluorescence. This provides a very useful tool to detect unsuccessful cloned colonies. The enzyme used for level 1 changes from BsmBI to BsaI to avoid any interference between different steps.

The level 2 plasmid combines two or more genes of interest with their respective promoters, terminators and tags. The resistance changes from ampicillin to kanamycin. The enzyme of this step is BsmBI again. This level is useful, if the construct you are designing requires multiple genes to be transformed into one yeast strain.

Yeast nomenclature

To make it fast and easy to differentiate between endogenous and heterologous genes and gene products we decided to use S. cerevisiae nomenclature according to yeastgenome.org.

Below nomenclature at the example of your favorite gene 1, YFG1 is explained.

Letter code	Meaning
YFG1	Your favorite gene S. cerevisiae wild type allele
yfg1Δ	Gene deletion of your favorite gene
Yfg1	Protein product of YFG1
YFG2	A heterologous gene product from mammalian cells

Design of our sub-projects

Engineering of Pex5 and PTS1

Designing our receptors

To achieve the engineering of an orthogonal import pathway, we followed two approaches regarding Pex5. The first is based on targeted mutagenesis based on educated guesses which is first verified by molecular dynamics and later experimentally in the laboratory. The second approach is based on a recently published paper: We searched for literature dealing with the modification of the peroxisomal import machinery. During our research we came across a paper of Cross et al., published in 2017, in which they present a synthetic construct of the Pex5 protein, partly Arabidopsis thaliana and partly Physcomitrella patens. Compared to the wild type Pex5, this one shows different binding affinities since it interacts with a PTS1* variant that does not interact with the wild type Pex5 Cross et al. . Since the protein sequences of yeast's and plants's Pex5 differ quite a lot, we aligned both sequences to understand where the mutations were set.

**Figure 1.1:** Alignment of the *Arabidopsis thaliana's* Pex5 and *Saccharomyces cerevisiae*Pex5

The alignment shows three red marked amino acids we changed in our receptor sequence. Interestingly, these mutations are located within the TPR motifs of our Pex5 protein and this persuaded us to try out this receptor, we call it R19. Due to lack of time we tested this Pex5 variant in silico and in vivo simultaneously. We started molecular dynamics simulations with a couple of PTS variants that we already tested with our previous designs − one of them was actually the variant they used in the paper (YQSYY). The details and results of our structural modeling can be found in the modeling section.
Furthermore, we synthesized this variant and together with two receptors we designed based on educated guesses we got three receptors for our experimental work.

Experimental design

Verification of peroxisomal protein import was performed by tagging the fluorescent protein mTurquoise with our designed PTS variants. Additionally, a peroxisomal membrane protein was used to ensure peroxisomal localization. For that reason, we chose the transmembrane domain of Pex13 tagged with the fluorescent protein mRuby.

Pex13−mRuby

We used the peroxisomal membrane protein Pex13 as a fluorescent marker − by just using the transmembrane domain of Pex13 with a short linker, we make sure that it has no influence on the peroxisomal features. To obtain a higher differentiation from mTurquoise, which we use for another construct, we chose to work with mRuby. Literature research revealed that such constructs have been tested before − Erdmann et al. (2004) described a construct containing only Pex13_200-310 with a C-terminal GFP.

**Figure 1.3:** Pex13 construct with C-terminal mRuby.

Pex5 variant

In order to achieve an orthogonal peroxisomal protein import machinery we used a Pex5 knockout yeast strain in which we transformed our artificial Pex5 variant containing a modified PTS1 binding pocket. Our variation facilitates the detection of a non native PTS1 variant instead of the wild type PTS1. The construct contains a medium strength promoter, the Pex5 gene and a terminator. The remaining plasmid parts can be seen in the plasmid map below.

mTurquoise−PTS

Our approach for import verification is based on the fluorescent protein mTurquoise tagged with our modeled PTS variants. After several promoter tests with different strengths, we decided to express this construct only in low amounts, since this was the most suitable possibility to detect potential mTurquoise localization.
Our construct is depicted in the figure below.

**Figure 1.5:** Fluorescent protein mTurquoise tagged with the PTS variant.

Combination of our constructs

To combine our constructs, we cloned our Pex5 and mTurquoise constructs into a level 2 plasmid portrayed below.

**Figure 1.6:** Level 2 plasmid containing the Pex5 gene and the fluorescent protein.

Subsequently, co-transformation of the Pex13−mRuby plasmid and the level 2 plasmid was performed in order to verify peroxisomal colocalization.

PTS screening

Trusting on our targeted approach alone seemed risky − that is why we planned a PTS screening to find the most favorable PTS for our three receptors. Dueber et al. (2016) used the Violacein assay for a similar purpose. They screened for the best PTS for the wild type receptor and were successful. Hence another subproject of our team is the integration of the Violacein pathway into the peroxisome (Violacein), we were already supplied with all necessary enzymes − VioA, VioB and VioE.

**Figure 1.8:** Pathway leading to the production of prodeoxyviolacein − the assay is based on the green color to identify colonies without functional import mechanism.

The figure above shows the principles of the assay. VioA and VioB are localized in the cytosol and lead to the production of the IPA imine dimer while VioE is tagged with a PTS1 variant. Successful import leads to white colonies whereas missing import results in green colonies due to the cytosolic production of prodeoxyviolacein.
Our rests upon the following two plasmids which are co-transformed into yeast.

**Figure 1.9:** Plasmids used for Violacein assay. The left one carries the coding sequence for the Pex5 variant, VioA and VioB whereas the right one contains the gene for VioE plus the attached PTS1 variant.

As shown above, we created one plasmid containing VioA, VioB and one of our Pex5 variants while the other plasmid only contained VioE. We then designed primers which bind to the VioE plasmid to amplify the whole plasmid except the terminator − random PTS1 variants were attached to VioE with the help of a random primer library. Following up, we did the ligation with the corresponding terminator and obtained a mix of several different VioE-PTS1 plasmids.
After plasmid amplification in Escherichia coli we then co-transformed yeast with the two constructs and waited for the colonies to grow. With the yeast growing, prodeoxyviolacein should be produced in yeast cells with absent import (green color) and the IPA imine dimer (white color) should be produced in those with functional import.
Plasmid preparation of those with white color and subsequent sequencing leads to the identification of functional PTS1 variants. Afterwards, we repeat the cloning steps described before to obtain a mTurquoise−PTS1* construct and co-transform it with the corresponding Pex5 variant. Eventually, the correct localization of mTurquoise tagged with these PTS1 variants provides proof for its function.

Mutagenesis of PTS2

Imagine you need different protein concentrations in your artificial compartment. What to do? Take our modified PTS2 sequences with varying import efficiencies.

To characterize the import efficiency for the site-directed PTS2 firefly luciferase was used. Luciferase is a luminescent protein which can be split in a C- and a N-terminal part. Only when combined, luminescence can be detected. To measure the import efficiency the two parts will be expressed and imported into the peroxisome in a separated way. The smaller part (Split2) of the split luciferase will be brought into the peroxisome first via the PTS1 dependent pathway. The other part is imported via the respective modified PTS2 sequence. The better this sequence is recognized by Pex7, the stronger the luminescence of the assembled luciferase can be detected in the peroxisome. There is a chance of split parts of the luciferase assembling in the cytosol if the import is too slow. To avoid wrong conclusions of the luciferase localisation, we designed a negative control experiment. It includes a split luciferase similar to the one used in the initial experiment, but without the peroxisomal targeting sequence. Consequently there will be no import into the peroxisome. If we subtract the luminescence of the negative control experiment from the luminescence of the main experiment we can define the degree of import.

In addition to a directed approach according to Kunze and colleagues we also want to perform a random mutagenesis experiment to alter the five variable amino acids of the core region of the PTS2 sequence in an unbiased manner. The aim is to generate a library of different peroxisomal PTS2. The 15 nucleotides are assembled by chance. In the DNA synthesis this sequence will either be described as [NNN]₅ or [DNK]₅. N stands for all four nucleotides mixed, K for either G or T and D for A,G or T. The “DNK” composition prohibits two out of three termination codons. Additionally with this library the amino acid frequency is improved towards a balanced ratio in between the different kinds (Dueber).

Each approach could generate up to 415 DNA sequences, which is roughly 1,07 billion. On the level of the amino acid sequence there are 3,2 million possibilities, since each residue can be taken by 20 different amino acids. For the assay we therefore need a high throughput method.

We adapted work of DeLoache, Russ and Dueber using the violacein pathway to measure the import effectiveness of tripeptides. The pathway consists of Violacein A (VioA), Violacein B (VioB) and Violacein E (VioE). It converts tryptophan into the green product prodeoxyviolacein (PDV). The first two enzymes, VioA and VioB, are expressed in the cytosol, and the third one, VioE, is targeted to the peroxisome with a PTS1 sequence. The degree of import can be measured by the intensity of green colour of the colonies. An efficient import signal leads to a strong import of the VioE into the peroxisome and subsequently to white colonies, because the intermediates cannot diffuse into the peroxisome to its respective enzyme. DeLoache et al. showed that there is a proportional correlation between the concentration of the green product PDV and a red fluorescent substance. The concentration of this product displays the import efficiency of the respective sequence.

This assay has been used for the evaluation of the generated PTS2 sequences. The VioE-PTS2 plasmids are harvested and cotransformed with a VioA-VioB plasmid. Each plasmid contains a specific auxotrophy marker. Consequently every growing colony contains both plasmids. To evaluate the respective sequence the concentration of the red fluorescence is measured. The more fluorescence is detected the more VioE is in the cytosol. Therefore the respective PTS2 is not that efficient. The other way around a low concentration of the fluorescent substance correlates with an efficient import via the respective PTS2.

Scientific background

Peroxisomal membrane proteins are synthesized on free polysomes in the cytosol and afterwards integrated into the membrane via two major pathways: one dependent on the endoplasmatic reticulum and one dependent on PEX19 and PEX3.

PEX19-dependent

ER-dependent

Experimental Work/Design

In order to test our hypothesis we fused the last 59 amino acids of the C-terminus of human PEX26 (AA 246-305) to a red fluorescent protein, to further elucidate the Pex3/Pex19-dependent import. mRuby is generally used as a marker in combination with a fluorescent microscope to visualize the localization of the fusion protein. The C-terminus of PEX26 contains a helical signal-anchor, which serves as both, a mPTS and transmembrane domain. We designed our construct with mRuby2 fused to the N-terminal side of the PEX26-C-terminus, this way the mRuby should face the cytosolic side of the peroxisomal membrane. Quite similar to our mRuby-PEX26 approach, we designed a construct for the ER-dependent import. Therefore, we fused the mRuby2 fluorescent protein to the N-terminus of Pex3 (AA 1-39). This construct should be N-terminally anchored in the peroxisomal membrane, with mRuby2 again facing the cellular lumen.

Our main goal is to introduce a rather complex membrane protein to the peroxisome that can alter specific traits. For that we fused the Pex3 N-terminus (AA 1-39) to a Halobacterium salinarum bacteriorhodopsin protein (AA 16-262), replacing the first 16 amino acids (Pex3-BacR). The original archaeal bacteriorhodopsin acts as a proton pump by capturing light energy to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy. Our assumption is that the first transmembrane segment determines the orientation of the following protein and that therefore due to the N-terminal anchoring signal the bacteriorhodopsin will be inserted in reverse orientation, pumping the protons into the peroxisome. This way the pH of the peroxisomal lumen could actively be controlled and adjusted.

Finally, our project involved combining the work of other subteams to verify the localization of our constructs in the peroxisome and analyze the effects they have on the import. Therefore, we are using the superfolded-GFP protein, another fluorescent marker, which is in our case fused to the peroxisomal import sequence PTS1, and a version of Pex11 that is fused to the fluorescent marker Venus. Both markers emit light in the green light spectrum, were as mRuby2 emits light in the red part of the spectrum, giving us a strong contrast and an easy way of differentiating between the two under the fluorescent microscope.

To physically create our constructs, we researched the DNA sequences of bacteriorhodopsin, Pex3 and PEX26 via UniProt and pre-designed our fusion constructs with the software tool „Geneious“. We ordered the synthesis of three separate parts (Pex3, PEX26 and Pex3-BacR) from IDT. To ease out the process of assembling our plasmids, we used the „Dueber Toolbox", containing various parts such as promoters and terminators, to tailor the plasmids specific to your needs. Finally, to combine all the selected parts, we used the „Golden Gate” assembly method.

Experimental Design

We will adapt the system of Sagt and colleagues to secrete the content of our modified compartments (Sagt et al, 2009) .
For the application of this system in S. cerevisiae we use a truncated version of the v-SNARE Snc1 to decorate our compartments (Figure 3.1) (Gerst et al, 1997) .

**Figure 3.1 A diagram of the general domain structure of Snc1.** V is a variable domain which is not important for the binding to the t-SNARE. TM is the transmembrane domain. H1 and H2 are the a-helical segments, forming the SNAREpin with the t-SNARE (Gerst *et al*, 1997)

To decorate the compartments with the SNARE we use a peroxisomal transmembrane protein . In our case we use the proteins Pex15 or Pex26, which were further investigated in another sub project, and fuse Snc1 to the N-terminus. We expressed these constructs of membrane anchor and Snc1 constitutively under control of the RPL18B promotor. In case of Pex15 we used a truncated version, lacking a large part of the N-terminus, only consisting of the transmembrane domain (315-383) (Figure 3.1). For Pex26 we use the truncated version published in Halbach et al. (Halbach et al, 2006) .

**Figure 3.2 Concept of secreting peroxisomal contents to the supernatant.** For the secretion, the membrane anchor Pex15 or Pex26 is used. This anchor is used to decorate peroxisomes or our modified compartments with the v-SNARE Snc1. For the secretion Snc1 interacts with the t-SNAREs in the cell membrane. Induced from this interaction the vesicle and cell membrane fuse and the content of the compartment is secreted to the supernatant.

We verified our secretion using beta-glucuronidase (GUS) as a reporter protein. In 2012 Stock and colleagues described the GUS reporter assay for unconventional secretion (Stock et al, 2012) . With it, it is possible to determine whether a protein is secreted conventional and is N-glycosylated or secreted unconventional and not N-glycosylated. GUS is a bacterial protein with an N-glycosylation-site, which is active only if the protein is not N-glycosylated. The GUS-activity can be measured with different reagents in plate or liquid assays. Liquid assays can be applied qualitatively as well as quantitatively to measure differences in activity. If GUS is secreted by the conventional pathway the N-glycosylation leads to inactivation of the enzyme (Stock et al, 2012) (Fig 3.2).

**Figure 3.3 The GUS Assay.** GUS secreted with an unconventional secreted protein like Cts1 from Ustilago maydis active in the supernatant. GUS secreted with a conventional Signal peptide (Sp) inactive in the supernatant. If GUS is in the cytoplasm there is also no activity (Lysis control) (Feldbrügge *et al*, 2013) .

GUS will be imported to the peroxisome with the PTS1 sequence and measured quantitatively in the supernatant. We will use a coexpression of GUS-PTS1 and Snc1-Pex15 or Snc1-Pex26 to identify the secretion of the compounds. Furthermore, we will use GUS-PTS1 expressed in S. cerevisiae without Snc1 fused to a membrane anchor for a control. We will measure the active GUS in the supernatant with a liquid assay based on the turnover of 4-methylumbelliferyl-beta-D-glucuronide to 4-methyl umbelliferone (4-MU) (Blázquez et al, 2007) . Here we expect a higher activity of GUS in the supernatant of cultures with Snc1 decorated peroxisomes.
To increase the variability of our constructs we also designed vectors with and without a GS-Linker connecting the Snc1 with the Pex15. Additionally we tested our constructs in strains with a deletion of Pex11 . This deletion leads to formation of larger peroxisomes and may increase the efficiency of our secretion mechanism.

Heading

Sensors

To enrich our toolbox we decided to measure four essential physiological factors: ATP, NADPH, Glutathione and pH.
ATP/ADP conversion is used as an energy currency in many cellular processes like translocation of proteins and metabolites or anabolic and catabolic turnovers. It is assumed that ANT1P, an ATP/AMP antiporter, is located in the peroxisomal membrane. Furthermore, the shuttle mechanism of the peroxisomal protein import machinery is ATP dependent (Palmieri L. et al., 2001). NADPH plays an important role in anabolic pathways and is also indispensable to our desired pathways of nootkatone and violacein. Glutathione is an antioxidant and redox buffer which is also found in yeast peroxisomes. It is used as cofactor by at least two types of peroxisomal proteins, the glutathione peroxidases and glutathione transferases, which reduce lipid- and hydrogen peroxides or transfer glutathione to lipid peroxides for the purpose of detoxification (Horiguchi H. et al., 2001). Furthermore, it partly represents the redox state of the peroxisome. Knowing the pH of a compartment is important for predicting the activities of almost all enzymatic processes inside and to follow up acidification and basification upon conversion of metabolites.

We finally chose two ratiometric sensors to perform measurements with. The pH sensitive and glutathione redox state reporting green fluorescent proteins pHLuorin2 (Mahon M. J. et al., 2011) and roGFP2 (Schwarzländer M. et al., 2016). Our aim was to target them either to the peroxisomal lumen or the cytosol. To achieve peroxisomal targeting we attached the peroxisomal targeting signal 1 via Golden Gate cloning.

Figure 5.1 Level 1 plasmids with medium promoters and uracil auxotrophy. Top left: cytosolic roGFP2. Top right: peroxisomal roGFP2. Bottom left: cytosolic pHLourin2. Bottom right: peroxisomal pHLourin2.

Using promoters with different expression strength in order to find optimal measurement conditions is of high interest. There is a trade-off between a high signal-to-noise ratio and self-induced effects which are both dependent on expression levels (Schwarzländer M. et al., 2016) . This is not generally the case for each sensor. For example, pHlourin2 has only a minor influence on the existing pH because of the buffer effect of proteins.
Validation of the peroxisomal localization can be achieved via fluorescence overlap of the sensor and a peroxisomal marker, in our case pex13-mRuby (import mechanism).

Figure 5.2 Level 2 plasmids with peroxisomal marker Peroxin13-mRuby and uracil auxotrophy for colocalization. Left roGFP2 with strong promoter. Right pHLourin2 with strong promoter.

It can also be achieved by transforming the sensors attached to the PTS1 sequence into a $\Delta$Pex5 yeast strain. The sensor is expected to show no specific localisation due to the missing import sequence. We calibrated the sensors in living yeast cells and physiological ranges so that we can not only perform relative but also quantitative measurements. We aim to confirm our hypothesis of a more oxidized redox state of roGFP2 in peroxisomes with violacein pathway activity and want to measure differences in pH within yeast strains with peroxisomal membrane anchored pex3-bacteriorhodopsin protein (membrane proteins) .

Once expression and localization of the sensor is proven by microscopy, measurements with a plate reader or a fluorometer are acceptable. This allows a high number of replicates to be measured accurately in reasonable time. Microscopy is performed with a filter based Nikon Eclipse TI fluorescence microscope at 100-fold magnification and plate reader measurements are performed with a Tecan infinite plate reader (Mahon M. J. et al., 2011) . For pHLuorin2 emission intensity is measured at 535 nm upon excitation at 405 nm and 485 nm. Same settings were used for roGFP2 (Schwarzländer M. et al., 2016) . Evaluation of the reported signals is done by the excitation ratio of the the corresponding excitation wavelength.

The initial step is to find a reliable source to prove the abundance of our precursor Farnesyl pyrophosphate (FPP) in yeast peroxisomes. So far there is no proof of existence of FPP inside yeast peroxisomes yet. However it is predicted to be present, as it was detected in mammalian and plant peroxisomes Olivier et al. (2000).

**Figure 7.1:** The structure of FPP the precursor for nootkatone.

The precursor FPP is converted into valencene by a valencene synthase (ValS). We chose the one from Callitropsis nootkatensis because of its comparably high efficiency in microorganisms. It achieves greater yields in yeast than the citrus valencene synthase. Furthermore, the product specificity is relatively high, while production of byproducts is low Beekwilder et al. (2014). The valencene synthase was also chosen because of its robustness towards pH and temperature changes Beekwilder et al. (2014). Our modelling approach revealed that for optimal yields an overexpression of valencene synthase is necessary because of its slow conversion rate (Model). This is why we chose the strongest promoter of the yeast toolbox (Dueber Toolbox) for this attempt.

Figure 7.3: The structure of valencene, an intermediate of nootkatone

The intermediate valencene is then converted into nootkatol by a P450 monooxygenase as well as into small amounts of our desired product nootkatone. The P450 monooxygenase we chose for this project was taken from the bacterium Bacillus megaterium. In this case it is not only a simple P450 monooxygenase, but an entire P450 system, consisting of a soluble P450 fused to a cytochrome P450 reductase (CPR) enzyme, making an additional reductase obsolete De Mot et al. (2002). Unlike eukaryotic P450s, which are mostly membrane bound, this prokaryotic BM3-P450 is located in the cytosol facilitating an easier transport into the peroxisome, as membrane integration of proteins is a more difficult task to achieve than import of cytosolic proteins Girvan et al. (2006). BM3 normally catalyzes the hydroxylation of long chain fatty acids Narhi et al. (1986), which in our case could inhibit the conversion of valencene. For that reason we used a mutated version of BM3, which is called AIPLF. This variant is an enhanced version of the BM3 AIP version, which has a ten times better substrate oxidation rate for valencene than the wildtype BM3 and produces less byproduct when valencene concentration is saturated. Additionally to previous named benefits the AIPLF variant with 5 point mutations in the active side has a significantly lower binding affinity towards long chain fatty acids and therefore increase the transposition rate of valencene. Schulz et al. (2015), Lehmann (2016).

The alcohol dehydrogenase from Pichia pastoris subsequently converts nootkatol into nootkatone by oxidation. It uses NAD⁺ as a cofactor which is reduced in the reaction. The regeneration of this cofactor is facilitated by the BM3, which oxidizes NADH Schulz et al. (2015).

The first milestone to achieve our goal is the separate integration of each of our three enzymes ValS, BM3 and ADH into level 1 vectors (Dueber Toolbox) in yeast and to verify their expression by Western Blot analysis. Therefore, a 3xFlag/6xHis-tag was added to the N-terminus of each of the proteins. It enables us to use an anti-His antibody followed by an anti-mouse-antibody to make protein abundances visible. Subsequently, the two enzymes ADH and ValS were combined in a level 2 cassette plasmid. BM3 is designed as a level 1 plasmid and will be co-expressed with the level 2 plasmid to achieve a nootkatone production. The expression of the enzymes in the cytoplasm is again verified by Western Blot analysis. The further approach aims to provide the enzymes with C-terminal peroxisomal targeting signals type one, which finally converts the nootkatone pathway into our artificial compartment.

Since the production of nootkatone does not lead to a change of colour we need to apply different methods for verifying substrates. Once we succeed with the qualitative validation via Western Blot analysis, we can verify the presence of nootkatone by using high performance liquid chromatography and mass spectrometry, respectively.

Model influence on Nootkatone expression

We modeled the nootkatone biosynthesis pathway using ordinary differential equations in order to optimize nootkatone production. We found two hard and an easy problem, all of which we could find a solution for. The easy problem is optimization of the enzyme concentrations of the biosynthesis pathway. According to our model of the Nootkatone pathway we found that overexpression of Valencene Synthase is necessary to maximize the Nootkatone yield, while both alcohol dehydrogenase and p450-BM3 have only minor effects on the yield.

One of the hard problems, as shown in our penalty model is the toxicity of nootkatone and nootkatol. Since the toxicity most likely stems from both nootkatone and nootkatol clogging up the cell wall we present our peroxisome as a solution for this problem. When comparing the cytosolic model to our peroxisomal model we found that, if our assumption that neither Nootkatone nor Nootkatol are able to pass the peroxisomal membrane holds up, we can greatly increase Nootkatone production.

A further problem is the influx of the pathway precursor farnesyl pyrophosphate (FPP). We used OptKnock analysis to design yeast strains with optimized FPP production. With this analysis we got hints that growing the yeast cells on a fatty acid medium might be a simple alternative to knocking out the desired genes.

Based on described of advantages of violacein this pathway was chosen. Violacein is naturally produced in numerous bacterial strains, most popular in the gram-negative Chromobacterium violaceum . It is related to biofilm production and shows typical activities of a secondary metabolite (Seong Yeol Choi et al., 2015).

