Latest revision as of 02:20, 2 November 2017

Results

Introduction

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 paid off! Below you can find the results of our efforts.

Sub-projects

MD simulations

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.

**Figure 1.1:** 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*−PTS1* combinations, we show just two exemplary results.

The fluorescent signal of mTurquoise was detected in the whole cell of each modelled receptor−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.

Pex5 variant R19

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.

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 a Pex5 deficient yeast strain either carrying the wild type Pex5 receptor and the fluorescence protein mTurquoise tagged with our designed PTS1* or the modified R19 receptor and the fluorescence protein mTurquoise with the wild type PTS1 sequence. Both negative controls did not reveal any localized fluorescence signal, indicating that the wild type Pex5 receptor is not capable to recognize our modified PTS1* sequence and on the other hand our R19 receptor does not recognize the wild type PTS1 sequence either. Albeit, this experiment did not verify whether the localized fluorescence signal are indeed the peroxisomes. For that reason, we validated these results by coexpressing the import machinery with a peroxisomal marker protein as can be seen in the following.

**Figure 1.3:** Pex5 variant R19 with the PTS1* and PTS*, co-transformed with the Pex13-mRuby construct − 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.

**Figure 1.4:** Wild type Pex5 with the PTS1* and wild type PTS1.

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 experimental design, 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. 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.
Conclusively, all our negative controls were not able to import mTurquoise into the peroxisomes, confirming the orthogonality of our artificial protein import mechanism.
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.

Conclusion

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. Our results show that we designed and established a new and orthogonal peroxisomal import system in Saccharomyces cerevisiae. We modified one of the most conserved import machineries within the domain of eukaryotes - no matter if it is plants, mammals or fungi. This enables 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 increases the yield and efficiency of rare biomolecule production in vivo. Furthermore, we managed to make a big step 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. This is why our new import machinery shows the potential for biotechnology and real world applications.

Violacein assay

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.

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 (Horstman). Luciferase is highly efficient because almost all energy is converted into light, the protein is thus very sensitive (Azad). 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. 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 (Leskinen).

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 (Cramer). This workflow can be used to characterize the content of the purified peroxisomes by Western blot analysis.

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. ´

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.

Figure 2.1: Colonies of the PTS2 library show a colour range of white to green. 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.

Therefore we were able to generate targeting sequences of different effectivities. Subsequently the OD₆₀₀ and the fluorescence with an excitation wavelength of 535 nm and emission wavelength of 585 nm were measured. According to (DeLoache) 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 OD₆₀₀. A wide distribution of different values were observed indicating a broad variety of different PTS2 versions.

Figure 2.2: Fluorescence per OD₆₀₀ of VioABE transformants are shown. For all tested transformants, fluorescence with an excitation wavelength of 535 nm (λ_Ex: 535 nm) and emission wavelength of 585 nm (λ_Em: 585 nm) per OD₆₀₀, referring on yeast cell count, was calculated. Results show a large distribution of import efficiency and therefore different PTS2 versions.

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 OD₆₀₀ indicates a strong targeting sequence resulting in a low VioE concentration in the cytoplasm and no conversion of Tryptophan to PDV.

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.

Figure 1: PEX26 expression and integration

Fluorescent microscopy of bacteriorhodopsin coexpressed with sf-GFP:.
Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). The green fluorescent spots (on the right) shows a typical peroxisomal shape. The signal for membrane marker mRuby-PEX26 is shown in red (on the left). Both signals show colocalization when merged (middle), which indicates that the protein gets integrated into the membrane.

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 Pex3 and PEX26 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.

Figure 1: Bacteriorhodopsin expression and integration

**Fluorescent microscopy of bacteriorhodopsin coexpressed with sf-GFP:**.
Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). The green fluorescent spots (on the right) shows a typical peroxisomal shape. The signal for the membrane marked bacteriorhodopsin is shown in red (on the left). Both signals show colocalization when merged (middle), which indicates that the protein gets integrated into the membrane.

Finally we used the same approach to direct a mRuby-tagged bacteriorhodopsin to our compartment. In coexpressing it with the same GFP as in the previous steps, we could show that the bacteriorhodopsin as well as Pex3 and PEX26 were successfully integrated into the membrane of our compartment. Since bacteriorhodopsin is a rather complex protein, we're very optimistic about integrating other proteins into the membrane using the same approach.

Outlook

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 bacteriorhodopsin, 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.

Heading

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

**Figure 3.1 Microscopic validation of the peroxisomal membrane anchor Pex15.** 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

**Figure 3.2 Validation of the membrane anchor Peroxisomal membrane anchor Pex26.** 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.

Next we measured secretion of compounds that are inside our artificial compartment, using a liquid GUS-assay . 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 Pex11 deleted.
The fluorescence increase over time of the samples which are decorated with SNAREs 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 Pex11 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.)

**Figure 3.3 Relative fluorescence units per minute (RFU/min) measured for supernatants of different *S. cerevisiae* strains.** 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.