**Figure 8.1** The synthesis of Violacein requires five enzymes encoded by the VioABCDE operon. VioA, a flavin-dependent L-tryptophan oxidase and VioB, a heme protein, work in combination to oxidize and dimerize L-tryptophan to an IPA imine dimer. Hydrogen peroxide is released as a by-product of the VioA reaction. Next step by VioE is the rearrangement of the IPA imine dimer to protodeoxyviolaceinic acid, which can non-enzymatically oxidize to prodeoxyviolacein or, by VioC via deoxyviolaceinic acid, oxidize to pink deoxyviolacein. The flavin-dependent oxygenases VioC and VioD require interaction with the oxidized form of flavin-adenine dinucleotide (FAD) (uniprot, uniprot) . The two enzymes act sequentially: first, VioD hydroxylates protodeoxyviolaceinic acid, leading to protoviolaceinic acid. Second, VioC creates the oxindole at the 2-position of one indole ring, leading to violet violacein (Balibar CJ *et al.*, 2006) (Janis J. Füller *et al.*, 2016).

Relocating the pathway into the peroxisome enables proximity of the enzymes and substrates. Furthermore the yeast cell is protected from the toxic substance hydrogen peroxide. Yeast peroxisomes have no problem with this as their main function is the beta-oxidation of fatty acids and the detoxification of the thereby produced H₂O₂ (Erdmann R. et al., 2007) . Because VioC and VioD are FAD-dependent, it is additionally an evidence for FAD availability inside of the peroxisome, if the synthesis of Violacein works. Otherwise the two enzymes would not be able to catalyze the reaction.
The genes for VioA, VioB and VioE were amplificated via PCR with Golden Gate compatible overhangs from the biobrick VioABCE (Part: BBa_K274004).

By Golden Gate cloning the peroxisomal targeting sequence (PTS1) was attached to the C-terminus of every pathway protein. Combined with the other necessary parts of the toolbox they represent the level 1 plasmids.

The PTS-tag marks the proteins for the import into peroxisomes. This should first of all point out the functionality of the yeast’s natural import mechanism and also be the basis for demonstrating our own modeled PTS*, proving our designed orthogonal import mechanism. Furthermore we also aim to optimize the working conditions for the enzymes inside of the reaction room - the peroxisomes. For example to vary the pH with new membrane proteins such as bacteriorhodopsin. To secure this change, we can also check the current conditions by our designed sensors.

There are several methods to verify the pathway’s enzymes. First of all, violacein and several intermediates (prodeoxyviolacein, deoxyviolacein, proviolacein) are colorful and the production in yeast can be visualized easily. Furthermore we added a His-/Flag-tag to the N-terminus of every protein (see geneious plasmid cards) to confirm their expression via SDS page and western blot. After verifying the presence of the enzymes the next step is to test their functionality. Before performing in vivo experiments in yeast an in vitro assay was implemented. For this the three enzyme pathway leading to PDV was reconstructed, testing VioA, VioB and VioE. To enable the best conditions for the enzymes, the pathway was studied intensively and all needed cofactors were calculated and added to the in vitro reaction (see protocol prodeoxyviolacein in vitro assay). This included FAD, MgCl₂, catalase for decomposition of hydrogen peroxide, and the substrate L-tryptophan. The in vitro reaction was followed by qualitative analysis via HPLC and mass spectrometry.

Introduction

Our optogenetic toolbox enhancements can be divided into three subgroups: controllable protein import via Pex5, controllable protein import via Pex7 and controllable gene expression. Each of these sub-projects are of different design which will be illustrated in the following.

Pex5 import with LOV2

LOV2 is an optogenetic protein derived from Avena sativa’s Phototropin 1. In its dark state the J$_{\alpha}$-helix located at the C-terminus is bound to the core of the protein. Upon irradiation with blue light (~460 nm), a covalent bond between a cysteine residue on the LOV2 protein and a flavin mononucleotide chromophore causes the J$_{\alpha}$-helix to unfold, which in turn exposes the C-terminus (Spiltoir et al, 2016) . This property is very useful, as short amino acid sequences can be attached to this end of the LOV-protein, for example a PTS1. The idea for this project was to attach PTS1 to the C-terminus and the protein of interest to the N-terminus. Upon irradiation with blue light, the fusion protein would be imported into our compartment. We used a mutated version of LOV2 whose C-terminus has an increased dark-state binding affinity. This is caused by the substitutions and T406A and T407A. These mutations greatly reduce the possibility of the J$_{\alpha}$-helix being exposed in the dark state. Our PTS1 sequence consists of the amino acids LQSKL. As a proof of concept for this construct we fused sfGFP to its N-terminus:

**Figure 9.1** Level 1 plasmid containing the sequence coding for the GFP-LOV2-PTS1 fusion protein

This was done in order to visualize our experiment’s results during microscopy: upon successful import of the fusion protein, one would observe GFP fluorescence localized to our compartment. Otherwise, the whole cell would be illuminated.

Pex7 import

The idea behind this project is to initially block the protein of interest’s PTS2 with a fluorescent protein which can be removed by an optogenetically activated TEV-protease. For this project we use the protein Phytochrome-B from Arabidopsis thaliana and its interaction partner PIF6. These two proteins, also derived from Arabidopsis thaliana, bind together upon irradiation by red light (660 nm) and separate upon irradiation with far-red light (780 nm). We used this property to activate a split version of a TEV-protease whose split halves were each fused to one of the two optogenetic proteins.

The TEV-protease was obtained from the Biobrick BBa_K1319004. This variant contains the anti self-cleavage mutation S219V. Using overhang-PCR we created a split version of the Biobrick protease based on work done by Wehr et al, 2016 and the iGEM team Munich 2013. The split was made between amino acid 118 and 119.
Our construct for attaching proteins N-and C-terminally is highly variable: it consists only of a TEV-cleavage site, the PTS2 sequence and a short linker and was designed as a 3b-part for the yeast-toolbox. This means that we can attach any protein to its N- or C-terminus we desire. We planned on attaching different fluorescent proteins to each sides of the 3b-part in a level 1 ligation. For our experiment we planned on using the pairs mTurquoise-mVenus and GFP-mRuby.

**Figure 9.2** level 1 plasmid containing the sequence coding for the GFP-PTS2-Ruby fusion protein

The two other constructs were planned as follows: Phytochrome B was fused to the C-terminal TEV-half, PIF6 was fused to the N-terminal TEV-half. The PhyB-TEV2 part and the TEV1-PIF6 part were supposed to be inserted into a shared plasmid via a level 2 Golden Gate ligation. Finally, the level 2 plasmid and the remaining level 1 construct were to be co transformed into S. cerevisiae. Our experiment consisted of illuminating one sample with red light (660 nm) while keeping another sample in the dark. Fluorescence microscopy would then be used to check whether the import was successful [3]. If cleavage and subsequent protein import was successful, fluorescence of one protein would be localized to the compartment while that of the other would be observed throughout the cell.

Optogenetically controlled gene expression

This project is based on work done by Weber et al, 2016 . Using the interaction between Phytochrome B and PIF6 they designed an optogenetic switch for enabling and disabling transcription of a chosen gene. It is based on the tetracycline operon and the transcription factor VP16. The tetO operator is located upstream of a minimal promoter which in turn is located upstream of the gene of interest. The tetR repressor binds to the tetO sequence. Fused to it is PIF6. Phytochrome B is fused to the transcription factor VP16. Upon illumination with red light, Phytochrome binds to the tetR-PIF6 complex. VP16 is now located in close proximity to the minimal promoter, which enables the RNA-polymerase-2 to start transcription of the gene of interest.
We designed a promoter part for the Dueber toolbox which consists of tetO and the minimal promoter region. This can be used as a promoter in a level 1 ligation for any desired gene of interest (a GFP-tagged Pex11 in our example).

**Figure 9.3** Level 1 plasmid containing the sequence coding for the GFP-Pex11 fusion protein with the tetO-pmin promoter

Transformation into S. cerevisiae is accompanied by co-transformation of a level 2 plasmid containing both the tetR-PIF6 and PhyB-VP16 constructs:

**Figure 9.4** Level 2 plasmid containing the sequences coding for the PhyB-VP16 and tetR-PIF6 proteins respectively

Verification methods depend on which gene is expressed. See size and number and secretion for details.

For the future one could think of much more radical strategies for peroxisomal engineering with a final goal of a “minimal peroxisome” by redirecting metabolic pathways through changing the protein-localization-signal in the yeast genome. Additionally, endogenous metabolic pathways could be redirected to our novel artificial compartment for establishing a customized metabolism specifically tailored for the user's application.

Introduction

In order to achieve a fully controllable artificial compartment, the first step was to design a completely orthogonal import system. Next was the knockout of endogenous import systems. However, a few proteins are imported neither by the Pex5 nor the Pex7 import machinery. Therefore, specific genome engineering designs, such as knockouts, deleting or redirecting the protein localization could be utilized for the ultimate goal of creating a synthetic organelle.

Additionally, knockouts or genome integrations enable customization of the peroxisomal properties, such as membrane permeability, size/number, decoupling of peroxisomes from cytoskeleton and the peroxisomal metabolism.

All these strategies allow a rational design of an artificial compartment, which is fully engineerable regarding the proteome, metabolome and the entire peroxisomal environment.

Design of yeast multi -knockout strains

The Crispr Cas9 System

The demands on yeast engineering have significantly increased with the design of more complex systems and extensive metabolic pathways. Genetic techniques that have historically relied on marker recycling are unable to keep up with the ambitions of synthetic biologists. In recent years the Crispr Cas9 system has been used for several strain-engineering purposes, including:

Markerless integration of multiple genetic cassettes into selected genomic loci
Multiplexed and iterative gene knockouts without the need to recycle a marker
Precise genome editing – nucleotide substitutions, etc.

We utilized the Cas9 system as a tool for peroxisomal engineering and have adopted the existing toolbox from (Lee et al. 2015) and the complete cloning system which also provides the possibilities for genome integration and gene editing by Cas9. For this, two oligonucleotides have to be designed for targeting the Cas protein to the gene of interest.

Figure 1: Plasmid construction for the gRNA expression plasmid
Two oligos, containing the targeting sequence of the gRNA, have to be annealed and can then be integrated in the gRNA entry Vector by a Golden Gate reaction. Adapted from (Lee et al. 2015)

Several gRNA vectors can subsequently be assembled into one vector with a Cas9 expression cassette and then be transformed into yeast. The expression of Cas9 together with gene specific gRNA´s leads to double strand breakage followed by non-homologous end joining repair or homologous recombination, in case of added repair DNA (figure 3).

Figure 2: Plasmid construction for the expression plasmid containing Cas9 and gRNA´s
Vector for Cas9 and gRNA expression, assembled by a Golden Gate reaction, containing a URA marker, Cen6 yeast origin and a kanamycin resistance. Adapted from (Lee et al. 2015)

The combination of the Cas9 system with DNA repair sequences enables not only knockouts of peroxisomal proteins, but also allows redirecting protein localization by changing protein targeting signals or integration of linear DNA into yeast chromosomes. Genome engineering facilitates yeast strain development for customized peroxisomes.

Figure 3: Design of repair DNA sequences for homologous recombination after inducing double strand break by Cas9
Repair DNA sequences can be used to increase the efficiency for Cas9 guided knocking out of specific genes, but would also allow genomic integration of targeting signals or complete genes. Adapted from (Lee et al. 2015)

The peroxisomal proteome of yeast (saccharomyces cerevisiae)

The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (Kohlwein et al. 2013). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.

Gene	Required for growth on oleate	Expression induced by oleate	Enzyme/activity	Molecular mass (kDa)	Isoelectric point	Molecules per cell	Localization	Function
ß-Oxidation enzymes
PCS60 (FAT2)	No	Yes	Medium chain fatty acyl-CoA synthetase	60.5	9.98	8.770	Peripheral peroxisomal membrane and matrix	Activates fatty acids with a preference for medium chain lengths, C9-C13
FAT1	No	-	Very long chain fatty acyl-CoA synthetase and long chain fatty acid transporter	77.1	8.47	16,900	Lipid droplet, ER, peroxisome Three predicted TM	Activates fatty acids with a preference for very long chain lengths, C20–C26
POX1	Yes	Yes	Acyl-CoA- oxidase	84.0	8.73	ND	Peroxisomal matrix	Oxidation of acyl-CoA
CTA1	No	Yes	Catalase	58.6	7.46	623	Peroxisomal matrix	Degrades hydrogen peroxide produced by Pox1
FOX2 (POX2)	Yes	Yes	Multifunctional enzyme; 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase	98.7	9.75	ND	Peroxisomal matrix	-
POT1 (FOX3, POX3)	Yes	Yes	3-Ketoacyl-CoA thiolase	44.7	7.56	ND	Peroxisomal matrix	Cleaves 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA
DCI1 (ECI2)	-	-	Δ(3,5)-Δ(2,4)-dienoyl-CoA isomerase (putative)	30.1	8.83	ND	Peroxisomal matrix	Auxiliary enzyme of fatty acid β-oxidation; role in β-oxidation debated
SPS19 (SPX1)	Yes	Yes	2,4-Dienoyl-CoA reductase	31.1	9.67	ND	Peroxisomal matrix	Auxiliary enzyme of fatty acid β-oxidation
ECI1	Yes	Yes	Δ3, Δ2-enoyl-CoA isomerase	31.7	8.21	ND	Peroxisomal matrix	Auxiliary enzyme of fatty acid β-oxidation
TES1 (PTE1)	Yes	Yes	Acyl-CoA thioesterase	40.3	9.58	ND	Peroxisomal matrix	Auxiliary enzyme of fatty acid β-oxidation
MDH3	Yes	Yes	Malate dehydrogenase	37.3	10.00	3,300	Peroxisomal matrix	Required for the malate-oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+, part of the glyoxylate cycle
IDP3	Yes	Yes	NADP+ dependent isocitrate dehydrogenase	47.91	10.02	ND	Peroxisomal matrix	Required for the 2-ketoglutarate/isocitrate shuttle, exchanging peroxisomal NADP+ for cytosolic NADPH
CAT2	No	No	Carnitine acetyl-CoA transferase	77.2	8.34	470	Peroxisome mitochondria	Transfers activated acetyl groups to carnitine to form acetylcarnitine which can be shuttled across membranes
Glyoxylate cycle
CIT2	No	-	Citrate synthase	51.4	6.34	2,310	Peroxisomal matrix	Condensation of acetyl CoA and oxaloacetate to form citrate
MDH3	Yes	Yes	Malate dehydrogenase	37.3	10.00	3,300	Peroxisomal matrix	Required for the malate–oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+
MLS1	Yes	-	Malate synthase	62.8	7.18	ND	Peroxisomal protein	Required for utilization of nonfermentable carbon sources
Other peroxisome-associated enzyme activities
GPD1 (DAR1, HOR1, OSG1, OSR5	-	-	NAD-dependent glycerol-3-phosphate dehydrogenase	42.9	5.26	807	Peroxisome, cytosol, nucleus	Key enzyme of glycerol synthesis, essential for growth under osmotic stress
PNC1	-	-	Nicotinamidase	25.0	6.23	7,720	Peroxisome, cytosol	Converts nicotinamide to nicotinic acid as part of the NAD(+) salvage pathway
NPY1	-	-	NADH diphosphatase	43.5	6.26	846	Peroxisome cytosol	Hydrolyzes the pyrophosphate linkage in NADH and related nucleotides
STR3	-	-	Cystathionine β-lyase	51.8	7.96	ND	Peroxisome	Converts cystathionine into homocysteine
STR3	-	-	Cystathionine ß-lyase	51.8	7.96	ND	Peroxisome	Converts cystathionine into homocysteine
GTO1	-	-	ω-Class glutathione transferase	41.3	9.53	-	Peroxisome	Induced under oxidative stress
AAT2(ASP5)	-	Yes	Aspartate aminotransferase	46.1	8.50	7,700	Cytosol, peroxisome	Involved in nitrogen metabolism
PCD1	-	-	Nudix pyrophosphatase with specificity for coenzyme A and CoA derivatives	39.8	6.59	238	Peroxisome	May function to remove potentially toxic oxidized CoA disulfide from peroxisomes
LPX1	-	Yes	Triacylglycerol lipase	43.7	8.16	2,350	Peroxisomal matrix	-
Peroxisomal transporters
PXA1 (LPI1, PAL1, PAT2, SSH2	-	-	Subunit of a heterodimeric ATP-binding cassette transporter complex	100.0	10.34	ND	Peroxisomal membrane	Import of long-chain fatty acids into peroxisomes
PXA2 (PAT1)	-	-	Subunit of a heterodimeric ATP-binding cassette transporter complex	97.1	9.47	ND	Peroxisomal membrane	Import of long-chain fatty acids into peroxisomes
ANT1(YPR118C)	-	-	Adenine nucleotide transporter	36.4	10.6	2,250	Peroxisomal membrane	Involved in β-oxidation of medium-chain fatty acids
Peroxins
Pex1 (PAS1)	-	-	AAA ATPase	117.3	6.93	2,100	Peroxisomal membrane	Involved in recycling of Pex5, forms heterodimer with Pex6
Pex2 (RT1, PAS5)	-	-	E3 ubiquitin ligase	30.8	9.02	339	Peroxisomal membrane	RING finger protein, forms complex with Pex10 and Pex12. Involved in matrix protein import
Pex3 (PAS3)	-	-	-	50.7	6.29	1,400	Peroxisomal membrane	Required for proper localization of PMPs
Pex4 (PAS2, UBC10)	-	-	Ubiquitin conjugating enzyme	21.1	5.36	ND	Peroxisomal membrane	Involved in matrix protein import
Pex5 (PAS10)	-	-	Soluble PTS1 receptor	69.3	4.79	2,070	Cytosol and peroxisomal matrix	Required for import of PTS1-containing peroxisomal proteins, contains TPR domains
Pex6 (PAS8)	-	-	AAA ATPase	115.6	5.44	1,630	Peroxisomal membrane	Involved in recycling of Pex5, forms heterodimer with Pex1
Pex7 (PAS7, PEB1)	-	-	Soluble PTS2 receptor	42.3	8.34	589	Cytosol and peroxisomal matrix	Requires Pex18 and Pex21 for association to the receptor docking site, contains WD40 repeat
Pex8 (PAS6)	-	-	Intra peroxisomal organizer of the peroxisomal import machinery	68.2	7.62	538	Peroxisomal matrix and luminal membrane face	Pex5-cargo dissociation
Pex9	-	-	PTS-receptor	-	-	-	-	-
Pex10	-	-	E3 ubiquitin ligase	39.1	9.88	ND	Peroxisomal membrane	RING finger protein involved in Ubc4-dependent Pex5 ubiquitination. Forms complex with Pex2 and Pex12
Pex11 (PMP24, PMP 27)	-	-	-	26.9	10.65	1,630	Peroxisomal membrane	Involved in peroxisome fission, required for medium-chain fatty acid oxidation
Pex12 (PAS11)	-	-	E3 ubiquitin ligase	46.0	9.86	907	-	RING finger protein, forms complex with Pex2 and Pex10
Pex13 (PAS20)	-	-	Component of docking complex for Pex5 and Pex7	42.7	9.83	7,900	Peroxisomal membrane	Forms complex with Pex14 and Pex17
Pex14	-	-	Central component of the receptor docking complex	38.4	4.61	2,570	Peroxisomal membrane	Interacts with Pex13
Pex15 (PAS21)	-	-	-	43.7	8.42	1,070	Peroxisomal membrane	Recruitment of Pex6 to the peroxisomal membrane, tail anchored PMP
Pex17 (PAS9)	-	-	Component of docking complex for Pex5 and Pex7	23.2	10.24	656	Peroxisomal membrane	Forms complex with Pex13 and Pex14
Pex18	-	-	-	Required for PTS2 import	32.0	4.78	ND	Interacts with Pex7 partially redundant with Pex21
Pex19 (PAS12)	-	-	Chaperone and import receptor for newly synthesized PMP	38.7	4.08	5,350	Cytosol, peroxisome, farnesylated	Interacts with PMPs, involved in PMP sorting. Also interacts with Myo2 and contributes to peroxisome partitioning
Pex21	-	-	Required for PTS2 protein import	33.0	6.67	ND	Cytosol	Interacts with Pex7, partially redundant with Pex18
Pex22(YAF5)	-	-	Required for import of peroxisomal proteins	19.9	8.33	259	Peroxisomal membrane	Recruits Pex4 to the peroxisomal membrane
Pex25	-	-	Involved in the regulation of peroxisome size and maintenance, required for re-introduction of peroxisomes in peroxisome deficient cells	44.9	9.77	2,420	Peripheral peroxisomal membrane	Recruits GTPase RhoI to peroxisomes, interacts with homologous protein Pex27
Pex27	-	-	Involved in the regulation of peroxisome size and number	44.1	10.49	382	Peripheral peroxisomal membrane	Interacts with homologous protein Pex25
Pex28	-	-	Involved in the regulation of peroxisome size, number and distribution	66.1	7.09	ND	Peroxisomal membrane	May act upstream of Pex30, Pex31 and Pex 32
Pex29	-	-	63.5	6.8	5,040	Peroxisomal membrane	May act upstream of Pex30, Pex31 and Pex32
Pex30	-	-	Involved in the regulation of peroxisome number	59.5	5.59	4,570	Peroxisomal membrane	Negative regulator, partially functionally redundant with Pex31 and Pex32
Pex31	-	-	Involved in the regulation of peroxisome number	52.9	10.15	238	Peroxisomal membrane	Negative regulator, partially functionally redundant with Pex30 and Pex32
Pex32	-	-	Involved in the regulation of peroxisome number	48.6	9.14	ND	Peroxisomal membrane	Negative regulator partially functionally redundant with Pex30 and Pex31
Pex34	-	-	Involved in the regulation of peroxisome number	16.6	10.30	ND	Peroxisomal membrane	-
Peroxisome fission and inheritance
DYN2 (SLC1)	-	-	Light chain dynein	10.4	9.03	1,310	Cytosol	Microtubule motor protein
SEC20	-	-	v-SNARE	43.9	5.92	4,910	Golgi, ER	Involved in retrograde transport from the Golgi to the ER, interacts with the Dsl1 complex through Tip20
SEC39(DSL3)	-	-	Component of the Ds11p-tethering complex	82.4	4.65	1,840	ER, nuclear envelope	Proposed to be involved in protein secretion
DSL1 (RNS1)	-	-	Component of the ER target site that interacts with coatomer	88.1	4.69	8,970	Peripheral ER, Golgi membrane	Forms a complex with Sec39 and Tip20 that interacts with ER SNAREs, Sec20 and Use1
FIS1 (MDV2)	-	-	Required for peroxisome fission	17.7	9.87	2,410	Peroxisomal membrane mitochondria	Tail anchored protein recruits Dnm1 via Mdv1/Caf4; also involved in mitochondrial fission
DNM1	-	-	GTPase, dynamin like protein involved in peroxisome fission	85.0	5.25	9,620	-	Also involved in mitochondrial fission
VPS1 (GRD1, LAM1, SPO15, VPL1, VPT26)	-	-	GTPase, dynamin like protein involved in peroxisome fission	78.7	8.15	5,960	-	Also involved in vacuolar protein sorting
VPS34 (END12, PEP15, VPL7, VPT29, STT8, VPS7)	-	-	Phosphatidylinositol 3-kinase	100.9	7.79	1,080	-	Forms complex with Vps15
INP1	-	-	Involved in retention of peroxisomes in mother cells	47.3	8.34	639	Peroxisomal membrane	Recruited to the peroxisome by binding to Pex3
INP2	-	-	Myo2 receptor, involved in peroxisome inheritance	81.5	9.41	736	Peroxisomal membrane	-
RHO1	-	-	GTP binding protein of the Rho subfamily of Ras like proteins, involved in actin assembly at the peroxisome	23.2	6.07	ND	-	Involved in de novo peroxisome formation recruited to peroxisomes by Pex25

Knockout designs in our project

Pex 9

Pex9 is a recently discovered import receptor for PTS1 proteins, which is induced by oleate and is an import receptor for both malate synthase isoenzymes Mls1p and Mls2p. In order to get a completely empty reaction room, a Pex9 knockout was designed to prevent unintended protein import.

Pex 31 & Pex 32

It has been shown that knockouts of Pex31 and Pex32 leads to an increased Peroxisomal size, but additionally the membrane permeability was affected (Zhou et al. 2016). This effect can be used as a tool for engineering membrane permeability by knocking out or overexpress both genes. A knockout would lead to an increased permeability and one could think of an opposite effect in case of overexpression, but this has not been shown yet.

INP1

The INP1 knockout was designed after the skype call of Prof Dueber, who recommended us, decoupling of peroxisomes from cytoskeleton in order to improve the secretion efficiency. INP1 is responsible for the tethering of peroxisome, which would inhibit the secretion of peroxisomes.

POT1

The only protein, which is imported by the Pex7 import machinery in saccharomyces is the 3-ketoacyl-CoA thiolase (POT1). A knockout of POT1 would enable utilizing the Pex7 import for proteins of interest, which cannot be tagged at the C-terminus with pts1, without having unintended import of other enzymes.

Genomic integration of our novel Pex5 import receptor

After testing our new Pex5 import systems, which is completely orthogonal to the natural import, the next step would be to replace the endogenous system with our artificial import system. Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance (Figure 4). Afterwards the plasmid was transformed into the yeast strain which was created by our collaboration partner Aachen (double knockout strain Pex5 & Pex7).

Figure 4: Design of integration plasmid for integrating our orthogonal Pex5 import receptor.
Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance

The resulting yeast strain allows full control over the peroxisomal matrix proteome, by replacing the whole protein import machinery, which is the first step for creating our artificial compartment.

Outlook

Besides the genome engineering approaches, which were performed in our project one could think of more radical strategies for peroxisomal engineering. A final goal could be a “minimal peroxisome”, which contains only the proteins that are required for the biogenesis of the peroxisome and import of proteins and metabolites. On the one hand peroxisomal pathways could be redirected to cytosol or other organelles and one the other hand endogenous metabolic pathways could be redirected to our novel artificial compartment by changing the protein localization signal in the yeast genome with help of the Cas9 system. All these strategies would allow tremendous improvements for metabolic engineering applications by creating an artificial compartment, which can be rational designed and customized for specific metabolic pathways.

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-<article>
+    <article>
-<h1>Results</h1>
+        <div id="ToC"></div>
-<div id="ToC"></div>
+<h1>Design</h1>
+<p>We designed a novel toolbox to gain the complete control over all major functions of the peroxisome. The toolbox is our solution for improving the engineering workflow and predictability of synthetic constructs. Interested? Find out how.</p>
+<h2 id="Background">Scientific background</h2>
+<h3 id="RootProblem">The root problem</h3>
+<p>Synthetic biology is an engineering discipline. And while we are able to plan our constructs with tools like biobricks, a major difference to e.g. electrical engineering is that they are not nicely isolated on a chip, but surrounded by all types of interfering agents. One of the major issues regarding protein expression in a novel chassis is unwanted and unexpected crosstalk between engineered pathways and the native cellular processes of the production host. A further issue is toxicity of the products or intermediates of the pathway. Both can greatly change our system’s behaviour which in some cases leads us to having to trial-and-error find a solution, making our previously modeled optimization useless. </p>
+<h3 id="Approach">Our approach</h3>
+<p>The natural approach of organisms to deal with metabolic interference and toxic byproducts is subcellular compartmentalization. This has proven to be a functional solution in naturally occurring pathways in eukaryotes as well as new synthetic pathways for biotechnological application. Thus, the creation of a synthetic organelle presents a suitable strategy to increase the efficiency and yield of non-native pathways. A common approach is to build up artificial compartments from scratch. Many breakthroughs have been achieved in the last decade, however the creation of a fully synthetic compartment is yet a milestone to reach for. We on the contrary want to start by engineering artificial compartments through orthogonalization.</p>
-<h2>Introduction</h2>
+<p>The peroxisome is the ideal starting candidate as it has many advantages over other compartments, including it being able to import fully folded proteins and not being essential in yeast under optimal growth conditions. Our project’s aim is to create a toolbox for manipulating and creating customizable peroxisomes as a first step towards synthetic organelles.</p>
-<p>
-While there still is much that can be done to improve our toolbox further, we are nonetheless extremely proud of our achievements. The many months of lab work definitely payed off! Below you can find the results of our efforts.
-</p>
-<h2>Sub-projects</h2>
+<p>By modifying the import machinery of yeast peroxisomes only selected proteins will be imported into the peroxisomes leaving the researcher in full control over the content of the peroxisomal lumen. Furthermore our toolbox will include a secretion mechanism for the synthesized products, various intra-compartmental sensors, modules for the integration of proteins into the peroxisome membrane, as well as optogenetic control for some of these parts for a more precise spatiotemporal control.</p>
-<button class="accordion">
+<p>As a proof of concept for the functionality of the toolbox and the customizable compartment two metabolic pathways will be integrated into the altered peroxisome: (i) Violacein biosynthesis and (ii) nootkatone biosynthesis. Violacein, a bisindole formed by condensation of two tryptophan molecules, is a violet pigment and thus easy to quantify in the cell. Nootkatone on the other side is a natural compound found inside the peel of the grapefruit, which gives it its characteristic taste and smell. In addition, nootkatone is a natural mosquito and tick repellent that is already being commercially used and industrially manufactured. Unfortunately, the production costs are extremely high. Furthermore, the production of nootkatone inside yeast is challenging as it is toxic for yeast and thus, the production efficiency is rather low. A successful implementation of the synthetic compartment will show increased yields in the production of these compounds and showcase the potential of this approach for similar future applications.</p>
-  <h2 id="PEX5Import">PEX5 Import</h2>
-  <p>
-The orthogonalization of the Pex5 import mechanism was an ambitious and challenging task &minus; interested how we did? See our results!
-</p>
-</button>
-<div class="panel">
-<h3>MD simulations</h3>
-<p>
- The first experiments we performed in the wet lab are the tests of the receptors we modelled via molecular dynamics. As soon as we finished building our constructs, we transformed them into Pex5 knock out yeast cells. The results of this experiments can be seen in the following figure.
-</p>
-<div class="half-width">
-<figure>
-<img src="https://static.igem.org/mediawiki/2017/b/b9/Artico_815.png">
-<figcaption><strong>Figure 1.1: </strong>PEX5 variants R8 and R15 obtained in the course of molecular dynamics simulations. The figures show an unspecific localization of mTurquoise in the whole cytosol, meaning there is no functional import mechanism. Due to the indifferent results with any Pex5*&minus;PTS1* combinations, we show just two exemplary results.
-</figcaption>
-</figure>
-</div>
-<p>
+<!--
- The fluorescent signal of mTurquoise was detected in the whole cell of each modelled receptor&minus;peptide combination. This indicates that our receptors were not able to recognize the PTS-variants tagged to mTurquoise and thus we did not obtain any evidences regarding orthogonal peroxisomal protein import.
+<h3>Scientific background</h3>
-</p>
+<h4>Peroxisomal quick facts</h4>
+<
+ubiquitous single-membrane-bounded organelles
+assemble, multiply, or degrade in response to metabolic needs of the cell
+Can import folded and even oligomeric proteins
+Expendable in yeast under optimal growth conditions
+De novo biogenesis
+<h4>Peroxisome biogenesis and inheritance</h4>
+-->
-<h3>Pex5 variant R19</h3>
-<p>
+<h2 id="CloningStrategies">Cloning strategies and the Yeast Toolbox for Multipart-Assembly</h2>
- Our second approach for the modification of the importer Pex5 was our designed receptor R19. Based on published literature we built this receptor by replacing three amino acids within the Pex5 protein sequence of the wild type yeast. The corresponding modified PTS1* is characterized by its -SYY sequence at the very end of the peptide. Figure 1.2 displays a fluorescence microscopy image proving our artificial protein import system in a Pex5 deficient yeast strain.
-</p>
+<p>While describing our cloning strategies we mentioned several <i>levels</i>, which stand for different stages of our plasmids. They are further described in the work of J.M. Dueber and colleagues, who designed the well established yeast toolkit we used in this project
-<div class="max-width">
-<figure>
-<img src="https://static.igem.org/mediawiki/2017/0/02/Artico_lvl1results.png">
-<figcaption>
-	<strong>Figure 1.2: </strong>Pex5 variant R19 with the PTS1* and the two negative controls consisting of R19 with the wild-type PTS1 and the PTS1* cloned  in the wild-type strain
-</figcaption>
+  <a href="http://pubs.acs.org/doi/abs/10.1021/sb500366v ">  <abbr title="Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology, 4(9), 975-986.">
-</figure>
+        (Dueber)</abbr></a>.
-</div>
+The toolkit offers the possibility to design plasmids with desired antibiotic resistances, promoters as well as terminators from standardized parts. It also provides fluorescence proteins, protein-tags and many more useful components as part plasmids. These part plasmids are distinguished in different part types due to their specific overhangs to ensure their combination in the correct order (e.g. promoter - gene of interest - terminator) all in a versatile one-pot Golden Gate reaction without time-consuming conventional cloning steps.</p>
-<p>
-Our first results show that coexpression of R19 with mTurquoise tagged to PTS1* leads to import of the fluorescent reporter protein, indicated by localized fluorescence areas . The negative control consists of the wild type yeast strain carrying mTurquoise tagged with our designed PTS1* shows exactly the opposite: Fluorescence was detected in the whole cell, indicating that R19 is not capable to recognize and import the modified peroxisomal targeting signal with its cargo. Though, this figure does not prove that the reporter protein is located in the peroxisomes. Therefore we validated this results by coexpressing this import machinery with a peroxisomal marker protein as can be seen in the following.
-</p>
-<div class="max-width">
-<figure>
-<img src="https://static.igem.org/mediawiki/2017/2/20/Artico_r19lvl2col.png">
-<figcaption>
-	<strong>Figure 1.3: </strong>Pex5 variant R19 with the PTS1* and PTS*, co-transformed with the PEX13-mRuby construct &minus; the localization of mTurquoise tagged with PTS1* is clear and due to that import was successfully validated whereas the control did not show any signs of import.
-</figcaption>
+<div class="flex-row-2>
+<div><img src="https://static.igem.org/mediawiki/2017/8/81/T--Cologne-Duesseldorf--Toolbox_assembly.jpeg
 </div>
+<div><figure>
+ <img src="https://static.igem.org/mediawiki/2017/3/3d/T--cologne-duesseldorf--dueber_toolbox.png">
+    <figcaption><b>Figure 1: The yeast toolkit starter set comprises of 96 parts and vectors.</b> The eight primary part types can be further divided into subtypes.
-<div class="half-width">
+<a href="http://pubs.acs.org/doi/abs/10.1021/sb500366v "><abbr title="Lee, Michael E., et al. A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology 4.9 (2015): 975-986.">(Dueber)</abbr></a>
-<figure>
+</figcaption></figure></div>
-<img src="https://static.igem.org/mediawiki/2017/7/79/Artico_wtpwtpstar.png">
-<figcaption>
-	<strong>Figure 1.4: </strong>Wild type Pex5 with the PTS1* and wild type PTS1.
-</figcaption>
-</figure>
 </div>
+<p>The cloning steps regarding the plasmid <i>levels</i> are implemented in <i>E.coli</i> in order to reduce the required time to generate the final plasmids. The different <i>levels</i> are therefore defined by their part content and their antibiotic resistances. </p>
+<p>To generate a <i>level 0</i> plasmid, the gene of interest is ligated into the provided <i>level 0</i> backbone via Golden Gate assembly using the enzyme BsmBI. The backbone contains a resistance to Chloramphenicol, as well as an origin of replication, creating a very basic yet functional plasmid.</p>
-<p>
-Pex13, as an integral protein of the peroxisomal membrane, provides perfect features to mark the membrane in order to clarify whether the localized areas which were shown in figure 1.2 are indeed the peroxisomes. As described in our <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-3">experimental design</a>, we used Pex13's transmembrane domain and fused mRuby to it. Figure 1.3 above shows clearly the location of the peroxisomes as the fluorescent signal of mTurquoise is definitely located in the peroxisomes, which proves that R19 transports our cargo into the peroxisomes. On the contrary, mTurquoise is located in the whole cytosol in the strain with R19 and the natural Pts1, indicating that there is no functional protein import.
+<p>The <i>level 1</i> plasmid contains a promoter and terminator suited for <i>S. cerevisiae</i>. There is the possibility of including  a polyhistidine-tag if there is a need for Western blot analysis. The antibiotic resistance contained in the <i>level 1</i> plasmid changes from chloramphenicol to ampicillin which enables filtering out residual <i>level 0</i> plasmids contained in the Golden Gate product. Furthermore, the <i>Dueber toolbox</i> includes the possibility of designing GFP-Dropout cassettes. These are custom-built <i>level 1</i> backbones whose inserts are sfGFP as well as promoter and terminator suited for <i>E. coli</i>.  Upon a successful cloning step the GFP is replaced by the part(s) of interest, and correct colony shows a white colour. In case of a wrong ligation event colonies show a green fluorescence. This provides a very useful tool to detect unsuccessful cloned colonies. The enzyme used for <i>level 1</i> changes from BsmBI to BsaI to avoid any interference between different steps. </p>
-On the other hand we observed similar fluorescent signals in wild type yeasts that possess the modified PTS tagged to mTurquoise, as displayed in figure 1.4: Moreover, the figure shows the wild type yeast expressing mTurquoise tagged to the natural PTS1. Comparing both pictures, the wild type receptor is not capable to recognize our artificial PTS1* peptide and thus no import could be detected.
+<p>The <i>level 2</i> plasmid combines two or more genes of interest with their respective promoters, terminators and tags. The resistance changes from ampicillin to kanamycin. The enzyme of this step is BsmBI again. This level is useful, if the construct you are designing requires multiple genes to be transformed into one yeast strain.</p>
-<br>
-Conclusively, all our negative controls were not able to import mTurquoise into the peroxisomes, confirming the orthogonality of our artificial protein import mechanism.
-<br>
- <br>
-Finally, our results clarify that we established a synthetic protein import machinery, which works fully independent from the natural yeast peroxisomal protein import system. We were able to demonstrate the recognition of an artificial PTS1* sequence by our designed Pex5 receptor R19 and that the same receptor does no longer recognize the wild type PTS1 sequence. That means we accomplished to modify a highly conserved protein import mechanism without destroying its function but changing its affinity for our distinguished peptide sequence. This facilitates the possibility to utilize the primordial peroxisomes as an artificial cell compartment.
-</p>
+ <h3> Yeast nomenclature </h3>
+<p>To make it fast and easy to differentiate between endogenous and heterologous genes and gene products we decided to use <i> S. cerevisiae </i> nomenclature according to <a href="http://seq.yeastgenome.org/sgdpub/Saccharomyces_cerevisiae.pdf">yeastgenome.org</a>. </p>
+<p>Below nomenclature at the example of <b><u>y</u>our <u>f</u>avorite <u>g</u>ene 1, <i>YFG1</i></b> is explained. </p>
-<h3>Conclusion</h3>
+<table>
-<p>
+      <tr>
-As the relocalization of an enzymatic pathway like the nootkatone and violacein pathway depends on a working import machinery that selects specifically for certain cargo proteins, this subproject was and is a crucial part for our whole project.
+        <th>Letter code</th>
-				Our results show that we designed and established a new and orthogonal peroxisomal import system in <i>Saccharomyces cerevisiae</i>. We modified one of the most conserved import machineries within the domain of eukaryotes - no matter if it is plants, mammals or fungi. This opens up new possibilities for biotechnological applications since this import system can be used to shift toxic compound reactions into the natural stress-resistant peroxisomes and thereby increase the yield and efficiency of rare biomolecule production <em>in vivo</em>.
+        <th>Meaning</th>
-				Furthermore, we managed to make a big step further towards a synthetic cell: While many research groups try to build up a synthetic cell from scratch, we decided to build it up from the inside by subverting its natural functional systems and making it fully customizable and controllable.
+      </tr>
-				This is why our new import machinery shows the potential for biotechnology and real world applications.
+      <tr>
-</p>
+        <td><i>YFG1</i></td>
-<div class="callout">
+        <td><b>Y</b>our <b>f</b>avorite <b>g</b>ene <i>S. cerevisiae</i> wild type allele</td>
-<h3>Violacein assay</h3>
+       </tr>
-<p>
+     <tr>
- The Violacein assay would have been an easy and fast application to identify possible yeast colonies that possess a functional new import machinery. It would have eased  finding a fitting peroxisomal targeting signal for our Pex5 variants due to the huge number of different PTS1 variants we could have screened using this method. Because time ran out and we already found a fitting import machinery in our receptor R19 and our modified PTS1* P*, we decided to discontinue this experiment and focused on the validation of our previously generated results.
+        <td><i>yfg1</i>Δ</td>
-</p>
+        <td>Gene deletion of <b>y</b>our <b>f</b>avorite <b>g</b>ene</td>
-</div>
+       </tr>
-</div>
+      <tr>
+        <td>Yfg1</td>
+        <td>Protein product of <i>YFG1</1></td>
+       </tr>
+      <tr>
+        <td>YFG2</td>
+        <td>A heterologous gene product from mammalian cells</td>
+       </tr>
+</table>
+<h2>Design of our sub-projects</h2>
 <button class="accordion">
-   <h2 id="Pex7Import">Pex7 Import</h2>
+   <h2 id="ProteinImport">Protein Import</h2>
-   <p>Abstract</p>
+   <p>Control over the peroxisomal proteome is an essential requirement for our project. Our objective is the obtainment of a fully orthogonal import system by utilizing the two peroxins Pex5 and Pex7.</p>
    </button>
 <div class="panel">
-  <h3>Heading</h3>
+ <h3 id="Pex5">Engineering of Pex5 and PTS1</h4>
-<p>
+ <p>
-The biased mutagenesis of the PTS2 could be characterized with a split-variant of YFP ( yellow fluorescent protein) or a split-luciferase. YFP tends to self assemble, consequently appropriate internal controls have to be designed <a href="http://www.mdpi.com/1422-0067/15/6/9628 "><abbr title="Horstman, A., Tonaco I., Boutilier K. and Immink R. A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies. International journal of molecular sciences 15.6 (2014): 9628-9643">(Horstman)</abbr></a>. Luciferase is highly efficient because almost all energy is converted into light, the protein is thus very sensitive <a href="https://www.ncbi.nlm.nih.gov/pubmed/25002334 "><abbr title="Azad, T., Tashakor A., and Hosseinkhani S. Split-luciferase complementary assay: applications, recent developments, and future perspectives. Analytical and bioanalytical chemistry 406.23 (2014): 5541-5560.">(Azad)</abbr></a>. It offers a suitable alternative to YFP as a single readout protein. We expected to detect luminescence as well in the actual samples as in the negative control containing no peroxisomal targeting signals due to split assembly in the cytoplasm. Unfortunately no suitable method to measure luminescence in the peroxisomes was established in this project.
+ </p><h4 id="h5-1">Designing our receptors</h5>
-Prerequisite for detecting luminescence is the availability of the substrate luciferin. It does not diffuse into the peroxisome in concentrations high enough for the luminescence reaction and becomes the limiting factor <a href="https://www.ncbi.nlm.nih.gov/pubmed/14558144      "><abbr title="Leskinen, P., Virta M. and Karp M. One‐step measurement of firefly luciferase activity in yeast. Yeast 20.13 (2003): 1109-1113.">(Leskinen)</abbr></a>.</p>
+ <p>To achieve the engineering of an orthogonal import pathway, we followed two approaches regarding Pex5. The first is based on targeted mutagenesis based on educated guesses which is first verified by <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#h2-1">molecular dynamics</a> and later experimentally in the laboratory.
-<p>An alternative step to verify the localization of the assembled split-luciferase in the peroxisome is to extract and purify the organelles. Prof. Ralph Erdmann established this method: a cell-free homogenate is created and the organelles are pelleted by centrifugation steps <a href="https://www.ncbi.nlm.nih.gov/pubmed/26330630 "><abbr title="Cramer, J., Effelsberg, D., Girzalsky, W. and Erdmann, R. Isolation of Peroxisomes from Yeast. Cold Spring Harbor Protocols 2015.9 (2015): pdb-top074500.">(Cramer)</abbr></a>. This workflow can be used to characterize the content of the purified peroxisomes by Western blot analysis.</p>
+   The second approach is based on a recently published paper: We searched for literature dealing with the modification of the peroxisomal import machinery. During our research we came across a <a href="https://www.nature.com/articles/s41467-017-00487-7">paper</a> of Cross <i>et al.</i>, published in 2017, in which they present a synthetic construct of the Pex5 protein, partly <i>Arabidopsis thaliana</i> and partly <i>Physcomitrella patens</i>. Compared to the wild type Pex5, this one shows different binding affinities since it interacts with a PTS1* variant that does not interact with the wild type Pex5 <a href=" https://www.nature.com/articles/s41467-017-00487-7 "><abbr title=" Cross, Laura L.; Paudyal, Rupesh; Kamisugi, Yasuko; Berry, Alan; Cuming, Andrew C.; Baker, Alison; Warriner, Stuart L. (2017): Towards designer organelles by subverting the peroxisomal import pathway. In: Nature communications 8 (1), S. 454."> Cross <i>et al. </i> </abbr></a>. Since the protein sequences of yeast's and plants's Pex5 differ quite a lot, we aligned both sequences to understand where the mutations were set. </p>
-<p>To measure the import efficiency of a vast amount of targeting sequences via split-luciferase one needs to ensure a sufficient luciferin concentration in the peroxisome. Therefore luciferin importer have to be implemented in the peroxisomal membrane. Since this implies a huge cloning effort split-luciferase is not suitable for high throughput screening. ´</p>
+   <figure>
+<div class="flex-row-2">
+     <div>
+     <img src="https://static.igem.org/mediawiki/2017/b/be/Artico_atp5_yp5.png">
+     <figcaption>
+         <strong>Figure 1.1:</strong> Alignment of the <i>Arabidopsis thaliana's</i> Pex5 and <i>Saccharomyces cerevisiae</i>Pex5
+     </figcaption>
+	</div>
+     <div>
+     <img src="https://static.igem.org/mediawiki/2017/9/96/Artico_R19WT.png">
+     <figcaption>
+         <strong>Figure 1.2:</strong> Alignment of the yeast’s Pex5 with the Pex5 variant R19
+     </figcaption>
+	</div>
+</div>
+   </figure>
-<p>At the random mutagenesis approach one expected green and white colonies indicating varying import efficiencies.  The colonies containing “DNK” or “NNN” substitutions in the variable PTS2 region show a wide range of colours between white and dark green. The wild type PTS2 colonies depict a constant light green colour. The negative control containing VioE without a PTS2 shows a dark green colour in every colony. </p>
+<p>The alignment shows three red marked amino acids we changed in our receptor sequence. Interestingly, these mutations are located within the TPR motifs of our Pex5 protein and this persuaded us to try out this receptor, we call it R19. Due to lack of time we tested this Pex5 variant in silico and <i>in vivo</i> simultaneously. We started molecular dynamics simulations with a couple of PTS variants that we already tested with our previous designs &minus; one of them was actually the variant they used in the paper (<i>YQSYY</i>). The details and results of our structural modeling can be found in the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#h2-1">modeling section</a>.
+<br>
+Furthermore, we synthesized this variant and together with two receptors we designed based on educated guesses we got three receptors for our experimental work.</p>
-<img src="https://static.igem.org/mediawiki/2017/9/9d/T--cologne-duesseldorf--Platte_Sternchen_3.jpg  ">
+ <h4>Experimental design</h5>
-<figcaption><b>Figure 2.1: Colonies of the PTS2 library show a colour range of white to green.</b> It indicates targeting sequences of different import efficiencies. White colour correlates with a strong import, VioE is targeted to the peroxisome and hence no green product (PDV) is detectable.  </figcaption>
+ <p>
+   Verification of peroxisomal protein import was performed  by tagging the fluorescent protein mTurquoise with our designed PTS variants. Additionally, a peroxisomal membrane protein was used to ensure peroxisomal localization. For that reason, we chose the transmembrane domain of Pex13 tagged with the fluorescent protein mRuby.
+ </p>
+ <h5>Pex13−mRuby</h6>
+ <p>We used the peroxisomal membrane protein Pex13 as a fluorescent marker &minus; by just using the transmembrane domain of Pex13 with a short linker, we make sure that it has no influence on the peroxisomal features. To obtain a higher differentiation from mTurquoise, which we use for another construct, we chose to work with mRuby. Literature research revealed that such constructs have been tested before − <a href="https://www.ncbi.nlm.nih.gov/pubmed/15133130"><abbr title="Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals"> Erdmann<i> et al.</i> (2004)</abbr></a> described a construct containing only Pex13<sub>200-310</sub> with a C-terminal GFP.</p>
+   <figure>
+     <img src="https://static.igem.org/mediawiki/2017/d/d4/Artico_P13RUBY.png">
+     <figcaption>
+         <strong>Figure 1.3:</strong> Pex13 construct with C-terminal mRuby.
+     </figcaption>
+   </figure>
+   <h5>Pex5 variant</h6>
+   <p>In order to achieve an orthogonal peroxisomal protein import machinery we used a Pex5 knockout yeast strain in which we transformed our artificial  Pex5 variant containing a modified PTS1 binding pocket. Our variation facilitates the detection of a non native PTS1 variant instead of the wild type PTS1. The construct contains a medium strength promoter, the Pex5 gene and a terminator. The remaining plasmid parts can be seen in the plasmid map below.
+     </p>
-<p>Therefore we were able to generate targeting  sequences of different effectivities.  Subsequently the OD<sub>600</sub>  and the fluorescence with an  excitation wavelength of 535 nm and emission wavelength of 585 nm were measured. According to
+     <figure>
-<a href="https://www.nature.com/articles/ncomms11152"><abbr title="DeLoache, W. C., Russ, Z. N., & Dueber, J. E. (2016). Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways. Nature communications, 7.
+       <img src="https://static.igem.org/mediawiki/2017/d/da/Artico_pex5variant.png">
-">(DeLoache)</abbr></a>
+       <figcaption><strong>Figure 1.4:</strong> Pex5 gene variant.</figcaption>
+     </figure>
- production of PDV was associated with a yet unknown red fluorescent product, detectable at the described wavelength.  The import efficiency can be defined as the fluorescence per OD<sub>600</sub>.  A wide distribution of different values were observed indicating a broad variety of different PTS2 versions.</p>
+    <h5>mTurquoise−PTS</h6>
+   <p>
+     Our approach for import verification is based on the fluorescent protein mTurquoise tagged with our modeled PTS variants. After several promoter tests with different strengths, we decided to express this construct only in low amounts, since this was the most suitable possibility to detect potential mTurquoise localization.
+     <br>
+     Our construct is depicted in the figure below.
+		</p>
+     <figure>
+       <img src="https://static.igem.org/mediawiki/2017/b/bd/Artico_fppts.png">
+       <figcaption><strong>Figure 1.5:</strong> Fluorescent protein mTurquoise tagged with the PTS variant.</figcaption>
+     </figure>
+   <h5>Combination of our constructs</h6>
+   <p>To combine our constructs, we cloned our Pex5 and mTurquoise constructs into a level 2 plasmid portrayed below.</p>
+     <figure>
+       <img src="https://static.igem.org/mediawiki/2017/8/88/Artico_P5FP.png">
+       <figcaption><strong>Figure 1.6:</strong> Level 2 plasmid containing the Pex5 gene and the fluorescent protein.</figcaption>
+     </figure>
+     <p>Subsequently,  co-transformation of the Pex13−mRuby plasmid and the level 2 plasmid was performed in order to verify peroxisomal colocalization.</p>
+ <h5>PTS screening</h6>
+<p>Trusting on our targeted approach alone seemed risky − that is why we planned a PTS screening to find the most favorable PTS for our three receptors. <a href="https://www.nature.com/articles/ncomms11152"><abbr title="Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways.">Dueber <i>et al.</i> (2016)</abbr></a> used the Violacein assay for a similar purpose. They screened for the best PTS for the wild type receptor and were successful. Hence another subproject of our team is the integration of the Violacein pathway into the peroxisome (<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Violacein">Violacein</a>), we were already supplied with all necessary enzymes − VioA, VioB and VioE.</p>
-<img src="https://static.igem.org/mediawiki/2017/1/17/T--cologne-duesseldorf--PDV_test1.jpg">
+<!-- eigenen pathway (danke violacein team) oder das aus dem paper?
+<div class="half-width">
+	<figure>
+		<img src="https://static.igem.org/mediawiki/2017/1/13/Artico_dueberassay.jpg">
+			<figcaption><strong>Figure 1.7:</strong> Violacein Assay (<a href="https://www.nature.com/articles/ncomms11152"><abbr="Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways.">Dueber et al., 2016</a>)</figcaption>
+	</figure>
+</div>
+-->
+<figure>
+		<img src="https://static.igem.org/mediawiki/parts/a/a2/T--Cologne-Duesseldorf--PDVpathway.png">
+<figcaption>
+<strong>Figure 1.8:</strong> Pathway leading to the production of prodeoxyviolacein &minus; the assay is based on the green color to identify colonies without functional import mechanism.
+</figcaption>
+</figure>
-<figcaption> <b>Figure 2.2: Fluorescence per OD<sub>600</sub> of VioABE transformants are shown.</b> For all tested transformants, fluorescence with an  excitation wavelength of 535 nm (λ<sub>Ex</sub>: 535 nm) and emission wavelength of 585 nm (λ<sub>Em</sub>: 585 nm) per OD<sub>600</sub>, referring on yeast cell count, was calculated. Results show a large distribution of import efficiency and therefore different PTS2 versions.
+     <p>The figure above shows the principles of the assay. VioA and VioB are localized in the cytosol and lead to the production of the IPA imine dimer while VioE is tagged with a PTS1 variant. Successful import leads to white colonies whereas missing import results in green colonies due to the cytosolic production of prodeoxyviolacein.
-  </figcaption>
+     <br>
+     Our rests upon the following two plasmids which are co-transformed into yeast.</p>
+<figure>
+     <div class="flex-row-2">
+				<div>
+         <img src="https://static.igem.org/mediawiki/2017/2/20/Artico_vioabr.png">
+				</div>
+				<div>
+         <img src="https://static.igem.org/mediawiki/2017/5/5c/Artico_vioeassay.png">
+				</div>
+     </div>
+       <figcaption>
+         <strong>Figure 1.9:</strong> Plasmids used for Violacein assay. The left one carries the coding sequence for the Pex5 variant, VioA and VioB whereas the right one contains the gene for VioE plus the attached PTS1 variant.
+       </figcaption>
+</figure>
+     <p>As shown above, we created one plasmid containing VioA, VioB and one of our Pex5 variants while the other plasmid only contained VioE. We then designed primers which bind to the VioE plasmid to amplify the whole plasmid except the terminator − random PTS1 variants were attached to VioE with the help of a random primer library. Following up, we did the ligation with the corresponding terminator and obtained a mix of several different VioE-PTS1 plasmids.
+     <br>
+     After plasmid amplification in <i>Escherichia coli</i> we then co-transformed yeast with the two constructs and waited for the colonies to grow. With the yeast growing, prodeoxyviolacein should be produced in yeast cells with absent import (green color) and the IPA imine dimer (white color) should be produced in those with functional import.
+<br>
+Plasmid preparation of those with white color and subsequent sequencing leads to the identification of functional PTS1 variants. Afterwards, we repeat the cloning steps described before to obtain a mTurquoise&minus;PTS1* construct and co-transform it with the corresponding Pex5 variant. Eventually, the correct localization of mTurquoise tagged with these PTS1 variants provides proof for its function.</p>
-<p>A high value correlates with an inefficient targeting sequence since VioE is not imported into the peroxisome with the respective sequence. A low fluorescence per OD<sub>600</sub> indicates a strong targeting sequence resulting in a low VioE concentration in the cytoplasm and no conversion of Tryptophan to PDV.</p>
+<h3>Mutagenesis of PTS2</h4>
-<p>The next step would be to isolate the plasmids of promising yeast strains and sequence them. Subsequently mutations leading to an increased import can be characterized and organized in a library consisting of different parts with varying import efficencies. </p>
+ <p>Imagine you need different protein concentrations in your artificial compartment. What to do? Take our modified PTS2 sequences with varying import efficiencies. </p>
+<p>To characterize the import efficiency for the site-directed PTS2 firefly luciferase was used. Luciferase is a luminescent protein which can be split in a C- and a N-terminal part. Only when combined, luminescence can be detected. To measure the import efficiency the two parts will be expressed and imported into the peroxisome in a separated way. The smaller part (Split2) of the split luciferase will be brought into the peroxisome first via the PTS1 dependent pathway. The other part is imported via the respective modified PTS2 sequence. The better this sequence is recognized by Pex7, the stronger the luminescence of the assembled luciferase can be detected in the peroxisome. There is a chance of split parts of the luciferase assembling in the cytosol if the import is too slow. To avoid wrong conclusions of the luciferase localisation, we designed a negative control experiment. It includes a split luciferase similar to the one used in the initial experiment, but without the peroxisomal targeting sequence. Consequently there will be no import into the peroxisome. If we subtract the luminescence of the negative control experiment from the luminescence of the main experiment we can define the degree of import.</p>
+<p>In addition to a directed approach according to Kunze and colleagues we also want to perform a random  mutagenesis experiment to alter the five variable amino acids of the core region of the PTS2 sequence in an unbiased manner. The aim is to generate a library of different peroxisomal PTS2. The 15 nucleotides are assembled by chance. In the DNA synthesis this sequence will either be described as [NNN]<sub>5</sub> or [DNK]<sub>5</sub>. N stands for all four nucleotides mixed, K for either G or T and D for A,G or T. The “DNK” composition prohibits two out of three termination codons. Additionally with this library the amino acid frequency is improved towards a balanced ratio in between the different kinds <a href="https://www.ncbi.nlm.nih.gov/pubmed/27025684 "><abbr title="DeLoache, W. C., Russ, Z. N., & Dueber, J. E. (2016). Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways. Nature communications, 7.">(Dueber)</abbr></a>.</p>
+<p>Each approach could generate up to 415 DNA sequences, which is roughly 1,07 billion. On the level of the amino acid sequence there are 3,2 million possibilities, since each residue can be taken by 20 different amino acids.  For the assay we therefore need a high throughput method. </p>
+<p>We adapted work of DeLoache, Russ and Dueber using the violacein pathway to measure the import effectiveness of tripeptides. The pathway consists of Violacein A (VioA), Violacein B (VioB) and Violacein E (VioE). It converts tryptophan into the green product prodeoxyviolacein (PDV). The first two enzymes, VioA and VioB, are expressed in the cytosol, and the third one, VioE, is targeted to the peroxisome with a PTS1 sequence. The degree of import can be measured by the intensity of green colour of the colonies. An efficient import signal leads to a strong import of the VioE into the peroxisome and subsequently to white colonies, because the intermediates cannot diffuse into the peroxisome to its respective enzyme. DeLoache <i>et al.</i> showed that there is a proportional correlation between the concentration of the green product PDV and a red fluorescent substance. The concentration of this product displays the import efficiency of the respective sequence.</p>
+<p>This assay has been used for the evaluation of the generated PTS2 sequences. The VioE-PTS2 plasmids are harvested and cotransformed with a VioA-VioB plasmid. Each plasmid contains a specific auxotrophy marker. Consequently every growing colony contains both plasmids. To evaluate the respective sequence the concentration of the red fluorescence is measured. The more fluorescence is detected the more VioE is in the cytosol. Therefore the respective PTS2 is not that efficient. The other way around a low concentration of the fluorescent substance correlates with an efficient import via the respective PTS2. </p>
 </div>
 <button class="accordion">
-   <h2 id="SizeAndNumber">Size and Number</h2>
+   <h2 id=”Membrane_Integration”>Membrane Integration</h2>
-   <p>The big picture! Read through our results and find out how we achieved the full control of our compartment’s size and number via Pex34 overexpression!</p>
+   <p>In our design we worked with both,  the cytosolic import way and the the ER-dependant one. We first tested our hypothesis and then created membrane anchors that can be fused to you protein of interest C-terminally as well as N-terminal. This allows you to add proteins to the compartment that never been introduced to the peroxisomal membrane before.</p>
    </button>
 <div class="panel">
-   <h3>Heading</h3>
-    <p>
+<h3>Scientific background</h3>
+<p>Peroxisomal membrane proteins are synthesized on free polysomes in the cytosol and afterwards integrated into the membrane via two major pathways: one dependent on the endoplasmatic reticulum and one dependent on PEX19 and PEX3.
+<h3>PEX19-dependent</h3>
+<h3>ER-dependent</h3>
+<h3>Experimental Work/Design</h3>
+<p>In order to test our hypothesis we fused the last 59 amino acids of the C-terminus of human <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> (AA 246-305) to a red fluorescent protein, to further elucidate the  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>/Pex19-dependent import. mRuby is generally used as a marker in combination with a fluorescent microscope to visualize the localization of the fusion protein. The C-terminus of <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> contains a helical signal-anchor, which serves as both, a mPTS and transmembrane domain. We designed our construct with mRuby2 fused to the N-terminal side of the <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a>-C-terminus, this way the mRuby should face the cytosolic side of the peroxisomal membrane.
+Quite similar to our mRuby-<a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> approach, we designed a construct for the ER-dependent import. Therefore, we fused the mRuby2 fluorescent protein to the N-terminus of  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> (AA 1-39). This construct should be N-terminally anchored in the peroxisomal membrane, with mRuby2 again facing the cellular lumen.</p>
+<img src="https://static.igem.org/mediawiki/2017/4/4e/PMP_figures_Pex3_and_PEX26.png">
+<p>Our main goal is to introduce a rather complex membrane protein to the peroxisome that can alter specific traits. For that we fused the  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> N-terminus (AA 1-39) to a <i>Halobacterium salinarum</i> <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> protein (AA 16-262), replacing the first 16 amino acids (<a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>-BacR). The original archaeal <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> acts as a proton pump by capturing light energy to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy.
+Our assumption is that the first transmembrane segment determines the orientation of the following protein and that therefore due to the N-terminal anchoring signal the <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> will be inserted in reverse orientation, pumping the protons into the peroxisome. This way the pH of the peroxisomal lumen could actively be controlled and adjusted.</p>
+<p>Finally, our project involved combining the work of other subteams to verify the localization of our constructs in the peroxisome and analyze the effects they have on the import. Therefore, we are using the superfolded-GFP protein, another fluorescent marker, which is in our case fused to the peroxisomal import sequence PTS1, and a version of Pex11 that is fused to the fluorescent marker Venus. Both markers emit light in the green light spectrum, were as mRuby2 emits light in the red part of the spectrum, giving us a strong contrast and an easy way of differentiating between the two under the fluorescent microscope.  </p>
+<p>To physically create our constructs, we researched the DNA sequences of <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a>,  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> and <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> via UniProt and pre-designed our fusion constructs with the software tool „Geneious“. We ordered the synthesis of three separate parts (<a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>, <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> and  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>-BacR) from IDT. To ease out the process of assembling our plasmids, we used the „<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-2">Dueber Toolbox</a>", containing various parts such as promoters and terminators, to tailor the plasmids specific to your needs. Finally, to combine all the selected parts, we used the „Golden Gate” assembly method.</p>
-    </p>
 </div>
 <button class="accordion">
    <h2 id="Secretion">Secretion</h2>
-   <p>We are the first to implement  a completely new secretion mechanism in<i> S. cerevisiae</i>, which is able to secrete the peroxisomes` insides. This mechanism does not occur naturally in yeast. First we were able to show that our membran anchors Pex15 and PEX26 are integrated into the peroxisomal membrane. By fusing our v-SNARE Snc1 to different membrane anchor we were able to cause secretion of the content of our peroxisomes to the extracellular supernatant. When Snc1 is fused to Pex15 via a linker we measured the highest amount of secretion.</p>
+   <p>For the peroxisome secretion in <i>S. cerevisiae</i> we designed fusion proteins of the v-SNARE <a href="http://parts.igem.org/Part:BBa_K2271060">  Snc1 </a> with different peroxisomal membrane anchors.
+We tested the constructs using a GUS Assay. The assays were performed using transformants of the strain BY4742.</p>
    </button>
 <div class="panel">
-<p>To check whether our membrane anchors localize in the peroxisomal membrane we used a Zeiss Elyra PS microscope. For Pex15 we observed localization using a construct with mVenus fused to the C-terminus of the Pex15 version we used. The fluorescence in the cells showed the typical shape of a peroxisomal localization (Figure 3.1). Shown in figure 3.2 is the localization of PEX26, which was highlighted using an N-terminal fusion with the fluorescent Protein mRuby. The microscopy pictures also indicate peroxisomal localization and even an co-localization with sfGFP-PTS1</p>
+<h3>Experimental Design </h3>
+<p>We will adapt the system of  Sagt and colleagues  to secrete the content of our modified compartments <a href=" https://www.ncbi.nlm.nih.gov/pubmed/19457257">
+  <abbr title=" Sagt, C. M., ten Haaft, P. J., Minneboo, I. M., Hartog, M. P., Damveld, R. A., van der Laan, J. M., ... & van der Klei, I. (2009). Peroxicretion: a novel secretion pathway in the eukaryotic cell. BMC biotechnology, 9(1), 48.
+">
+      (Sagt <i> et al</i>, 2009)
+  </abbr>
+</a>. <br>
+For the application of this system in <i>S. cerevisiae</i> we use a truncated version of the v-SNARE Snc1 to decorate our compartments (Figure 3.1) <a href=" http://www.jbc.org/content/272/26/16591.short">
+  <abbr title=" Gerst, J. E. (1997). Conserved α-helical segments on yeast homologs of the synaptobrevin/VAMP family of v-SNAREs mediate exocytic function. Journal of Biological Chemistry, 272(26), 16591-16598.
-<figure>
+">
+      (Gerst <i> et al</i>, 1997)
+  </abbr>
+</a>. <br>
-        <img src="https://static.igem.org/mediawiki/2017/a/a4/T--Cologne-Duesseldorf--Pex15_mVenus.png">
+ <figure>
+   <img src="https://static.igem.org/mediawiki/2017/f/fe/T--Cologne-Duesseldorf--SecretionSNAREgerstwiki.png">
-        <figcaption><b> Figure 3.1 Microscopic validation of the peroxisomal membrane anchor Pex15. </b> Microscopy pictures were taken with a Zeiss Elyra PS. The signal for mVenus-Pex15 is shown in yellow. The picture validates the peroxisomal membrane localization of Pex15</figcaption>
+   <figcaption> <b>Figure 3.1 A diagram of the general domain structure of Snc1. </b> V is a variable domain which is not important for the binding to the t-SNARE. TM is the transmembrane domain. H1 and H2 are the a-helical segments, forming the SNAREpin with the t-SNARE  <a href=" http://www.jbc.org/content/272/26/16591.short">
+  <abbr title=" Gerst, J. E. (1997). Conserved α-helical segments on yeast homologs of the synaptobrevin/VAMP family of v-SNAREs mediate exocytic function. Journal of Biological Chemistry, 272(26), 16591-16598.
+">
+      (Gerst <i> et al</i>, 1997)
+  </abbr>
+</a></figcaption>
 </figure>
-<br>
-<br>
+<p>To decorate the compartments with the SNARE we use a <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#MembraneIntegration"> peroxisomal transmembrane protein </a>  . In our case we use the proteins Pex15 or Pex26, which were further investigated in another sub project, and fuse Snc1 to the N-terminus. We expressed these constructs of membrane anchor and Snc1 constitutively under control of the RPL18B promotor. In case of Pex15 we used a truncated version, lacking a large part of the N-terminus, only consisting of the transmembrane domain (315-383) (Figure 3.1). For Pex26 we use the truncated version published in Halbach et al. <a href=" http://jcs.biologists.org/content/119/12/2508">
+  <abbr title=" Halbach, A., Landgraf, C., Lorenzen, S., Rosenkranz, K., Volkmer-Engert, R., Erdmann, R., & Rottensteiner, H. (2006). Targeting of the tail-anchored peroxisomal membrane proteins Pex26 and Pex15 occurs through C-terminal Pex19-binding sites. Journal of cell science, 119(12), 2508-2517.
+">
+      (Halbach <i> et al</i>, 2006)
+  </abbr>
+</a>.
+</p>
 <figure>
-         <img src="https://static.igem.org/mediawiki/2017/3/3d/T--Cologne-Duesseldorf--Pex26_overlay.png">
+         <img src="https://static.igem.org/mediawiki/2017/7/78/T--Cologne-Duesseldorf--SecretionSNAREpicwiki.png">
-         <figcaption><b>Figure 3.2 Validation of the membrane anchor Peroxisomal membrane anchor PEX26.</b> Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). It shows a typical  peroxisomal shape. The signal for the membrane marker mRuby-PEX26 is shown in yellow. Both signals co localizing in the overlay. Which indicates that PEX26 is viable as a peroxisomal membrane marker.</figcaption>
+         <figcaption><b>Figure 3.2 Concept of secreting peroxisomal contents to the supernatant. </b>For the secretion, the membrane anchor Pex15 or Pex26 is used. This anchor is used to decorate peroxisomes or our modified compartments with the v-SNARE Snc1. For the secretion Snc1 interacts with the t-SNAREs in the cell membrane. Induced from this interaction the vesicle and cell membrane fuse and the content of the compartment is secreted to the supernatant.</figcaption>
 </figure>
+<p>We verified our secretion using beta-glucuronidase (GUS) as a reporter protein. In 2012 Stock and colleagues described the GUS reporter assay for unconventional secretion <a href=" https://www.ncbi.nlm.nih.gov/pubmed/22446315 ">
+  <abbr title=" Stock, J., Sarkari, P., Kreibich, S., Brefort, T., Feldbrügge, M., & Schipper, K. (2012). Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis. Journal of biotechnology, 161(2), 80-91.
-<p>Next we measured secretion of compounds that are inside our artificial compartment, using a liquid <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Secretion"> GUS-assay </a>. Towards this purpose we coexpressed GUS-PTS1 and  Snc1 fused to different membrane anchors. For lysis controls, GUS with PTS1 was expressed in the Strains BY4742 and BY4742 with the gene <em>PEX11</em> deleted. <br>
+">
-The fluorescence increase over time of the samples which are <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Secretion"> decorated with SNAREs </a> is higher in comparison to that of the lysis controls. The highest activity could be measured in the samples using the truncated Pex15 membrane anchor without a linker. The same construct in a background strain with a <em>PEX11</em> deletion showed a lower GUS activity in the supernatant. The strains expressing Snc1 linked to PEX26 or Snc1 directly fused to the N-terminus of Pex15 only showed minor increase of RFU over time. (Figure 3.3.) </p>
+      (Stock <i> et al</i>, 2012)
+  </abbr>
+</a>
+. With it, it is possible to determine whether a protein is secreted conventional and is <i>N</i>-glycosylated or secreted unconventional and not <i>N</i>-glycosylated. GUS is a bacterial protein with an <i>N</i>-glycosylation-site, which is active only if the protein is not <i>N</i>-glycosylated. The GUS-activity can be measured with different reagents in plate or liquid assays. Liquid assays can be applied qualitatively as well as quantitatively to measure differences in activity. If GUS is secreted by the conventional pathway the <i>N</i>-glycosylation leads to inactivation of the enzyme <a href=" https://www.ncbi.nlm.nih.gov/pubmed/22446315 ">
+  <abbr title=" Stock, J., Sarkari, P., Kreibich, S., Brefort, T., Feldbrügge, M., & Schipper, K. (2012). Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis. Journal of biotechnology, 161(2), 80-91.
-<div class="half-width">
+">
-<figure>
+      (Stock <i> et al</i>, 2012)
-<img src="https://static.igem.org/mediawiki/2017/e/e1/T--Cologne-Duesseldorf--Gus_Assay_Secret2.jpg">
+  </abbr>
+</a>
+ (Fig 3.2).</p>
-        <figcaption> <b> Figure 3.3 Relative fluorescence units per minute (RFU/min) measured for supernatants of different <em>S. cerevisiae</em> strains.</b> The fluorescence was measured for 12 hours in intervals of 10 minutes with an excitation of 365 nm and an emission of 465 nm. For the strain BY4742 (wt) which was used as the background strain the fluorescence did not increase over the measured time period. The lysis controls (GUS-PTS1; ∆Pex11 GUS-PTS1) show a lower activity than the samples of strains with Snc1-decorated peroxisomes. The highest activity could be measured in the strain using Pex15 with a linker as a membrane anchor (Pex15 L). The assay was performed in three technical replicates.
-</figcaption>
-</figure>
-</div>
-<!--
 <figure>
-         <img src="">
+         <img src="https://static.igem.org/mediawiki/2017/0/0e/T--Cologne-Duesseldorf--Gus_Erkl%C3%A4rung.png">
-         <figcaption>  </figcaption>
+         <figcaption><b>Figure 3.3 The GUS Assay. </b> GUS secreted with an unconventional secreted protein like Cts1 from Ustilago maydis active in the supernatant. GUS secreted with a conventional Signal peptide (Sp) inactive in the supernatant. If GUS is in the cytoplasm there is also no activity (Lysis control) <a href=" https://www.ncbi.nlm.nih.gov/pubmed/23455565">
+  <abbr title=" Feldbrügge, M., Kellner, R., & Schipper, K. (2013). The biotechnological use and potential of plant pathogenic smut fungi. Applied microbiology and biotechnology, 97(8), 3253-3265.
+">
+      (Feldbrügge <i> et al</i>, 2013)
+  </abbr>
+</a>. </figcaption>
 </figure>
--->
+<p>GUS will be imported to the peroxisome with the PTS1 sequence and measured quantitatively in the supernatant. We will use a coexpression of GUS-PTS1 and Snc1-Pex15 or Snc1-Pex26 to identify the secretion of the compounds. Furthermore, we will use GUS-PTS1 expressed in <i>S. cerevisiae</i> without Snc1 fused to a membrane anchor for a control. We will measure the active GUS in the supernatant with a liquid assay based on the turnover of  4-methylumbelliferyl-beta-D-glucuronide to 4-methyl umbelliferone (4-MU)<a href=" http://cshprotocols.cshlp.org/content/2007/2/pdb.prot4688.abstract">
+  <abbr title="Blázquez, M. (2007). Quantitative GUS activity assay of plant extracts. Cold Spring Harbor Protocols, 2007(2), pdb-prot4690.
+">
+      (Blázquez <i> et al</i>, 2007)
+  </abbr>
+</a>. Here we expect a higher activity of GUS in the supernatant of cultures with Snc1 decorated peroxisomes. <br>
+To increase the variability of our constructs we also designed vectors with and without a GS-Linker connecting the Snc1 with the Pex15. Additionally we tested our constructs in strains with a <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#SizeAndNumber"> deletion of Pex11 </a>. This deletion leads to formation of <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#SizeAndNumber"> larger peroxisomes</a> and may increase the efficiency of our secretion mechanism.</p>
 </div>
 <button class="accordion">
-   <h2 id="MembraneIntegration">Membrane Integration</h2>
+   <h2 id="SizeAndNumber">Size and Number</h2>
-   <p>Abstract</p>
+   <p>Size matters! Want to know how modifying out compartment’s morphology can benefit your reactions? Check out our design!!! </p>
    </button>
 <div class="panel">
- <div class="flex-row-3">
+  <h3>Heading</h3>
-<div><img src="https://static.igem.org/mediawiki/2017/d/d9/C2-PEX26-mRuby_red_Channel.jpeg"></div>
+    <p>
-<div><img src="https://static.igem.org/mediawiki/2017/a/a6/PMP_PEX26-mRuby_and_sfGFP-PTS1_merged.jpeg"></div>
-<div><img src="https://static.igem.org/mediawiki/2017/e/e9/C1-sfGFP-PTS1_Channel1.jpeg"></div>
-</div>
-<p>In order to have full control over the amount of expressed protein, we designed our plasmids with the inducible galactose promoter "pGAL1". Not only were we able to see that our fluorescent marked protein anchors from <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>  and  <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> would localize at specific points inside our cells but also to show that it was in deed the peroxisome they were accumulating at. For that we coexpressed each of our fluorescent membrane anchors together with a GFP protein that was fused to a PTS1 sequence and thus imported into the peroxisome.Fluorescent microscopy was used to colocalize both, the green fluorescing GFP and the red fluorescing mRuby and it is clearly visible, that our anchors integrated into the peroxisomal membrane.</p>
+    </p>
-<div class="flex-row-2">
-<div><img src="https://static.igem.org/mediawiki/2017/d/db/PMP_BACR-mRuby_and_gfp-pts1_merge.jpeg"></div>
-<div><img src="https://static.igem.org/mediawiki/2017/2/2d/BACR-mRuby_and_gfp-pts1_green_channel.jpeg"></div>
 </div>
-<p>
-Finally we used the same approach to direct a mRuby-tagged <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> to our compartment. In coexpressing it with the same GFP as in the previous steps, we could show that the <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> as well as <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> and <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> were successfully integrated into the membrane of our compartment. Since <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> is a rather complex protein, we're very optimistic about integrating other proteins into the membrane using the same approach.
-</p>
-<h4>Outlook</h4>
-<p>The ultimate goal of this subproject is, to have a complete set of ready to transform membrane proteins that could be combined with any promoter to create the optimal conditions for each desired situation. Besides <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a>, we also started to work with sugar translocators, since yeast does not posses the ability to import it into or export it from the peroxisome. This would open up a whole new chapter of peroxisomal usage, from example as a temporary storage compartment.
-</p>
-</div>
 <button class="accordion">
-   <h2 id="Sensors">Sensors</h2>
+   <h2 id="Sensors"><i>In Vivo</i> Sensors</h2>
-   <p>We successfully proved expression and peroxisomal localization of pHlourin2 and roGFP2 PTS1 constructs with fluorescence microscopy. Furthermore we performed  <i>in vitro</i> calibrations and in  <i>in vivo</i> measurements inside the cytosol and peroxisome. We provide a great outlook on application of sensoric measurements for testing and validating of synthetic metabolic pathways in our artificial compartment. </p>
+   <p>Surveillance of key physiology factors is indispensable for a good understanding and optimization of metabolic processes in compartments. In comparison to <i>in vitro</i> measurements, <i>in vivo</i> sensors have major advantages in terms of measuring relevant physiological properties in living systems. Former methods required destructive assays, expensive and time consuming preprocessing of cells or compartments to determining fluxes inside cells <a href="https://www.ncbi.nlm.nih.gov/pubmed/20854260"> <abbr title="2010, Frommer et al. -Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors."> Frommer<i>et al.</i> (2010)</abbr></a>. This leads to inexact measurements with poor spatial and temporal resolution. The usage of <i>in vivo</i> sensors tackles all of these problems at once. They allow detecting fluxes inside the cell and compartments in real time and supersede difficult and time consuming cell and organelle extraction. </p>
    </button>
 <div class="panel">
-    <p>
 <h3>Sensors</h3>
+<p>
+To enrich our toolbox we decided to measure four essential physiological factors: ATP, NADPH, Glutathione and pH.
+<br>ATP/ADP conversion is used as an energy currency in many cellular processes like translocation of proteins and metabolites or anabolic and catabolic turnovers. It is assumed that ANT1P, an ATP/AMP antiporter, is located in the peroxisomal membrane. Furthermore, the shuttle mechanism of the peroxisomal protein import machinery is ATP dependent
+<a
+href="https://www.ncbi.nlm.nih.gov/pubmed/11566870">
+<abbr title="Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter"> (Palmieri L. <i>et al.</i>, 2001)</abbr></a>. NADPH plays an important role in anabolic pathways and is also indispensable to our desired pathways of <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-5"> nootkatone </a>
+and <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Violacein">violacein.
+</a>Glutathione is an antioxidant and redox buffer which is also found in yeast peroxisomes. It is used as cofactor by at least two types of peroxisomal proteins, the glutathione peroxidases and glutathione transferases, which reduce lipid- and hydrogen peroxides or transfer glutathione to lipid peroxides for the purpose of detoxification
+<a href="http://www.jbc.org/content/276/17/14279.full.pdf">
+<abbr title="2001, Horiguchi H. et al.- Antioxidant System within Yeast Peroxisome">
+(Horiguchi H. <i>et al.</i>, 2001)</abbr></a>.
+Furthermore, it partly represents the redox state of the peroxisome. Knowing the pH of a compartment is important for predicting the activities of almost all enzymatic processes inside and to follow up acidification and basification upon conversion of metabolites.
+<br>
+<br>
+We finally chose two ratiometric sensors to perform measurements with. The pH sensitive and glutathione redox state reporting green fluorescent proteins pHLuorin2
+<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152828">
+<abbr title="2011, Mahon M. J. - pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein">
+(Mahon M. J. <i>et al.</i>, 2011)
+</abbr>
+</a>
+and roGFP2
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539">
+<abbr title="2016, Schwarzländer et al.- Dissecting Redox Biology Using Fluorescent Protein Sensors">
+(Schwarzländer M. <i>et al.</i>, 2016)</abbr></a>.
+Our aim was to target them either to the peroxisomal lumen or the cytosol. To achieve peroxisomal targeting we attached the peroxisomal targeting signal 1 via
+ <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-2"> Golden Gate cloning</a>.
+</p>
-<h4>Localization</h4>
-<p>pHlourin2 and roGFP2 was detectable by fluorescence microscopy and showed similar excitation spectrum as native GFP<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152828/"> <abbr title="2011, Mahon et al. - pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein"> Mahon <i>et al.</i> (2011)</abbr></a>. Sensors were compared to wild type with similar growth conditions. Cytosolic constructs were both visible and evenly distributed in the cell except for vacuole areas. </p>
 <div class="flex-row-2">
-	<div>
+ 	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/0/04/L1_ruc%2B_gfp-mcherry_200ms_str8green.png">
+   	<img src="https://static.igem.org/mediawiki/2017/c/cb/RoGFP2_lvl1_cytosolic_medium_promotor.png">
+ 	</div>
+ 	<div>
+   	<img src="https://static.igem.org/mediawiki/2017/8/8e/RoGFP2_lvl1_peroxisomal_medium_promotor.png">
+ 	</div>
 	</div>
-	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/e/e3/WT_GFP_mCherry_WT_GFP-mCherry_500ms_str4green.png">
-	</div>
-</div>
-<figcaption>
-<p>
-<font size="3"><strong>Figure5.1 </strong>Left cytosolic pHLuorin2 expression. Right wild type, without any fluorescence.
-</p>
-</font>
-</figcaption>
-<p>
-Carboxyl terminal fusion of the peroxisomal targeting signal 1 resulted in small high intens areas inside the cells. Cytosolic fluorescence was nearly silenced. The Pex5 receptor recognizes this signaling tag and ensures the import into the peroxisome, which is shown in the following figure. </p>
 <div class="flex-row-2">
-	<div>
+ 	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/e/ef/Pup%2B%2B_p13_gfp-mcherry_500ms_str4_m2green.png">
+   	<img src="https://static.igem.org/mediawiki/2017/e/ed/PHlourin2_lvl1_cytosolic_medium_promotor2.png
+">
+ 	</div>
+ 	<div>
+   	<img src="https://static.igem.org/mediawiki/2017/3/3a/PHlourin2_lvl1_peroxisomal_medium_promotor.png">
+ 	</div>
 	</div>
-	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/c/cb/L1_rup-_gfp-mcherry_200ms_str8green.png">
-	</div>
-</div>
-<figcaption>
+<figcaption><font size="2">
-<p>
+<strong> Figure 5.1 </strong> Level 1 plasmids with medium promoters and uracil auxotrophy. Top left: cytosolic roGFP2. Top right: peroxisomal roGFP2. Bottom left:  cytosolic pHLourin2. Bottom right: peroxisomal pHLourin2.</font>
-<font size="3"><strong>Figure5.2 </strong>Left pHLuorin2 localized in the peroxisome. Right roGFP2 localized to the peroxisome
-</p>
-</font>
 </figcaption>
 <p>
+Using promoters with different expression strength in order to find optimal measurement conditions is of high interest. There is a trade-off between a high signal-to-noise ratio and self-induced effects which are both dependent on expression levels
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539">
+<abbr title="2016, Schwarzländer et al.- Dissecting Redox Biology Using Fluorescent Protein Sensors">
+(Schwarzländer M. <i>et al.</i>, 2016)
+</abbr></a>.
+This is not generally the case for each sensor. For example, pHlourin2 has only a minor influence on the existing pH because of the buffer effect of proteins.
 <br>
-The peroxin13 mRuby construct also showed concentrated localization inside cellular areas. In comparison to our tested sensors with a comparable promoter parts <a href=" toolbox link einfügen ">016 and 017</a> it needed a higher gain for same fluorescence intensities. From our observations and literature we therefore considered peroxin13 mRuby construct as suitable peroxisomal marker. Expression of our level2 plasmids containing both a sensor and peroxin13 mRuby showed colocalization which leaded to yellow spots in merged channels<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655559/"> <abbr title="2009, Robert Yung-Liang Wang et al.- A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes"> Robert Yung-Liang Wang <i>et al.</i> (2009) </abbr> </a>.</p>
+Validation of the peroxisomal localization can be achieved via fluorescence overlap of the sensor and a peroxisomal marker, in our case pex13-mRuby (<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-4">import mechanism</a>).
-<div class="flex-row-2">
-	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/2/20/WT_GFP_mCherry_WT_GFP-mCherry_200ms_str8_red.tif_%28RGB%29.png">
-	</div>
-	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/d/df/L2_pup%2B_p12_gfp-mcherry_200ms_str8red.png">
-	</div>
-</div>
-<font size="3"><strong>Figure5.3 </strong>Left: wild type without any fluorescence. Right: Level 2 Plasmid containing peroxin13-mruby and pHLuorin2-PTS1 using the mcherry channel.
 </p>
-</font>
-</figcaption>
 <div class="flex-row-2">
-	<div>
+ 	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/9/94/Puc%2B%2B_p13_gfp-mcherry_500ms_str4_m2green.png">
+   	<img src="https://static.igem.org/mediawiki/2017/4/45/RoGFP2_lvl2_peroxisomal_strong_promotor_Peroxin13_mRuby.png">
+ 	</div>
+ 	<div>
+   	<img src="https://static.igem.org/mediawiki/2017/9/99/PHlourin2_lvl2_peroxisomal_strong_promotor_peroxin13_mRuby.png">
+ 	</div>
 	</div>
-	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/e/e5/L2_pup%2B_p12_gfp-mcherry_200ms_str8fgreen.png">
-	</div>
-</div>
-<font size="3"><strong>Figure5.4 </strong> Left:Level 2 Plasmid containing peroxin13-mruby and cytosolic pHLuorin2 using the GFP channel. Right: Level 2 Plasmid containing peroxin13-mruby and pHLuorin2-PTS1 using the GFP channel.
-</p>
-</font>
-</figcaption>
-<div class="flex-row-2">
-	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/e/e4/Puc%2B%2B_p13_gfp-mcherry_500ms_str4_m2merge.png">
-	</div>
-	<div>
-  	<img src="https://static.igem.org/mediawiki/2017/5/5b/Pup%2B%2B_p13_gfp-mcherry_500ms_str4_m2merge.png">
-	</div>
-</div>
-<figcaption>
-<p>
-<font size="3"><strong>Figure5.5 </strong> Left: Level 2 Plasmid containing peroxin13-mruby and cytosolic pHLuorin2. Images of the GFP and mcherry channel were merged.  Right: Level 2 Plasmid containing peroxin13-mruby and peroxisomal pHLuorin2. Images of the GFP and mcherry channel were merged.
-</p>
+<figcaption><font size="2">
-</font>
+<strong>Figure 5.2 </strong>Level 2 plasmids with peroxisomal marker Peroxin13-mRuby and uracil auxotrophy for colocalization. Left roGFP2 with strong promoter. Right pHLourin2 with strong promoter.</font>
 </figcaption>
-<h4>pHluorin2</h4>
+<p>
-<p> Initially we desired an in vivo calibration with pH values ranging from pH5.8 to 7.8. We therefore tried to equilibrate the pH of the cytosol with the supernatant. The cells were incubated in a potassium rich buffers containing the ionophore nigericin. Nigericin penetrates the cell membrane and acts as a potassium proton antiporter. Sadly we did not noticed any correlation between pH and the 405 to 485nm excitation ratio response even with 5 fold higher concentration.  For that reason we changed calibration method to an <i>in vitro</i> assay .
+It can also be achieved by transforming the sensors attached to the PTS1 sequence into a $\Delta$Pex5 yeast strain. The sensor is expected to show no specific localisation due to the missing import sequence. We calibrated the sensors in living yeast cells and physiological ranges so that we can not only perform relative but also quantitative measurements. We aim to confirm our hypothesis of a more oxidized redox state of roGFP2 in peroxisomes with <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Violacein">violacein pathway
-The cooled protein extracts of yeast strains containing pHlourin2 constructs and a wild type control were separately titrated to the desired pH values and measured at the plate reader afterwards.</p>
+</a> activity and want to measure differences in pH within yeast strains with peroxisomal membrane anchored pex3-bacteriorhodopsin protein
-<figure>
+<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#MembraneIntegration"> (membrane proteins) </a>.
-	<img src="https://static.igem.org/mediawiki/2017/4/47/Artico_pHLuorin_verlauf_results.svg">
-   <figcaption><font size="3"><strong>Figure5.4 </strong>pHLourin2 protein supernatant with different pH values between pH 5.8 and 7.8. Analysis were performed using the Infinite M2000 pro Tecan plate reader with dual excitation at 405/5 nm and 485/5 nm and emission at 535/25 nm.</font>
-</figcaption>
-</figure>
 <br>
-<p>As demonstrated above the sensor works perfectly fine. An ascending pH results in a shift of the excitation peaks from 485 nm to 405 nm over the entire considered pH area. </p>
-<figure>
-	<img src="https://static.igem.org/mediawiki/2017/f/fc/Artico_pHLuorin_ratioplot.svg">
-   <figcaption><font size="3"><strong>Figure5.6 </strong> The ratios between 405 nm and 485 nm at 535 nm emission, were obtained from the Infinite M2000 pro Tecan plate reader for the given pH values. </font
-</figcaption>
-</figure>
-<p>For the peroxisomal usage of our sensor we had to ensure that the PTS1 signal peptide had no effect on the direction or the fold change of the ratio response .
-<br> PTS1 tagged pHluorin2 shows a slightly smaller 405/485 nm ratio compared to the cytosolic located sensor. Despite the high standard deviation, experiments with a higher number of replicates are required examining significant differences. It seems that the linear correlation is not changed. Finally we performed <i>in vivo</i> measurements comparing peroxisomal and cytosolic pH.
 <br>
-For <i>in vivo</i> measurements yeast were grown on yeast nitrogen dropout medium at a pH of 6.0. A pH of 6.7/0.4 was measured in the peroxisomes, whereas in the cytosol we observed a slightly lower pH of 6,4/0,3. All measurements were obtained from yeast cultures with an OD<sub>600</sub> ranging from 0,8 up to 1.3. The large standard deviation might be rooted in the different OD<sub>600</sub>s. Literature does not agree about whether the pH inside the peroxisome is acidic or alkaline nor whether there are endogenous regulating mechanisms <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133865//"> <abbr title="2004, Francesco M. Lasorsa et al. - The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in ΔpH formation across peroxisomal membranes"> (Francesco M. Lasorsa<i>et al.</i>(2004), </abbr> </a><a href="http://jcs.biologists.org/content/117/18/4231"> <abbr title="2004, Carlo W. T. van Roermund et al. - The peroxisomal lumen in Saccharomyces cerevisiae is alkaline"> Carlo W. T. van Roermund<i>et al.</i> (2004))</abbr> </a>. Our result suggest a slight acid pH inside the peroxisomes and agreed with<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133865//"> <abbr title="2004, Francesco M. Lasorsa et al. - The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in ΔpH formation across peroxisomal membranes"> Francesco M. Lasorsa<i>et al.</i>(2004)</abbr> </a>.<p>
+Once expression and localization of the sensor is proven by microscopy, measurements with a plate reader or a fluorometer are acceptable. This allows a high number of replicates to be measured accurately in reasonable time. Microscopy is performed with a filter based Nikon Eclipse TI fluorescence microscope at 100-fold magnification and plate reader measurements are performed with a Tecan infinite plate reader
+<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152828">
+<abbr title="2011, Mahon et al.- pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein">
+(Mahon M. J. <i>et al.</i>, 2011)</abbr>
+</a>
+. For pHLuorin2 emission intensity is measured at 535 nm upon excitation at 405 nm and 485 nm. Same settings were used for roGFP2
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539">
+<abbr title="2016, Schwarzländer et al.- Dissecting Redox Biology Using Fluorescent Protein Sensors">(Schwarzländer M. <i>et al.</i>, 2016)</abbr>
+</a>
+. Evaluation of the reported signals is done by the excitation ratio of the the corresponding excitation wavelength.
+</p>
+</div>
-<br>
-In summary, stronger promoters promise to gain a better signal to noise ratio. Still pHlourin2 calibration does not dependent on promoter strength which supports the hypothesis that pHlourin2 has sparse effect on the existing pH level. The sensor characteristics are neither changed by the pts1 signal. The <i>in vivo</i> calibration might have failed due to the disability of penetrating the yeast cell wall. Nevertheless we were able to measure the pH <i>in vivo</i>.
-With this sensor we provide a part to iGEM which actually can detects pH changes inside our compartment purposed for pathway analyses or research. Data can be easily generated and examined. </p>
-<h3>roGFP2</h3>
+<button class="accordion">
-<p>After expression and correct localization to the peroxisome was validated we examined the function of roGFP2. We conducted an <i>in vitro</i> assay on fully oxidized and fully reduced roGFP2 and performed time measurements by subsequently adding  H<sub>2</sub>O<sub>2</sub>  and DTT to the protein extract. </p>
+  <h2>Nootkatone</h2>
-<figure>
+  <p>In order to have a proof of concept for our compartmentation strategy we intend to establish the nootkatone pathway inside the peroxisome. Therefore we aim to target the three enzymes for the nootkatone production to compartment. Before starting the actual lab work for the ambitious plan to establish a completely new pathway in the peroxisome, there are several aspects that have to be considered first.</p>
-	<img src="https://static.igem.org/mediawiki/2017/a/a2/Artico_rogfp_verlauf.svg">
+  </button>
-   <figcaption><font size="3"><strong>Figure5.7 </strong>In the beginning roGFP2 was either treated with 1 mM DTT reducing the sensor or with 1 mM  H<sub>2</sub>O<sub>2</sub> to oxidize the protein supernatant. Later the complete oxidation/reduction was achieved through adding additional
+<div class="panel">
-and DTT.</font
-</figcaption>
-</figure>
-<p>We could observe a functional sensor with a high dynamic range in the cytosolic and the PTS1 fused construct, which indicates high sensitivity. Further the  PTS1 Tag  does not seem to disturb the function of roGFP2(data not shown).
-The calibration was performed using the mid point calibration method, which was previously performed by assuming the midpoint potential to be at -280mV<a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2008.02030.x/full"> <abbr title="2008, Schwarzländer et al.- Confocal imaging of glutathione redox potential in living plant cells"> Schwarzländer  <i>et al.</i>(2008) </abbr> </a>.</p>
-<figure>
+<p>The initial step is to find a reliable source to prove the abundance of our precursor Farnesyl pyrophosphate (FPP) in yeast peroxisomes. So far there is no proof of existence of FPP inside yeast peroxisomes yet. However it is predicted to be present, as it was detected in mammalian and plant peroxisomes
-	<img src="https://static.igem.org/mediawiki/2017/f/f9/Artico_oxidized_rogfp.svg">
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/11108725">
-   <figcaption><font size="3"><strong>Figure5.8 </strong>405/485nm excitation ratio plotted against the oxidized proportion of roGFP2 </font
+<abbr title="2000, Olivier et al. - Identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes. AA-CoA thiolase, hmg-coa synthase, MPPD, and FPP synthase."> Olivier <i>et al.</i> (2000)</abbr></a>.
-</figcaption>
-</figure>
-<p>
-Based on the nernst equation we were now able to calculate the redox potential of roGFP2 regarding the oxidation of roGFP2<a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2008.02030.x/full"> <abbr title="2008, Schwarzländer et al.- Confocal imaging of glutathione redox potential in living plant cells"> Schwarzländer,  <i>et al.</i>(2008)</abbr></a>.
 </p>
 <figure>
-	<img src="https://static.igem.org/mediawiki/2017/f/fa/Artico_rogfp_mv.svg">
+     	<img src="https://static.igem.org/mediawiki/2017/b/b9/Farnesylpyrophosphat.png">
-   <figcaption><font size="3"><strong>Figure5.9 </strong>Oxidized roGFP2 proportion plotted against the redox potential of glutathione (mV)</font
+      	<figcaption><strong>Figure 7.1:</strong> The structure of FPP the precursor for nootkatone. </figcaption>
-</figcaption>
+  	</figure>
-</figure>.
-<p>
+<p>The precursor FPP is converted into valencene by a valencene synthase (ValS). We chose the one from <i>Callitropsis nootkatensis</i> because of its comparably high efficiency in microorganisms. It achieves greater yields in yeast than the citrus valencene synthase. Furthermore, the product specificity is relatively high, while production of byproducts is low
-Using our calibrated sensor we could compare the redox states within strains which differ in metabolic physiology.
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/24112147"><abbr title="Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.">Beekwilder <i>et al.</i> (2014)</abbr></a>.
+The valencene synthase was also chosen because of its robustness towards pH and temperature changes
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/24112147"><abbr title="2014, Beekwilder et al. - Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.">Beekwilder <i>et al.</i> (2014)</abbr></a>.
+Our modelling approach revealed that for optimal yields an overexpression of valencene synthase is necessary because of its slow conversion rate <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#MetabolicModel">(Model)</a>. This is why we chose the strongest promoter of the yeast toolbox
+<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox">(Dueber Toolbox)</a> for this attempt.
 </p>
 <figure>
-	<img src=" https://static.igem.org/mediawiki/2017/a/af/Artico_rogfp_living_cells.svg">
+         <img src="https://static.igem.org/mediawiki/parts/a/a8/ValS_lvl1_PTS1.png">
-   <figcaption><font size="3"><strong>Figure5.10 </strong>Cytosolic and peroxisomal 405/485 nm ratios of roGFP2 were obtained. We used a strong(+) and weaker(-) Promoter. Yeast were grown on yeast nitrogen dropout medium at pH 6.0. Cultures showed an OD<sub>600</sub> between 0.9 and 1.1.</font>
+          <figcaption> <strong>Figure 7.2:</strong>ValS lvl.1 PTS1 plasmid</figcaption>
-</figcaption>
+      </figure>
-</figure>
-<p> Comparisons of the cytosolic and peroxisomal Glutathione redox states showed no significant differences between the cytosol and the peroxisome. This result was surprising since varieties were reported in literature before. <a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539"> <abbr title="2015, Schwarzländer et al. -Dissecting Redox Biology Using Fluorescent
-Protein Sensors"> Schwarzländer <i>et al.</i> (2015)</abbr></a>.
-<h4>Outlook</h4>
+<img src="https://static.igem.org/mediawiki/2017/a/a7/Valencene.png">
-<p>We planned to calibrate our sensors <i>in vivo</i> as well and wanted to follow up changes induced by <a href=" link violacein einfügen ">violacein</a> and <a href=" link nootkatone einfügen ">nootkatone</a> pathways. Furthermore our objective was testing the expected acidification upon induced expression and integration of <a href=" link membrane ">bacteriorhodopsin</a>into the peroxisomal membrane with pHLuorin2. roGFP2 can be fused to numerous redox catalytic enzymes making it specific to certain redox pools <a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539"> <abbr title="2015, Schwarzländer et al. -Dissecting Redox Biology Using Fluorescent
+      	<figcaption><strong>Figure 7.3:</strong> The structure of valencene, an intermediate of nootkatone</figcaption>
-Protein Sensors"> Schwarzländer <i>et al.</i> (2015)</abbr></a>.This property makes it interesting to further usage for the iGEM community.
+  	</figure>
+<p>The intermediate valencene is then converted into nootkatol by a P450 monooxygenase as well as into small amounts of our desired product nootkatone.  The P450 monooxygenase we chose for this project was taken from the bacterium <i>Bacillus megaterium</i>. In this  case it is not only a simple P450 monooxygenase, but an entire P450 system, consisting of a soluble P450 fused to a cytochrome P450 reductase (CPR) enzyme, making an additional reductase obsolete
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/12419614">
+<abbr title="2002, De Mot et al. - A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes.">
+   	De Mot <i>et al.</i>  (2002)</abbr></a>.
-<br>
+Unlike eukaryotic P450s, which are mostly membrane bound, this prokaryotic BM3-P450 is located in the cytosol facilitating an easier transport into the peroxisome, as membrane integration of proteins is a more difficult task to achieve than import of cytosolic proteins
-In the future we want to expand our toolbox with an ATP and NADP<sup>+</sup> sensor. Both sensors are FRET (Förster Resonance energy Transfer) based sensors. They consist of two coupled fluorescence protein and a ligand- sensing domain. FRET is a distant depend process by which energy  transferred from an excited donor fluorophore to an acceptor molecule which is mostly a fluorophore as well. The NADP<sup>+</sup> sensor consists of two fluorophore proteins CFP and YFP and a indicator protein KBR. In the presence of NADP<sup>+</sup>, the distance between the two fluorophores is increased because of a conformational change of the sensing protein KBR. Exciting these complex by 440 nm results in a emission spectra with peaks at 478 nm and 526 nm. Thus, the 526/478 ratio between these wavelengths changes due to different NADP<sup>+</sup> concentrations<a href="https://www.ncbi.nlm.nih.gov/pubmed/26524720"> <abbr title="2015, Feng-Lan Zhao et al. -A genetically encoded biosensor for<i> in vitro</i> and <i>in vivo</i> detection of NADP<sup>+</sup>"> Feng-Lan Zhao <i>et al.</i> (2015)</abbr></a>.
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/17073779">
-<br>
+<abbr title="2006, Girvan et al. - Flavocytochrome P450 BM3 and the origin of CYP102 fusion species.">Girvan <i>et al.</i> (2006)</abbr></a>.
-ATP will be measureable using a Fret based ATP-sensor which consists of the two Fluorescent Proteins CFP and mVenus, derived from the YFP, which are both linked to the 𝜺 subunit of the F0F1-ATP synthase. Upon binding of ATP to the 𝜺 subunit a conformational change happens, which is detectable through fluorescence ratio measurements<a href="http://www.pnas.org/content/106/37/15651.full"><abbr title="2009, Hiromo Imamura et al.-Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators"> Hiromo Imamura <i>et al.</i> (2009)</abbr></a>. </p>
+BM3 normally catalyzes the hydroxylation of long chain fatty acids
-    </p>
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/3086309"><abbr title="1986, Narhi et al. - Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium.">Narhi <i>et al.</i> (1986)</abbr></a>, which in our case could inhibit the conversion of valencene. For that reason we used a mutated version of BM3, which is called AIPLF. This variant is an enhanced version of the BM3 AIP version, which has a ten times better substrate oxidation rate for valencene than the wildtype BM3 and produces less byproduct when valencene concentration is saturated. Additionally to previous named benefits the AIPLF variant with
-</div>
+<abbr title="R47L/Y51F/F87A/A328I/I401P">5 point mutations</abbr> in the active side has a significantly lower binding affinity towards long chain fatty acids and therefore increase the transposition rate of valencene.
-<button class="accordion">
+<a href="http://onlinelibrary.wiley.com/doi/10.1002/cctc.201402952/full">
+<abbr title="2015, Schulz et al. - Selective Enzymatic Synthesis of the Grapefruit Flavor (+)-Nootkatone">Schulz <i>et al.</i> (2015)</abbr></a>,
-  <h2 id="Nootkatone">Nootkatone</h2>
+<a href="https://docserv.uni-duesseldorf.de/servlets/DerivateServlet/Derivate-43193/Dissertation_SvenCarstenLehmann.pdf">
-  <p>After successfully integrating all our plasmids into yeast, we were able to verify the expression of each enzyme of the nootkatone pathway. </p>
+<abbr title="2016, Lehmann - Entwicklung eines P450-basierten Ganzzellkatalysators
-  </button>
+für die selektive Oxyfunktionalisierung von α-Pinen">Lehmann (2016)</abbr></a>.
-<div class="panel">
+</p>
-<p>In order to verify the cytosolic expression of ValS, BM3 and ADH we performed a Western Blot analysis for each of the enzymes. We were able to verify the expression of ValS, BM3 and ADH with and without PTS1 in the yeast cytoplasm and peroxisomes, respectively.</p>
+<figure>
+     	<img src="https://static.igem.org/mediawiki/parts/f/fe/BM3_lvl1_PTS1.png">
+      	<figcaption><strong>Figure 7.4:</strong> BM3 lvl.1 PTS1 plasmid </figcaption>
+  	</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2017/7/72/Nootkatol.png">
+      	<figcaption><strong>Figure 7.5:</strong> structure Nootkatol </figcaption>
+  	</figure>
+<p>The alcohol dehydrogenase from <i>Pichia pastoris</i> subsequently converts nootkatol into nootkatone by oxidation. It uses NAD<sup>+</sup> as a cofactor which is reduced in the reaction. The regeneration of this cofactor is facilitated by the BM3, which oxidizes NADH
+<a href="http://onlinelibrary.wiley.com/doi/10.1002/cctc.201402952/full">
+<abbr title="2015, Schulz et al. - Selective Enzymatic Synthesis of the Grapefruit Flavor (+)-Nootkatone">Schulz <i>et al.</i> (2015)</abbr></a>.
+</p>
+<figure>
+     	<img src="https://static.igem.org/mediawiki/2017/e/e2/ADH_lvl1_PTS1.png">
+      	<figcaption><strong>Figure 7.6:</strong> ADH lvl.1 PTS1 plasmid </figcaption>
+  	</figure>
 <figure>
-     	<img src="https://static.igem.org/mediawiki/2017/2/21/T--Cologne-Duesseldorf--western-blot-nootkatone.png" ;="" style="width: 70%; height: 70%">
+<img class="half-width" src="https://static.igem.org/mediawiki/2017/5/5f/Nootkatone.png">
-       	<figcaption> <strong>Figure 7.1 </strong> Protein abundance in WT and transformed cells in S. cerevisiae: Protein abundance was detected using 6xHis Tag Antibody in whole cell lysates. WT = wild type, ValS = Valencene Synthase, PTS1 = Peroxisome Targeting Signal 1, BM3 = Cytochrome P450 from Bacillus megaterium, ADH = Alcohol Dehydrogenase </figcaption>
+       	<figcaption><strong>Figure 7.7:</strong> structure Nootkatone </figcaption>
    	</figure>
-<p>Since protein abundance of ValS, BM3 and ADH, both with and without a peroxisome targeting signal, was verified in the cytosol, a mass spectrometry analysis (MS analysis) of nootkatone and its precursor valencene was performed.
+<p>The first milestone to achieve our goal is the separate integration of each of our three enzymes ValS, BM3 and ADH into level 1 vectors <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox">(Dueber Toolbox)</a>  in yeast and to verify their expression by Western Blot analysis. Therefore, a 3xFlag/6xHis-tag was added to the N-terminus of each of the proteins.  It enables us to use an anti-His antibody followed by an anti-mouse-antibody to make protein abundances visible. Subsequently, the two enzymes ADH and ValS were combined in a  level 2 cassette plasmid.  BM3 is designed as a level 1 plasmid and will be co-expressed with the level 2 plasmid to achieve a nootkatone production. The expression of the enzymes in the cytoplasm is again verified by Western Blot analysis.
-<br>There are three approaches in MS analysis. The first one is the qualitative approach in which is only determined if the substance is present or not. The second and third kinds are the quantitative or semi-quantitative approach in which the absolute or relative amount of a substance is investigated.
-First, we tried to validate nootkatone and valencene with the first approach and screened our samples for the existence of the first intermediate valencene and our final product nootkatone.
-<br>
-We could not show the synthesis of nootkatone nor valencene in our yeast yet, but we could smell its characteristic scent. The lack of proof via MS could result from an inefficient sample extraction or to low concentrations of product in the sample. The latter could also explain a peak in the MS analysis where nootkatone was expected. Unfortunately the peak was below detection limits and therefore can not be assumed to be a definite proof of nootkatone production. </p>
-<h3>Outlook</h3>
+The further approach aims to provide the enzymes with C-terminal peroxisomal targeting signals type one, which finally converts the nootkatone pathway into our artificial compartment. </p>
-<p>For further investigation we plan to do a semi-quantitative analysis by comparing the yield of samples of cytosolic synthesized nootkatone and peroxisomal nootkatone. To do so we need to perform a peroxisome purification in order to compare the amount of product produced. With this comparison we hope to proof that compartmentation, and thereby bypassing the problem of toxicity of substances for yeast, is the key for better yield of nootkatone. Also we intend to do a quantitative MS analysis to clarify if the yield of our nootkatone pathway is anywhere near the yield pathways with other enzymes/ enzyme-combinations could achieve. There was also the idea of exchanging cytosolic enzymes with membrane bound ones to see if there is any change in yield.</p>
-<p> Already planned but not implemented, due to lack of time, we have also a proof of localization of the pathway enzymes. Therefore we exchange the 3a part <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox">(Dueber Toolbox)</a> 3xFlag/6xHis of the plasmid with an other fluorescent 3a part, namely mRuby2. We can then show the localization of the enzymes via microscopy.
-<p>Another factor to be considered in further studies will be the available amount of FPP in peroxisomes. FPP is the essential precursor of nootkatone synthesis. But we cannot say yet if there is enough FPP in the peroxisome to justify an expression of the pathway in it. If there is not enough FPP available to generate nootkatone over the concentration of  100 mg/L it does not matter that beta-nootkatol and nootkatone is toxic to the cell. </p>
+<p>Since the production of nootkatone does not lead to a change of colour we need to apply different methods for verifying substrates. Once we succeed with the qualitative validation via Western Blot analysis, we can verify the presence of nootkatone by using high performance liquid chromatography and mass spectrometry, respectively.</p>
-<p>To tackle this problem there are three methods reported to increase the amount of FPP in the peroxisome for nootkatone production. The first one is to introduce a knockout mutation of squalene synthase and obtaining a mutant that is capable of efficient, aerobic uptake of ergosterol to limit the use of FPP for the sterol biosynthesis. The second approach is to knock out a phosphatase activity to limit the endogenous dephosphorylation of FPP. Third is to upregulate the catalytic activity of HMGR.
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/17013941">
-<abbr title="2007, Takahashi et al. - Metabolic engineering of sesquiterpene metabolism in yeast.">
-   	Takahashi <i>et al.</i> (2007)
-</abbr>
-</a>.</p>
-</div>
+<h3>Model influence on Nootkatone expression</h3>
+<p>We modeled the nootkatone biosynthesis pathway using ordinary differential equations in order to optimize nootkatone production. We found two hard and an easy problem, all of which we could find a solution for. The easy problem is optimization of the enzyme concentrations of the biosynthesis pathway. According to our <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#MetabolicModel">model of the Nootkatone pathway</a> we found that overexpression of Valencene Synthase is necessary to maximize the Nootkatone yield, while both alcohol dehydrogenase and p450-BM3 have only minor effects on the yield. </p>
+<p>One of the hard problems, as shown in our <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#Penalty-Model">penalty model</a> is the toxicity of nootkatone and nootkatol. Since the toxicity most likely stems from both nootkatone and nootkatol clogging up the cell wall we present our peroxisome as a solution for this problem. When comparing the cytosolic model to our <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#Peroxisome-model">peroxisomal model</a> we found that, if our assumption that neither Nootkatone nor Nootkatol are able to pass the peroxisomal membrane holds up, we can greatly increase Nootkatone production.</p>
+<p>A further  problem is the influx of the pathway precursor farnesyl pyrophosphate (FPP). We used <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model#OptKnock">OptKnock analysis</a> to design yeast strains with optimized FPP production. With this analysis we got hints that growing the yeast cells on a fatty acid medium might be a simple alternative to
+knocking out the desired genes.</p>
+</div>
 <button class="accordion">
    <h2 id="Violacein">Violacein</h2>
-  <p>
+<p>Working on a project is a process of well planned steps. It starts with an idea and theoretical research to create a design. When finally demonstrating the mechanism of the project it is important to point out the benefits with a suitable application.
-In the following the results of the essential experiments will be presented and discussed. The expression of the enzymes could be proven with SDS PAGE and western blot experiments. The following conducted <i>in vitro</i> assay was analyzed via HPLC-MS.
+<br>Synthetic biology offers countless numbers of new opportunities, especially in the field of metabolic engineering. To testify some main parts of our <strong>artico</strong> project, we decided to relocate a metabolic pathway into yeast peroxisomes. There are several reasons why this approach fits perfectly as a proof for our concept.
 </p>
-  </button>
+</button>
 <div class="panel">
-  <h3>Violacein</h3>
+<p> Based on described of<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Violacein"> advantages </a> of violacein this pathway was chosen. Violacein is naturally produced in numerous bacterial strains, most popular in the gram-negative <i> Chromobacterium violaceum </i>. It is related to biofilm production and shows typical activities of a secondary metabolite <a href="https://www.hindawi.com/journals/bmri/2015/465056/">
-<p>
+<abbr title="Violacein: Properties and Production of a Versatile Bacterial Pigment">
-The experiments were implemented following the protocols for
+(Seong Yeol Choi <i>et al.</i>, 2015).</abbr>
-<a href="https://static.igem.org/mediawiki/2017/8/8a/T--Cologne-Duesseldorf--western-blot-protocol.pdf">western blot</a>
+</a>
-and
-<a href="https://static.igem.org/mediawiki/2017/a/aa/T--Cologne-Duesseldorf--prodeoxyviolacein_assay.pdf">
-<i>in vitro</i> assay prodeoxyviolacein
-</a>.
 </p>
 <figure>
-<img
+<img src="https://static.igem.org/mediawiki/2017/0/09/T--Cologne-Duesseldorf--Violacein_Pathway_komplett.png">
-src="https://static.igem.org/mediawiki/parts/7/75/T--Cologne-Duesseldorf--Violacein_WB.png">
 <figcaption>
-<strong>Figure 8.1</strong> Western blot analysis of Vio enzyme expression in yeast lysate with anti-His-antibody. The cell cultures were harvested and cells were lysed at the following OD<sub>600</sub>: wild type (WT) control: 1.4; VioA_pts: 1.13; VioA: 1.49; VioB: 1.37; VioB_pts: 1.5; VioE: 1.17. The expressed enzymes have the following predicted molecular weights: VioA_pts: 48.9 kDa; VioA: 47.7 kDa; VioB: 112 kDa; VioB_pts: 113.4 kDa; VioE: 22.7 kDa. The pictures are assembled for better analysis, each panel was merged with the protein ladder to allow exact comparison.
+<strong>Figure 8.1</strong> The synthesis of Violacein requires five enzymes encoded by the VioABCDE operon. VioA, a flavin-dependent L-tryptophan oxidase and VioB, a heme protein, work in combination to oxidize and dimerize L-tryptophan to an IPA imine dimer. Hydrogen peroxide is released as a by-product of the VioA reaction. Next step by VioE is the rearrangement of the IPA imine dimer to protodeoxyviolaceinic acid, which can non-enzymatically oxidize to prodeoxyviolacein or, by VioC via deoxyviolaceinic acid, oxidize to pink deoxyviolacein. The flavin-dependent oxygenases VioC and VioD require interaction with the oxidized form of flavin-adenine dinucleotide (FAD)
+<a href="http://www.uniprot.org/uniprot/Q9S3U9">
+<abbr title="UniProtKB - Q9S3U9 (VIOC_CHRVO)">
+(uniprot,
+</abbr>
+</a>
+<a href="http://www.uniprot.org/uniprot/Q9S3U8">
+<abbr title="UniProtKB - Q9S3U8 (VIOD_CHRVO)">
+uniprot)
+</abbr>
+</a>
+. The two enzymes act sequentially: first, VioD hydroxylates protodeoxyviolaceinic acid, leading to protoviolaceinic acid. Second, VioC creates the oxindole at the 2-position of one indole ring, leading to violet violacein
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/17176066">
+<abbr title="In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum">
+(Balibar CJ <i> et al.</i>, 2006)
+</abbr>
+</a>
+<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025692/">
+<abbr title="Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum">
+(Janis J. Füller <i> et al.</i>, 2016).
+</abbr>
+</a>
 </figcaption>
 </figure>
-<p>The level 1 constructs show the predicted molecular weight, whereas the different intensities of the protein bands correlate with the different optical densities (ODs) and different expression levels in the yeast culture replicates. The unspecific bands in VioA, VioA_pts and VioB_pts have a lower molecular weight than our predicted bands. One reason for this might be to protein degradation by carboxy exonuclease activity
+<p> Relocating the pathway into the peroxisome enables proximity of the enzymes and substrates. Furthermore the yeast cell is protected from the toxic substance hydrogen peroxide. Yeast peroxisomes have no problem with this as their main function is the beta-oxidation of fatty acids and the detoxification of the thereby produced H<sub>2</sub>O<sub>2</sub>
-<a href="https://advansta.com/wikis/multiple-bands-in-western-blots-causes-and-solutions-2/">
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/17445803">
-<abbr title="Multiple Bands in Western Blots – Causes and Solutions">
+<abbr title="The peroxisomal protein import machinery">
-(Hurley A, 2017)
+(Erdmann R. <i>et al.</i>, 2007)
 </abbr>
-</a>.
+</a>. Because VioC and VioD are FAD-dependent, it is additionally an evidence for FAD availability inside of the peroxisome, if the synthesis of Violacein works. Otherwise the two enzymes would not be able to catalyze the reaction.
-<br>The protein extracts of VioA and VioA_pts were frozen before continuing the SDS PAGE on the next day. This, as well as poor handling of samples can lead to degradation. Also the liquid culture of VioB_pts grew over two days to reach our desired OD<sub>600</sub>, however the culture may already have reached stationary phase.
+<br>
-To decrease protein degradation in the future, protease inhibitors should be added to the lysis solution and all samples should be kept on ice.
+The genes for VioA, VioB and VioE were amplificated via PCR with Golden Gate compatible overhangs from the biobrick VioABCE
-<br> For further analysis of the enzyme functionality an assay followed by HPLC-MS analysis was implemented.
+<a href="http://parts.igem.org/Part:BBa_K274004">
+<abbr title="Part:BBa_K274004">
+(Part: BBa_K274004).
+</abbr>
+</a>
 </p>
+<div  class="half-width">
 <figure>
-<img
+<img src="https://static.igem.org/mediawiki/parts/0/00/T--Cologne-Duesseldorf--BioBrickViolaceinPlatte.png">
-src="https://static.igem.org/mediawiki/2017/2/2c/Artico_PDV_MS.png">
-<figcaption>
-<strong>Figure 8.2</strong> Overlaid extracted ion chromatograms (EIC) m/z 312.1131 of cell suspension 1 (CS1) time course experiment analyzed via LC-MS (Dionex Ultimate 3000, Thermofisher, USA; Maxis 4G, Bruker, Germany). From this given sample, $10\,\mu l$ were injected and measured in positive ionization mode. The signal intensity is given in counts per second. The section from 5.2 min to 6.2 min is shown. The peaks are corresponding to samples of the time course experiment taken at t=0 min, t=30, t=60, t=90 and t=120 past reaction start. The coloring of the peaks is increasing over time starting at light green for t=0 min to dark green for t=120 min. The prodeoxyviolacein (PDV) peak is shifting over time between 5.62 min from samples taken at 30 min after reaction start up to 5.57 min from samples taken at 120 min after reaction start. The counts increase continuously from about 0.01x10<sup>5</sup> to about 0.9x10<sup>5</sup>.
-</figcaption>
 </figure>
+</div>
 <p>
-<strong>Figure 8.2 </strong>shows the results of the mass spectrometry analysis of the PDV <i>in vitro</i> assay. Over a period of 120 min, samples were taken every 30 minutes. The cell suspension reaction mixture 1 (CS1) shows increase of the PDV production over time. PDV has a molecular weight of 312.1131, confirming the peak as the possible expected molecule. The mass spectrometry analysis of the wild type control did not show any peaks at the retention time of the potential PDV (data not shown).
+By Golden Gate cloning the peroxisomal targeting sequence (PTS1) was attached to the C-terminus of every pathway protein. Combined with the other necessary parts of the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-2"> toolbox </a> they represent the level 1 plasmids.
-<br>The shown data is from the cell suspension reaction. The LC-MS signals obtained with extracts from the protein suspension were too low to identify any possible molecule. A possible reason is that in contrast to the cell suspension, the protein extract lacks cofactors we did not consider in our master mix. Although the protein extraction was done precise and well-planned, we cannot guarantee a native protein state, which is needed for the enzymes to catalyse the reaction. Also the second reaction mix, containing a higher VioB concentration did not show a higher PDV production (data not shown). VioB is supposed to be the limiting factor of the reaction
+</p>
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/17176066">
-<abbr title="In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum">
+<div class="flex-row-2">
-(Balibar CJ <i> et al.</i>, 2006)
+    <div>
-</abbr>
+    <img src="https://static.igem.org/mediawiki/2017/b/b2/T--Cologne-Duesseldorf--VioA_pts1_plasmid.jpg">
-</a>
+    </div>
-, therefore we did the second reaction mixtures with a higher concentration of VioB.
+    <div>
+    <img src="https://static.igem.org/mediawiki/2017/1/1b/T--Cologne-Duesseldorf--VioB_lvl1_pts1_plasmid.jpg">
+    </div>
+</div>
+<div class="flex-row-2">
+    <div>
+ <img src="https://static.igem.org/mediawiki/2017/6/63/VioC_lvl1_pts1_plasmid.jpg">
+    </div>
+    <div>
+    <img src="https://static.igem.org/mediawiki/2017/4/43/T--Cologne-Duesseldorf--VioE_lvl1_pts1_plasmid.jpg">
+    </div>
+</div>
+<p>
+The PTS-tag marks the proteins for the import into peroxisomes. This should first of all point out the functionality of the yeast’s natural import mechanism and also be the basis for demonstrating our own modeled PTS*, proving our designed <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Pts1Import">orthogonal import mechanism</a>.
+Furthermore we also aim to optimize the working conditions for the enzymes inside of the reaction room - the peroxisomes. For example to vary the pH with new <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#MembraneIntegration"> membrane proteins </a> such as bacteriorhodopsin. To secure this change, we can also check the current conditions by our designed <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Sensors"> sensors</a>.
+</p>
+<p>
+There are several methods to verify the pathway’s enzymes. First of all, violacein and several intermediates (prodeoxyviolacein, deoxyviolacein, proviolacein) are colorful and the production in yeast can be visualized easily. Furthermore we added a His-/Flag-tag to the N-terminus of every protein (see geneious plasmid cards) to confirm their expression via SDS page and western blot. After verifying the presence of the enzymes the next step is to test their functionality. Before performing <i>in vivo</i> experiments in yeast an <i>in vitro</i> assay was implemented. For this the three enzyme pathway leading to PDV was reconstructed, testing VioA, VioB and VioE. To enable the best conditions for the enzymes, the pathway was studied intensively and all needed cofactors were calculated and added to the <i>in vitro</i> reaction (see protocol <a href="https://static.igem.org/mediawiki/2017/a/aa/T--Cologne-Duesseldorf--prodeoxyviolacein_assay.pdf"> prodeoxyviolacein <i>in vitro </i>assay</a>). This included FAD, MgCl<sub>2</sub>, catalase for decomposition of hydrogen peroxide, and the substrate L-tryptophan. The <i>in vitro</i> reaction was followed by qualitative analysis via HPLC and mass spectrometry.
 </p>
-<p>To identify PDV as accumulating compound at a retention time of about 5.6 min in LS-MS analysis, MS/MS experiments with standards were conducted afterwards. For further identification of the accumulating compound, fragmentation experiments are essential to exclude the accumulation of other compounds with the same molecular weight. Comparing the potential compound with measurements of standards enables its identification.
+</div>
+<button class="accordion"> <h2 id="optogenetics">Optogenetic enhancements</h2>
+<p>
+Optogenetics provide a useful tool for controlling cellular processes with high spatial and temporal precision. These include the expression of certain genes as well as the interaction or separation of two proteins. As many of our toolbox’s aspects benefit from precise control, we wanted to include optogenetics as a way of increasing its variability. Our plans included optogenetically controlled protein import, control of compartment size and number, and control of product secretion.
+</p>
+ </button>
+<div class="panel">
+<h3>Introduction</h3>
+<p>
+Our optogenetic toolbox enhancements can be divided into three subgroups: controllable protein import via Pex5, controllable protein import via Pex7 and controllable gene expression. Each of these sub-projects are of different design which will be illustrated in the following.
+</p>
+<h3>Pex5 import with LOV2</h3>
+<p>
+LOV2 is an optogenetic protein derived from <i>Avena sativa’</i>s Phototropin 1. In its dark state the J$_{\alpha}$-helix located at the C-terminus is bound to the core of the protein. Upon irradiation with blue light (~460 nm), a covalent bond between a cysteine residue on the LOV2 protein and a flavin mononucleotide chromophore causes the J$_{\alpha}$-helix to unfold, which in turn exposes the C-terminus
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/26513473">
+  <abbr title="Spilltor J., Strickland D, Glotzer M., Tucker CL (2016).Optical Control of Peroxisomal Trafficking.
+">
+      (Spiltoir <i> et al</i>, 2016)
+  </abbr>
+</a>.
+This property is very useful, as short amino acid sequences can be attached to this end of the LOV-protein, for example a PTS1. The idea for this project was to attach PTS1 to the C-terminus and the protein of interest to the N-terminus. Upon irradiation with blue light, the fusion protein would be imported into our compartment.
+We used a mutated version of LOV2 whose C-terminus has an increased dark-state binding affinity. This is caused by the substitutions and T406A and T407A. These mutations greatly reduce the possibility of the J$_{\alpha}$-helix being exposed in the dark state.
+Our PTS1 sequence consists of the amino acids LQSKL.
+As a proof of concept for this construct we fused sfGFP to its N-terminus:</p>
 </p>
-<div class="max-width">
 <figure>
-<img
+    <img src="https://static.igem.org/mediawiki/2017/c/c1/T--Cologne-Duesseldorf--GFP-LOV-PTS1.png">
-src="https://static.igem.org/mediawiki/2017/e/ef/T--Cologne-Duesseldorf--PDV_MSMS.jpg">
+    <figcaption><a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox"><strong>Figure 9.1</strong></a> Level 1 plasmid containing the sequence coding for the GFP-LOV2-PTS1 fusion protein</figcaption>
-<figcaption>
-<strong>Figure 8.3</strong> Fragment spectra of violacein/deoxyviolacein standard mix and potential prodeoxyvioalcein measured by direct infusion MS and LC-MS (Dionex Ultimate 3000, Thermofisher, USA; Maxis 4G, Bruker, Germany). Highlighted sub molecular structures represent the corresponding masses caused by fragmentation of the parent ion. The mass differences between the highlighted peaks are caused by the loss of a carbonyl group or nitrogen resulting of the fragmentation. Similar masses between the fragment spectra are marked with yellow/green dotted lines. <strong>A</strong>: fragment spectrum of violacein standard measured with direct infusion. <strong>B</strong>: fragment spectrum of deoxyviolacein standard measured with direct infusion. <strong>C</strong>: fragment spectrum of potential PDV measured with LC-MS; sample CS1 from <a href="https://static.igem.org/mediawiki/2017/a/aa/T--Cologne-Duesseldorf--prodeoxyviolacein_assay.pdf"> prodeoxyviolacein <i> in vitro </i> assay </a>.
-</figcaption>
 </figure>
-</div>
+<p>
+ This was done in order to visualize our experiment’s results during microscopy: upon successful import of the fusion protein, one would observe GFP fluorescence localized to our compartment. Otherwise, the whole cell would be illuminated.
-<p><strong>Figure 8.3 </strong>shows the fragment spectra for violacein (<strong>A</strong>), deoxyviolacein (<strong>B</strong>) and PDV (<strong>C</strong>). To verify if the produced compound with a mass of 312.1132 is PDV, the fragment spectrum of this compound is compared with its structurally similar precursors violacein and deoxyviolacein. These compounds are commercially available. All compounds have in common that they lose CO (-28 Da), PDV based on its structure only one CO-group. All show the loss of 15 Da, corresponding to Nitrogen. Deoxyviolacein and PDV share the same indole system. Both show peaks at m/z 143 Da and 167 Da. Violacein, does not show these signals lacking this indole ring. On the other hand violacein and deoxyviolacein share the same oxo-indole ring, resulting in a signal of 211 Da.
+</p>
-<br>This measurements and analysis of the <i>in vitro</i> prodeoxyvioalcein assay prove the functionality of the enzymes VioA, VioB and VioE which are necessary to produce PDV from L-tryptophan (<a href="https://static.igem.org/mediawiki/2017/0/09/T--Cologne-Duesseldorf--Violacein_Pathway_komplett.png">violacein pathway</a>). The results therefore show, that the integration of the bacterial pathway into <i>Saccharomyces cerevisiae</i> has been successful.
+<h3>Pex7 import</h3>
+<p>
+The idea behind this project is to initially block the protein of interest’s PTS2 with a fluorescent protein which can be removed by an optogenetically activated TEV-protease. For this project we use the protein Phytochrome-B from <I>Arabidopsis thaliana</I> and its interaction partner PIF6. These two proteins, also derived from Arabidopsis thaliana, bind together upon irradiation by red light (660 nm) and separate upon irradiation with far-red light (780 nm). We used this property to activate a split version of a TEV-protease whose split halves were each fused to one of the two optogenetic proteins.
+</p>
 <br>
-<br>
+<p>
-<font size="2">
+The TEV-protease was obtained from the Biobrick BBa_K1319004. This variant contains the anti self-cleavage mutation S219V. Using overhang-PCR we created a split version of the Biobrick protease based on work done by
-Many thanks to Felix Büchel and the MS platform, Cologne for extraordinary support.</font>
-</p>
-<h4>Outlook </h4>
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/17072307">
+  <abbr title="Wehr MC., Laage R., Bolz U., Fischer TM., Grünewald S., Scheek S., Bach A., Nave KA., Rossner MJ. (2016). Monitoring regulated protein-protein interactions using split TEV
-<p> Our outlook is characterized by the vision to use our own modeled PTS* import sequence for a real world application. But first we want to further investigate our <i>in vitro</i> strategy including the missing enzymes VioC and VioD and move on towards already developed <i>in vivo</i> tests. It would be great to qualify a statement about the efficiency of different import combinations into the peroxisome. To do so, different level 2 plasmids were planned. On each plasmid the combination of enzymes being peroxisomal or cytosolic is different.
+">
+      Wehr <i>et al</i>, 2016
+  </abbr>
+</a>
+ and the <a href="https://2013.igem.org/Team:TU-Munich/Project/Killswitch">iGEM team Munich 2013</a>. The split was made between amino acid 118 and 119.
+<br>
+Our construct for attaching proteins N-and C-terminally is highly variable: it consists only of a TEV-cleavage site, the PTS2 sequence and a short linker and was designed as a 3b-part for the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#CloningStrategies">yeast-toolbox</a>. This means that we can attach any protein to its N- or C-terminus we desire.
+We planned on attaching different fluorescent proteins to each sides of the 3b-part in a level 1 ligation. For our experiment we planned on using the pairs mTurquoise-mVenus and GFP-mRuby.
 </p>
 <figure>
-<img
+    <img src="https://static.igem.org/mediawiki/2017/f/f7/T--Cologne-Duesseldorf--GFP-PTS2-Ruby.png ">
-src="https://static.igem.org/mediawiki/parts/b/bc/T--Cologne-Duesseldorf--Violacein-Level2Kombis.png">
+    <figcaption><strong> Figure 9.2 </strong><a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox">level 1</a> plasmid containing the sequence coding for the GFP-PTS2-Ruby fusion protein</figcaption>
-<figcaption>
-<strong>Figure 8.4 </strong>To each one of the five pathway enzymes a peroxisomal targeting signal (PTS1) can be added, leading to the import of this enzyme. For example in the left panel of this figure, a yeast cell with the import of VioE is pictured, whereas VioA, VioB, VioC and VioE remain cytosolic. Depending on PTS1 being attached to the enzyme or not, 20 different enzyme combinations are possible. Also more than one enzyme can be imported into the peroxisome as you can see in the middle or right panel. It is known that some intermediates can pass the peroxisomal membrane including its precursor L-tryptophan
-<a href="http://www.nature.com/articles/ncomms11152">
-<abbr title="Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways">
-(John E. Dueber <i>et al.</i>, 2015)
-</abbr>
-</a>
-.
-</figcaption>
 </figure>
 <p>
-To assure the import of the enzymes and for further analysis it is indispensable to perform peroxisomal purification for an overall quanti- and qualification. Also measurements of the pathway intermediates and their fluxes across the peroxisomal membrane have to further be analyzed. The final step would be the comparison of the differences in the yield level, depending on the localization of the pathway enzymes (cytosolic or peroxisomal). With this the assumed better production in peroxisomes could be shown.
+The two other constructs were planned as follows: Phytochrome B was fused to the C-terminal TEV-half, PIF6 was fused to the N-terminal TEV-half.
+The PhyB-TEV2 part and the TEV1-PIF6 part were supposed to be inserted into a shared plasmid via a level 2 Golden Gate ligation.
+Finally, the level 2 plasmid and the remaining level 1 construct were to be co transformed into <i>S. cerevisiae</i>.
+Our experiment consisted of illuminating one sample with red light (660 nm) while keeping another sample in the dark. Fluorescence microscopy would then be used to check whether the import was successful [3]. If cleavage and subsequent protein import was successful, fluorescence of one protein would be localized to the compartment while that of the other would be observed throughout the cell.
 </p>
-</div>
+<h3>Optogenetically controlled gene expression</h3>
-<button class="accordion"> <h3 id="optogenetics">Optogenetic enhancements</h3>
 <p>
-Here you can find our results and discussion regarding the optogenetics subtopic
+This project is based on work done by
+<a href="https://www.ncbi.nlm.nih.gov/pubmed/24556785">
+  <abbr title="Müller K., Zurbriggen MD. Weber W. (2016).Control of gene expression using a red- and far-red light-responsive bi-stable toggle switch.
+">
+      Weber <i>et al</i>, 2016
+  </abbr>
+</a>
+. Using the interaction between Phytochrome B and PIF6 they designed an optogenetic switch for enabling and disabling transcription of a chosen gene. It is based on the tetracycline operon and the transcription factor VP16. The tetO operator is located upstream of a minimal promoter which in turn is located upstream of the gene of interest. The tetR repressor binds to the tetO sequence. Fused to it is PIF6. Phytochrome B is fused to the transcription factor VP16. Upon illumination with red light, Phytochrome binds to the tetR-PIF6 complex. VP16 is now located in close proximity to the minimal promoter, which enables the RNA-polymerase-2 to start transcription of the gene of interest.
+<br>
+We designed a promoter part for the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#CloningStrategies">Dueber toolbox</a> which consists of tetO and the minimal promoter region. This can be used as a promoter in a level 1 ligation for any desired gene of interest (a GFP-tagged Pex11 in our example).
 </p>
- </button>
+<figure>
-<div class="panel">
+    <img src="https://static.igem.org/mediawiki/2017/1/1d/T--Cologne-Duesseldorf--TetO-GFP-Pex11-Lv1.png">
+    <figcaption> <strong> Figure 9.3 </strong><a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox"></a>Level 1 plasmid containing the sequence coding for the GFP-Pex11 fusion protein with the tetO-pmin promoter</figcaption>
+</figure>
-<h3>Pex5 import with LOV2</h3>
-<p>
+<p> Transformation into <I>S. cerevisiae</I> is accompanied by co-transformation of a level 2 plasmid containing both the tetR-PIF6 and PhyB-VP16 constructs:
-Our GFP-LOV-PTS1 construct was successfully cloned and transformed into <I>S. Cerevisiae</I>. Following a lightbox experiment, GFP-fluorescence was observed throughout the cells in both the illuminated sample and the dark control, indicating unsuccessful import (data not shown here).
-</p>
-<h3>Pex7 import</h3>
-<p>
-All three constructs of our Split-TEV PTS2 subproject have been successfully cloned to Lv1 in regards to the Dueber toolbox. A Lv2 plasmid containing the PhyB-TEV2 and TEV1-PIF6 constructs is required in order to transform all constructs into S. Cerevisiae. This has not been created so far.
-</p>
-<h3>Optogenetically controlled gene expression</h3>
-<p>
-The TetO-Pmin promoter construct has been brought to Lv1 with <I>mRuby</I> and <I>Pex11</I> as genes of interest. The TetR-PIF6 construct has also been brought to Lv1. The PhyB-VP16 construct has not been successfully integrated into the Dueber toolbox.
 </p>
+<figure>
+    <img src="https://static.igem.org/mediawiki/2017/d/d3/T--Cologne-Duesseldorf--PhyB-TetR-Lv2.png">
+    <figcaption><strong>Figure 9.4</strong><a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox"></a> Level 2 plasmid containing the sequences coding for the PhyB-VP16 and tetR-PIF6 proteins respectively</figcaption>
+</figure>
-<h2>Outlook</h2>
-<p>
-The variability of our compartment toolbox could be greatly increased by using optogenetics. We planned on using constructs suited for optogenetic control of protein import via both pathways as well as constructs designed for optogenetically controlled gene expression. Even though we did not get to finish our work  on this sub project in this year’s project, we still want to underline its importance for future applications and improvements of our toolbox.
-As mentioned above, we were not successful in demonstrating optogenetically controlled protein import via PTS1.
-Our theory here is that the LOV2-variant obtained from <i>Avena Sativa</i> does not correctly function in <i>S. Cerevisiae</i>, possibly due to the different cytosolic conditions, such as pH, ion- or enzyme concentrations. Future tests would involve using the LOV2-variant from <i>Arabidopsis Thaliana</i>.
-Another aspect we considered was the possibility of steric inhibition of the protein of interests function by the LOV2 attached to it. A future idea we came up for solving this problem would be to add a TEV-protease cleavage site between the protein of interest and the LOV2-protein. The corresponding TEV-protease could be fitted with a PTS, leading to cleavage of the fusion protein upon it being imported into the compartment.
-<br>
-<br>
-Unfortunately we were unable to test our TEV-protease construct, as we did not finish the cloning process. However, we think that upon further development of the toolbox it is an aspect which should be considered, especially since there is only one more cloning step which needs to be completed in order for it to be eligible for transformation into <I>S. Cerevisiae </I>.</p>
-<br>
-<br>
 <p>
-The optogenetic control of our <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#Secretion">secretion mechanism</a> via gene expression also still awaits testing due to unfinished cloning. If successful, it would enable secretion of our compartments content within a few hours after illumination.
+Verification methods depend on which gene is expressed. See <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#SizeAndNumber">size and number</a> and <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Secretion">secretion</a> for details.
-The system we worked on in the lab is not the only idea we thought about. Another approach we haven’t pursued yet is attaching the vSNARE-proteins we are using to PIF6 and insert Phytochrome B into the peroxisomal membrane via our Pex26 anchor(LINK ZU PMP). In theory, illumination with red light would then lead to instant secretion.
 </p>
 </div>
+<button class="accordion">
+ <h2 id="designrules">Design Rules For Genome Engineering Regarding Customization of Peroxisome Properties</h2>
+ <p>In order to reach the ultimate goal of creating a fully controllable artificial compartment, genome engineering can be utilized for customizing the compartment's properties,.These include aspects such as membrane permeability, size/number, or decoupling of peroxisomes from the cytoskeleton, the peroxisomal proteome or metabolome. In our project we used the Crispr Cas9 system for knocking out several genes (Pex9, Pex31&Pex32, INP1, POT1) at the same time in order to engineer the previously mentioned properties. Furthermore, we designed a yeast strain  with a completely replaced protein-import machinery for controlling the entire peroxisomal lumen.</p>
+  </button>
+<div class="panel">
+<p>For the future one could think of much more radical strategies for peroxisomal engineering with a final goal of a “minimal peroxisome” by redirecting metabolic pathways through changing the protein-localization-signal in the yeast genome. Additionally, endogenous metabolic pathways could be redirected to our novel artificial compartment for establishing a customized metabolism specifically tailored for the user's application.</p>
+  <h3>Introduction</h3>
+<p>In order to achieve a fully controllable artificial compartment, the first step was to design a completely orthogonal import system. Next was the knockout of endogenous import systems. However, a few proteins are imported neither by the Pex5 nor the Pex7 import machinery. Therefore, specific genome engineering designs, such as knockouts, deleting or redirecting the protein localization could be utilized for the ultimate goal of creating a synthetic organelle.</p>
+<p>Additionally, knockouts or genome integrations enable customization of the peroxisomal properties, such as membrane permeability, size/number, decoupling of peroxisomes from cytoskeleton and the peroxisomal metabolism.</p>
+<p>All these strategies allow a rational design of an artificial compartment, which is fully engineerable regarding  the proteome, metabolome and the entire peroxisomal environment.</p>
+<h3>Design of yeast multi -knockout strains</h3>
+<h4>The Crispr Cas9 System</h4>
+<p>The demands on yeast engineering have significantly increased with the design of more complex systems and extensive metabolic pathways. Genetic techniques that have historically relied on marker recycling are unable to keep up with the ambitions of synthetic biologists. In recent years the Crispr Cas9 system has been used for several strain-engineering purposes, including:</p>
+<ul>
+  <li>Markerless integration of multiple genetic cassettes into selected genomic loci</li>
+  <li>Multiplexed and iterative gene knockouts without the need to recycle a marker</li>
+  <li>Precise genome editing – nucleotide substitutions, etc.</li>
+</ul>
+<p>We utilized the Cas9 system as a tool for peroxisomal engineering and have adopted the existing toolbox from <abbr title="Lee, Michael E.; DeLoache, William C.; Cervantes, Bernardo; Dueber, John E. (2015): A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. In: ACS synthetic biology 4 (9), S. 975–986. DOI: 10.1021/sb500366v.">(Lee et al. 2015)</abbr> and the complete cloning system which also provides the possibilities for genome integration and gene editing by Cas9. For this, two oligonucleotides have to be designed for targeting the Cas protein to the gene of interest.</p>
+<figure>
+  <img src="https://static.igem.org/mediawiki/2017/3/3b/--T--cologne-duesseldorf--Cas9_1.PNG">
+  <figcaption>Figure 1: Plasmid construction for the gRNA expression plasmid<br>Two oligos, containing the targeting sequence of the gRNA, have to be annealed and can then be integrated in the gRNA entry Vector by a Golden Gate reaction. Adapted from (<abbr title="Lee, Michael E.; DeLoache, William C.; Cervantes, Bernardo; Dueber, John E. (2015): A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. In: ACS synthetic biology 4 (9), S. 975–986. DOI: 10.1021/sb500366v.">Lee et al. 2015</abbr>)</figcaption>
+</figure>
+<p>Several gRNA vectors can subsequently be assembled into one vector with a Cas9 expression cassette and then be transformed into yeast. The expression of Cas9 together with gene specific gRNA´s leads to double strand breakage followed by non-homologous end joining repair or homologous recombination, in case of added repair DNA (figure 3).</p>
+<figure>
+  <img src="https://static.igem.org/mediawiki/2017/6/69/--T--cologne-duesseldorf--Cas9_2.PNG">
+  <figcaption>Figure 2: Plasmid construction for the expression plasmid containing Cas9 and gRNA´s<br>Vector for Cas9 and gRNA expression, assembled by a Golden Gate reaction, containing a URA marker, Cen6 yeast origin and a kanamycin resistance. Adapted from (<abbr title="Lee, Michael E.; DeLoache, William C.; Cervantes, Bernardo; Dueber, John E. (2015): A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. In: ACS synthetic biology 4 (9), S. 975–986. DOI: 10.1021/sb500366v.">Lee et al. 2015</abbr>)</figcaption>
+</figure>
+<p>The combination of the Cas9 system with DNA repair sequences enables not only knockouts of peroxisomal proteins, but also allows redirecting protein localization by changing protein targeting signals or integration of linear DNA into yeast chromosomes. Genome engineering facilitates yeast strain development for customized peroxisomes.</p>
+<figure>
+  <img src="https://static.igem.org/mediawiki/2017/9/9b/--T--cologne-duesseldorf--Cas9_3.PNG">
+  <figcaption>Figure 3: Design of repair DNA sequences for homologous recombination after inducing double strand break by Cas9<br>
+  Repair DNA sequences can be used to increase the efficiency for Cas9 guided knocking out of specific genes, but would also allow genomic integration of targeting signals or complete genes.  Adapted from (<abbr title="Lee, Michael E.; DeLoache, William C.; Cervantes, Bernardo; Dueber, John E. (2015): A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. In: ACS synthetic biology 4 (9), S. 975–986. DOI: 10.1021/sb500366v.">Lee et al. 2015</abbr>)</figcaption>
+</figure>
+<h3>The peroxisomal proteome of yeast (saccharomyces cerevisiae)</h3>
+<p>The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (<abbr title="Kohlwein, Sepp D.; Veenhuis, Marten; van der Klei, Ida J. (2013): Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat--store 'em up or burn 'em down. In: Genetics 193 (1), S. 1–50. DOI: 10.1534/genetics.112.143362.">Kohlwein et al. 2013</abbr>). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.</p>
+<table>
+  <thead>
+    <tr>
+      <th>Gene</th>
+      <th>Required for growth on oleate</th>
+      <th>Expression induced by oleate</th>
+      <th>Enzyme/activity</th>
+      <th>Molecular mass (kDa)</th>
+      <th>Isoelectric point</th>
+      <th>Molecules per cell </th>
+      <th>Localization</th>
+      <th>Function</th>
+    </tr>
+  </thead>
+  <tbody>
+    <tr>
+      <td colspan=”9”>ß-Oxidation enzymes</td>
+    </tr>
+    <tr>
+      <td>PCS60 (FAT2)</td>
+      <td>No</td>
+      <td>Yes</td>
+      <td>Medium chain fatty acyl-CoA synthetase</td>
+      <td>60.5</td>
+      <td>9.98</td>
+      <td>8.770</td>
+      <td>Peripheral peroxisomal membrane and matrix</td>
+      <td>Activates fatty acids with a preference for medium chain lengths, C9-C13</td>
+    </tr>
+    <tr>
+      <td>FAT1</td>
+      <td>No</td>
+      <td>-</td>
+      <td> Very long chain fatty acyl-CoA synthetase and long chain fatty acid transporter</td>
+      <td>77.1</td>
+      <td>8.47</td>
+      <td>16,900</td>
+      <td>Lipid droplet, ER, peroxisome Three predicted TM</td>
+      <td>Activates fatty acids with a preference for very long chain lengths, C20–C26</td>
+    </tr>
+    <tr>
+      <td>POX1</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Acyl-CoA- oxidase</td>
+      <td>84.0</td>
+      <td>8.73</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Oxidation of acyl-CoA</td>
+    </tr>
+    <tr>
+      <td>CTA1</td>
+      <td>No</td>
+      <td>Yes</td>
+      <td>Catalase</td>
+      <td>58.6</td>
+      <td>7.46</td>
+      <td>623</td>
+      <td>Peroxisomal matrix</td>
+      <td>Degrades hydrogen peroxide produced by Pox1</td>
+    </tr>
+    <tr>
+      <td>FOX2 (POX2)</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Multifunctional enzyme; 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase</td>
+      <td>98.7</td>
+      <td>9.75</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td>POT1 (FOX3, POX3)</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>3-Ketoacyl-CoA thiolase</td>
+      <td>44.7</td>
+      <td>7.56</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Cleaves 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA</td>
+    </tr>
+    <tr>
+      <td>DCI1 (ECI2)</td>
+      <td>-</td>
+      <td>-</td>
+      <td> Δ(3,5)-Δ(2,4)-dienoyl-CoA isomerase (putative)</td>
+      <td>30.1</td>
+      <td>8.83</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Auxiliary enzyme of fatty acid β-oxidation; role in β-oxidation debated</td>
+    </tr>
+    <tr>
+      <td>SPS19 (SPX1)</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>2,4-Dienoyl-CoA reductase</td>
+      <td>31.1</td>
+      <td>9.67</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
+    </tr>
+    <tr>
+      <td>ECI1</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Δ3, Δ2-enoyl-CoA isomerase</td>
+      <td>31.7</td>
+      <td>8.21</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
+    </tr>
+    <tr>
+      <td>TES1 (PTE1)</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Acyl-CoA thioesterase</td>
+      <td>40.3</td>
+      <td>9.58</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Auxiliary enzyme of fatty acid β-oxidation</td>
+    </tr>
+    <tr>
+      <td>MDH3</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Malate dehydrogenase</td>
+      <td>37.3</td>
+      <td>10.00</td>
+      <td>3,300</td>
+      <td>Peroxisomal matrix</td>
+      <td>Required for the malate-oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+, part of the glyoxylate cycle
+      </td>
+    </tr>
+    <tr>
+      <td>IDP3</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>NADP+ dependent isocitrate dehydrogenase</td>
+      <td>47.91</td>
+      <td>10.02</td>
+      <td>ND</td>
+      <td>Peroxisomal matrix</td>
+      <td>Required for the 2-ketoglutarate/isocitrate shuttle, exchanging peroxisomal NADP+ for cytosolic NADPH</td>
+    </tr>
+    <tr>
+      <td>CAT2</td>
+      <td>No</td>
+      <td>No</td>
+      <td>Carnitine acetyl-CoA transferase</td>
+      <td>77.2</td>
+      <td>8.34</td>
+      <td>470</td>
+      <td>Peroxisome mitochondria</td>
+      <td>Transfers activated acetyl groups to carnitine to form acetylcarnitine which can be shuttled across membranes</td>
+    </tr>
+    <tr>
+      <td colspan=”9”>Glyoxylate cycle</td>
+    </tr>
+    <tr>
+      <td>CIT2</td>
+      <td>No</td>
+      <td>-</td>
+      <td>Citrate synthase</td>
+      <td>51.4</td>
+      <td>6.34</td>
+      <td>2,310</td>
+      <td>Peroxisomal matrix</td>
+      <td>Condensation of acetyl CoA and oxaloacetate to form citrate</td>
+    </tr>
+    <tr>
+      <td>MDH3</td>
+      <td>Yes</td>
+      <td>Yes</td>
+      <td>Malate dehydrogenase</td>
+      <td>37.3</td>
+      <td>10.00</td>
+      <td>3,300</td>
+      <td>Peroxisomal matrix</td>
+      <td>Required for the malate–oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+</td>
+    </tr>
+    <tr>
+      <td>MLS1</td>
+      <td>Yes</td>
+      <td>-</td>
+      <td>Malate synthase</td>
+      <td>62.8</td>
+      <td>7.18</td>
+      <td>ND</td>
+      <td>Peroxisomal protein</td>
+      <td>Required for utilization of nonfermentable carbon sources</td>
+    </tr>
+    <tr>
+      <td colspan=”9”> Other peroxisome-associated enzyme activities</td>
+    </tr>
+    <tr>
+      <td> GPD1 (DAR1, HOR1, OSG1, OSR5</td>
+      <td>-</td>
+      <td>-</td>
+      <td> NAD-dependent glycerol-3-phosphate dehydrogenase</td>
+      <td>42.9</td>
+      <td>5.26</td>
+      <td>807</td>
+      <td>Peroxisome, cytosol, nucleus</td>
+      <td> Key enzyme of glycerol synthesis, essential for growth under osmotic stress</td>
+    </tr>
+    <tr>
+      <td>PNC1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Nicotinamidase</td>
+      <td>25.0</td>
+      <td>6.23</td>
+      <td>7,720</td>
+      <td>Peroxisome, cytosol</td>
+      <td>Converts nicotinamide to nicotinic acid as part of the NAD(+) salvage pathway</td>
+    </tr>
+    <tr>
+      <td>NPY1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>NADH diphosphatase</td>
+      <td>43.5</td>
+      <td>6.26</td>
+      <td>846</td>
+      <td>Peroxisome cytosol</td>
+      <td>Hydrolyzes the pyrophosphate linkage in NADH and related nucleotides</td>
+    </tr>
+    <tr>
+      <td>STR3</td>
+      <td>-</td>
+      <td>-</td>
+      <td> Cystathionine β-lyase</td>
+      <td>51.8</td>
+      <td>7.96</td>
+      <td>ND</td>
+      <td>Peroxisome</td>
+      <td>Converts cystathionine into homocysteine</td>
+    </tr>
+    <tr>
+      <td>STR3</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Cystathionine ß-lyase</td>
+      <td>51.8</td>
+      <td>7.96</td>
+      <td>ND</td>
+      <td>Peroxisome</td>
+      <td>Converts cystathionine into homocysteine</td>
+    </tr>
+    <tr>
+      <td>GTO1</td>
+      <td>-</td>
+      <td>-</td>
+      <td> ω-Class glutathione transferase </td>
+      <td>41.3</td>
+      <td>9.53</td>
+      <td>-</td>
+      <td>Peroxisome</td>
+      <td>Induced under oxidative stress</td>
+    </tr>
+    <tr>
+      <td>AAT2(ASP5)</td>
+      <td>-</td>
+      <td>Yes</td>
+      <td>Aspartate aminotransferase</td>
+      <td>46.1</td>
+      <td>8.50</td>
+      <td>7,700</td>
+      <td>Cytosol, peroxisome</td>
+      <td>Involved in nitrogen metabolism</td>
+    </tr>
+    <tr>
+      <td>PCD1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Nudix pyrophosphatase with specificity for coenzyme A and CoA derivatives</td>
+      <td>39.8</td>
+      <td>6.59</td>
+      <td>238</td>
+      <td>Peroxisome</td>
+      <td>May function to remove potentially toxic oxidized CoA disulfide from peroxisomes</td>
+    </tr>
+    <tr>
+      <td>LPX1</td>
+      <td>-</td>
+      <td>Yes</td>
+      <td>Triacylglycerol lipase</td>
+      <td>43.7</td>
+      <td>8.16</td>
+      <td>2,350</td>
+      <td>Peroxisomal matrix</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td colspan=”9”>Peroxisomal transporters</td>
+    </tr>
+    <tr>
+      <td>PXA1 (LPI1, PAL1, PAT2, SSH2</td>
+      <td>-</td>
+      <td>-</td>
+      <td> Subunit of a heterodimeric ATP-binding cassette transporter complex</td>
+      <td>100.0</td>
+      <td>10.34</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>Import of long-chain fatty acids into peroxisomes</td>
+    </tr>
+    <tr>
+      <td>PXA2 (PAT1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Subunit of a heterodimeric ATP-binding cassette transporter complex</td>
+      <td>97.1</td>
+      <td>9.47</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>Import of long-chain fatty acids into peroxisomes</td>
+    </tr>
+    <tr>
+      <td>ANT1(YPR118C)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Adenine nucleotide transporter</td>
+      <td>36.4</td>
+      <td>10.6</td>
+      <td>2,250</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in β-oxidation of medium-chain fatty acids</td>
+    </tr>
+    <tr>
+      <td colspan=”9”>Peroxins</td>
+    </tr>
+    <tr>
+      <td>Pex1 (PAS1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>AAA ATPase</td>
+      <td>117.3</td>
+      <td>6.93</td>
+      <td>2,100</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in recycling of Pex5, forms heterodimer with Pex6</td>
+    </tr>
+    <tr>
+      <td>Pex2 (RT1, PAS5)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>E3 ubiquitin ligase</td>
+      <td>30.8</td>
+      <td>9.02</td>
+      <td>339</td>
+      <td>Peroxisomal membrane</td>
+      <td>RING finger protein, forms complex with Pex10 and Pex12. Involved in matrix protein import</td>
+    </tr>
+    <tr>
+      <td>Pex3 (PAS3)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>50.7</td>
+      <td>6.29</td>
+      <td>1,400</td>
+      <td>Peroxisomal membrane</td>
+      <td>Required for proper localization of PMPs</td>
+    </tr>
+    <tr>
+      <td>Pex4 (PAS2, UBC10)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Ubiquitin conjugating enzyme</td>
+      <td>21.1</td>
+      <td>5.36</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in matrix protein import</td>
+    </tr>
+    <tr>
+      <td>Pex5 (PAS10)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Soluble PTS1 receptor</td>
+      <td>69.3</td>
+      <td>4.79</td>
+      <td>2,070</td>
+      <td>Cytosol and peroxisomal matrix</td>
+      <td>Required for import of PTS1-containing peroxisomal proteins, contains TPR domains</td>
+    </tr>
+    <tr>
+      <td>Pex6 (PAS8)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>AAA ATPase</td>
+      <td>115.6</td>
+      <td>5.44</td>
+      <td>1,630</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in recycling of Pex5, forms heterodimer with Pex1</td>
+    </tr>
+    <tr>
+      <td>Pex7 (PAS7, PEB1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Soluble PTS2 receptor</td>
+      <td>42.3</td>
+      <td>8.34</td>
+      <td>589</td>
+      <td>Cytosol and peroxisomal matrix</td>
+      <td>Requires Pex18 and Pex21 for association to the receptor docking site, contains WD40 repeat </td>
+    </tr>
+    <tr>
+      <td>Pex8 (PAS6)</td>
+      <td>-</td>
+      <td>-</td>
+      <td> Intra peroxisomal organizer of the peroxisomal import machinery </td>
+      <td>68.2</td>
+      <td>7.62</td>
+      <td>538</td>
+      <td>Peroxisomal matrix and luminal membrane face</td>
+      <td>Pex5-cargo dissociation</td>
+    </tr>
+    <tr>
+      <td>Pex9</td>
+      <td>-</td>
+      <td>-</td>
+      <td>PTS-receptor</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td>Pex10</td>
+      <td>-</td>
+      <td>-</td>
+      <td>E3 ubiquitin ligase</td>
+      <td>39.1</td>
+      <td>9.88</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>RING finger protein involved in Ubc4-dependent Pex5 ubiquitination. Forms complex with Pex2 and Pex12 </td>
+    </tr>
+    <tr>
+      <td>Pex11 (PMP24, PMP 27)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>26.9</td>
+      <td>10.65</td>
+      <td>1,630</td>
+      <td>Peroxisomal membrane</td>
+      <td>Involved in peroxisome fission, required for medium-chain fatty acid oxidation </td>
+    </tr>
+    <tr>
+      <td>Pex12 (PAS11)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>E3 ubiquitin ligase</td>
+      <td>46.0</td>
+      <td>9.86</td>
+      <td>907</td>
+      <td>-</td>
+      <td>RING finger protein, forms complex with Pex2 and Pex10</td>
+    </tr>
+    <tr>
+      <td>Pex13 (PAS20)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Component of docking complex for Pex5 and Pex7</td>
+      <td>42.7</td>
+      <td>9.83</td>
+      <td>7,900</td>
+      <td>Peroxisomal membrane</td>
+      <td>Forms complex with Pex14 and Pex17</td>
+    </tr>
+    <tr>
+      <td>Pex14</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Central component of the receptor docking complex</td>
+      <td>38.4</td>
+      <td>4.61</td>
+      <td>2,570</td>
+      <td>Peroxisomal membrane</td>
+      <td>Interacts with Pex13</td>
+    </tr>
+    <tr>
+      <td>Pex15 (PAS21)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>43.7</td>
+      <td>8.42</td>
+      <td>1,070</td>
+      <td>Peroxisomal membrane</td>
+      <td>Recruitment of Pex6 to the peroxisomal membrane, tail anchored PMP</td>
+    </tr>
+    <tr>
+      <td>Pex17 (PAS9)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Component of docking complex for Pex5 and Pex7</td>
+      <td>23.2</td>
+      <td>10.24</td>
+      <td>656</td>
+      <td>Peroxisomal membrane</td>
+      <td>Forms complex with Pex13 and Pex14</td>
+    </tr>
+    <tr>
+      <td>Pex18</td>
+      <td>-</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Required for PTS2 import</td>
+      <td>32.0</td>
+      <td>4.78</td>
+      <td>ND</td>
+      <td>Interacts with Pex7 partially redundant with Pex21</td>
+    </tr>
+    <tr>
+      <td>Pex19 (PAS12)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Chaperone and import receptor for newly synthesized PMP</td>
+      <td>38.7</td>
+      <td>4.08</td>
+      <td>5,350</td>
+      <td>Cytosol, peroxisome, farnesylated</td>
+      <td>Interacts with PMPs, involved in PMP sorting. Also interacts with Myo2 and contributes to peroxisome partitioning</td>
+    </tr>
+    <tr>
+      <td>Pex21</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Required for PTS2 protein import</td>
+      <td>33.0</td>
+      <td>6.67</td>
+      <td>ND</td>
+      <td>Cytosol</td>
+      <td>Interacts with Pex7, partially redundant with Pex18</td>
+    </tr>
+    <tr>
+      <td>Pex22(YAF5)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Required for import of peroxisomal proteins</td>
+      <td>19.9</td>
+      <td>8.33</td>
+      <td>259</td>
+      <td>Peroxisomal membrane</td>
+      <td>Recruits Pex4 to the peroxisomal membrane</td>
+    </tr>
+    <tr>
+      <td>Pex25</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome size and maintenance, required for re-introduction of peroxisomes in peroxisome deficient cells</td>
+      <td>44.9</td>
+      <td>9.77</td>
+      <td>2,420</td>
+      <td>Peripheral peroxisomal membrane</td>
+      <td>Recruits GTPase RhoI to peroxisomes, interacts with homologous protein Pex27</td>
+    </tr>
+    <tr>
+      <td>Pex27</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome size and number</td>
+      <td>44.1</td>
+      <td>10.49</td>
+      <td>382</td>
+      <td>Peripheral peroxisomal membrane</td>
+      <td>Interacts with homologous protein Pex25</td>
+    </tr>
+    <tr>
+      <td>Pex28</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome size, number and distribution</td>
+      <td>66.1</td>
+      <td>7.09</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>May act upstream of Pex30, Pex31 and Pex 32</td>
+    </tr>
+    <tr>
+      <td>Pex29</td>
+      <td>-</td>
+      <td>-</td>
+      <td>63.5</td>
+      <td>6.8</td>
+      <td>5,040</td>
+      <td>Peroxisomal membrane</td>
+      <td>May act upstream of Pex30, Pex31 and Pex32</td>
+    </tr>
+    <tr>
+      <td>Pex30</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome number</td>
+      <td>59.5</td>
+      <td>5.59</td>
+      <td>4,570</td>
+      <td>Peroxisomal membrane</td>
+      <td>Negative regulator, partially functionally redundant with Pex31 and Pex32</td>
+    </tr>
+    <tr>
+      <td>Pex31</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome number</td>
+      <td>52.9</td>
+      <td>10.15</td>
+      <td>238</td>
+      <td>Peroxisomal membrane</td>
+      <td>Negative regulator, partially functionally redundant with Pex30 and Pex32 </td>
+    </tr>
+    <tr>
+      <td>Pex32</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome number</td>
+      <td>48.6</td>
+      <td>9.14</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>Negative regulator partially functionally redundant with Pex30 and Pex31</td>
+    </tr>
+    <tr>
+      <td>Pex34</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in the regulation of peroxisome number</td>
+      <td>16.6</td>
+      <td>10.30</td>
+      <td>ND</td>
+      <td>Peroxisomal membrane</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td colspan=”9”>Peroxisome fission and inheritance</td>
+    </tr>
+    <tr>
+      <td>DYN2 (SLC1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Light chain dynein</td>
+      <td>10.4</td>
+      <td>9.03</td>
+      <td>1,310</td>
+      <td>Cytosol</td>
+      <td>Microtubule motor protein</td>
+    </tr>
+    <tr>
+      <td>SEC20</td>
+      <td>-</td>
+      <td>-</td>
+      <td>v-SNARE</td>
+      <td>43.9</td>
+      <td>5.92</td>
+      <td>4,910</td>
+      <td>Golgi, ER</td>
+      <td>Involved in retrograde transport from the Golgi to the ER, interacts with the Dsl1 complex through Tip20</td>
+    </tr>
+    <tr>
+      <td>SEC39(DSL3)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Component of the Ds11p-tethering complex</td>
+      <td>82.4</td>
+      <td>4.65</td>
+      <td>1,840</td>
+      <td>ER, nuclear envelope</td>
+      <td>Proposed to be involved in protein secretion</td>
+    </tr>
+    <tr>
+      <td>DSL1 (RNS1)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Component of the ER target site that interacts with coatomer</td>
+      <td>88.1</td>
+      <td>4.69</td>
+      <td>8,970</td>
+      <td>Peripheral ER, Golgi membrane</td>
+      <td>Forms a complex with Sec39 and Tip20 that interacts with ER SNAREs, Sec20 and Use1</td>
+    </tr>
+    <tr>
+      <td>FIS1 (MDV2)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Required for peroxisome fission</td>
+      <td>17.7</td>
+      <td>9.87</td>
+      <td>2,410</td>
+      <td>Peroxisomal membrane mitochondria</td>
+      <td>Tail anchored protein recruits Dnm1 via Mdv1/Caf4; also involved in mitochondrial fission</td>
+    </tr>
+    <tr>
+      <td>DNM1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>GTPase, dynamin like protein involved in peroxisome fission</td>
+      <td>85.0</td>
+      <td>5.25</td>
+      <td>9,620</td>
+      <td>-</td>
+      <td>Also involved in mitochondrial fission</td>
+    </tr>
+    <tr>
+      <td> VPS1 (GRD1, LAM1, SPO15, VPL1, VPT26)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>GTPase, dynamin like protein involved in peroxisome fission</td>
+      <td>78.7</td>
+      <td>8.15</td>
+      <td>5,960</td>
+      <td>-</td>
+      <td>Also involved in vacuolar protein sorting</td>
+    </tr>
+    <tr>
+      <td> VPS34 (END12, PEP15, VPL7, VPT29, STT8, VPS7)</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Phosphatidylinositol 3-kinase</td>
+      <td>100.9</td>
+      <td>7.79</td>
+      <td>1,080</td>
+      <td>-</td>
+      <td>Forms complex with Vps15</td>
+    </tr>
+    <tr>
+      <td>INP1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Involved in retention of peroxisomes in mother cells</td>
+      <td>47.3</td>
+      <td>8.34</td>
+      <td>639</td>
+      <td>Peroxisomal membrane</td>
+      <td>Recruited to the peroxisome by binding to Pex3</td>
+    </tr>
+    <tr>
+      <td>INP2</td>
+      <td>-</td>
+      <td>-</td>
+      <td>Myo2 receptor, involved in peroxisome inheritance</td>
+      <td>81.5</td>
+      <td>9.41</td>
+      <td>736</td>
+      <td>Peroxisomal membrane</td>
+      <td>-</td>
+    </tr>
+    <tr>
+      <td>RHO1</td>
+      <td>-</td>
+      <td>-</td>
+      <td>GTP binding protein of the Rho subfamily of Ras like proteins, involved in actin assembly at the peroxisome</td>
+      <td>23.2</td>
+      <td>6.07</td>
+      <td>ND</td>
+      <td>-</td>
+      <td>Involved in <em>de novo</em> peroxisome formation recruited to peroxisomes by Pex25</td>
+    </tr>
+  </tbody>
+</table>
+<h3>Knockout designs in our project</h3>
+<h4>Pex 9</h4>
+<p>Pex9 is a recently discovered import receptor for PTS1 proteins, which is induced by oleate
+and is an import receptor for both malate synthase isoenzymes Mls1p and Mls2p. In order to get a completely empty reaction room, a Pex9 knockout was designed to prevent unintended protein import.</p>
+<h4>Pex 31 & Pex 32</h4>
+<p>It has been shown that knockouts of Pex31 and Pex32 leads to an increased Peroxisomal size, but additionally the membrane permeability was affected (<abbr title="Zhou, Yongjin J.; Buijs, Nicolaas A.; Zhu, Zhiwei; Gómez, Diego Orol; Boonsombuti, Akarin; Siewers, Verena; Nielsen, Jens (2016): Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition. In: Journal of the American Chemical Society 138 (47), S. 15368–15377. DOI: 10.1021/jacs.6b07394.">Zhou et al. 2016</abbr>). This effect can be used as a tool for engineering membrane permeability by knocking out or overexpress both genes. A knockout would lead to an increased permeability and one could think of an opposite effect in case of overexpression, but this has not been shown yet.</p>
+<h4>INP1</h4>
+<p>The INP1 knockout was designed after the skype call of Prof Dueber, who recommended us, decoupling of peroxisomes from cytoskeleton in order to improve the secretion efficiency. INP1 is responsible for the tethering of peroxisome, which would inhibit the secretion of peroxisomes.</p>
+<h4>POT1</h4>
+<p>The only protein, which is imported by the Pex7 import machinery in saccharomyces is the 3-ketoacyl-CoA thiolase (POT1). A knockout of POT1 would enable utilizing the Pex7 import for proteins of interest, which cannot be tagged at the C-terminus with pts1, without having unintended import of other enzymes.
+<h3>Genomic integration of our novel Pex5 import receptor</h3>
+<p>After testing our new Pex5 import systems, which is completely orthogonal to the natural import, the next step would be to replace the endogenous system with our artificial import system. Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance (Figure 4). Afterwards the plasmid was transformed into the yeast strain which was created by our collaboration partner Aachen (double knockout strain Pex5 & Pex7).</p>
+<figure>
+  <img src="https://static.igem.org/mediawiki/2017/2/27/--T--cologne-duesseldorf--Cas9_4.PNG">
+  <figcaption>Figure 4: Design of integration plasmid for integrating our orthogonal Pex5 import receptor.<br>Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance</figcaption>
+</figure>
+<p>The resulting yeast strain allows full control over the peroxisomal matrix proteome, by replacing the whole protein import machinery, which is the first step for creating our artificial compartment.</p>
+<h3>Outlook</h3>
+<p>Besides the genome engineering approaches, which were performed in our project one could think of more radical strategies for peroxisomal engineering. A final goal could be a “minimal peroxisome”, which contains only the proteins that are required for the biogenesis of the peroxisome and import of proteins and metabolites. On the one hand peroxisomal pathways could be redirected to cytosol or other organelles and one the other hand endogenous metabolic pathways could be redirected to our novel artificial compartment by changing the protein localization signal in the yeast genome with help of the Cas9 system. All these strategies would allow tremendous improvements for metabolic engineering applications by creating an artificial compartment, which can be rational designed and customized for specific metabolic pathways.</p>
+</div>
-</article>
+  </article>
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Difference between revisions of "Team:Cologne-Duesseldorf/Eric"

Revision as of 02:22, 2 November 2017

Design

Scientific background

The root problem

Our approach

Cloning strategies and the Yeast Toolbox for Multipart-Assembly

Yeast nomenclature

Design of our sub-projects

Protein Import

Engineering of Pex5 and PTS1

Designing our receptors

Experimental design

Pex13−mRuby

Pex5 variant

mTurquoise−PTS

Combination of our constructs

PTS screening

Mutagenesis of PTS2

Membrane Integration

Scientific background

PEX19-dependent

ER-dependent

Experimental Work/Design

Secretion

Experimental Design

Size and Number

Heading

In Vivo Sensors

Sensors

Nootkatone

Model influence on Nootkatone expression

Violacein

Optogenetic enhancements

Introduction

Pex5 import with LOV2

Pex7 import

Optogenetically controlled gene expression

Design Rules For Genome Engineering Regarding Customization of Peroxisome Properties

Introduction

Design of yeast multi -knockout strains

The Crispr Cas9 System

The peroxisomal proteome of yeast (saccharomyces cerevisiae)

Knockout designs in our project

Pex 9

Pex 31 & Pex 32

INP1

POT1

Genomic integration of our novel Pex5 import receptor

Outlook