Sensors

Localization

pHlourin2 and roGFP2 was detectable by fluorescence microscopy and showed a similar excitation spectrum as native GFP Mahon et al. (2011). 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.

Figure5.1 Left: cytosolic pHLuorin2 expression. Right: wild type, without any fluorescence.

Carboxyl terminal fusion of the peroxisomal targeting signal 1 resulted in small areas with high intensity 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.

Figure5.2 Left: pHLuorin2 localized in the peroxisome. Right: roGFP2 localized to the peroxisome

peroxin13 mRuby construct showed concentrated localization inside cellular areas too. In comparison to our tested sensors with the comparable promoter parts 016 and 017 peroxin13 mRuby 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 level 2 plasmids containing both a sensor and peroxin13 mRuby showed colocalization which leaded to yellow spots in merged channels Robert Yung-Liang Wang et al. (2009) .

Figure5.3 Left: wild type without any fluorescence. Right: Level 2 Plasmid containing peroxin13-mruby and pHLuorin2-PTS1 using the mcherry channel.

Figure5.4 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.

Figure5.5 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.

pHluorin2

Initially we desired an in vivo calibration with pH values ranging from pH 5.8 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. The ionophore 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 in vitro assay . 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.

**Figure5.4** 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.

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.

**Figure5.6** 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.

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 .
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 in vivo measurements comparing peroxisomal and cytosolic pH.
For in vivo 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 OD₆₀₀ ranging from 0,8 up to 1.3. The large standard deviation might be rooted in the different ODs₆₀₀. Literature does not agree about whether the pH inside the peroxisome is acidic or alkaline nor whether there are endogenous regulating mechanisms (Francesco M. Lasorsaet al.(2004), Carlo W. T. van Roermundet al. (2004)) . Our result suggest a slight acid pH inside the peroxisomes and agreed with Francesco M. Lasorsaet al.(2004) .

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 in vivo calibration might have failed due to the disability of penetrating the yeast cell wall. Nevertheless we were able to measure the pH in vivo. 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.

roGFP2

After expression and correct localization to the peroxisome was validated we examined the function of roGFP2. We conducted an in vitro assay on fully oxidized and fully reduced roGFP2 and performed time measurements by subsequently adding H₂O₂ and DTT to the protein extract.

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 Schwarzländer et al.(2008) .

**Figure5.8** 405/485nm excitation ratio plotted against the oxidized proportion of roGFP2

Based on the nernst equation we were now able to calculate the redox potential of roGFP2 regarding the oxidation of roGFP2 Schwarzländer, et al.(2008).

**Figure5.9** Oxidized roGFP2 proportion plotted against the redox potential of glutathione (mV)

.

Using our calibrated sensor we could compare the redox states within strains which differ in metabolic physiology.

**Figure5.10** 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 OD₆₀₀ between 0.9 and 1.1.

We conducted two Mann-Whitney-Wilcoxen test on the different targets and on different promoters strength used(n₁=6, n₂=6) resulting in a p value of 0,69 and 0,82. Neither comparisons of cytosolic and peroxisomal targeting nor comparison of the different promoters showed significant differences in glutathione redox states. This result was surprising since varieties were reported in literature before. Schwarzländer et al. (2015).

Outlook

We planned to calibrate our sensors in vivo as well and wanted to follow up changes induced by violacein and nootkatone pathways. Furthermore our objective was testing the expected acidification upon induced expression and integration of bacteriorhodopsininto the peroxisomal membrane with pHLuorin2. roGFP2 can be fused to numerous redox catalytic enzymes making it specific to certain redox pools Schwarzländer et al. (2015).This property makes it interesting to further usage for the iGEM community.
In the future we want to expand our toolbox with an ATP and NADP⁺ 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⁺ sensor consists of two fluorophore proteins CFP and YFP and a indicator protein KBR. In the presence of NADP⁺, 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⁺ concentrations Feng-Lan Zhao et al. (2015).
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 Hiromo Imamura et al. (2009).

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.

**Figure 7.1** 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

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.
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.
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.

Outlook

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.

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 (Dueber Toolbox) 3xFlag/6xHis of the plasmid with an other fluorescent 3a part, namely mRuby2. We can then show the localization of the enzymes via microscopy.

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.

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. Takahashi et al. (2007) .

Violacein

The experiments were implemented following the protocols for western blot and in vitro assay prodeoxyviolacein .

**Figure 8.1** 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 OD₆₀₀: 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.

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 (Hurley A, 2017) .
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 OD₆₀₀, however the culture may already have reached stationary phase. To decrease protein degradation in the future, protease inhibitors should be added to the lysis solution and all samples should be kept on ice.
For further analysis of the enzyme functionality an assay followed by HPLC-MS analysis was implemented.

**Figure 8.2** 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 0.01x10⁵ to about 0.9x10⁵.

Figure 8.2 shows the results of the mass spectrometry analysis of the PDV in vitro 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).
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 catalyze 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 (Balibar CJ et al., 2006), therefore we did the second reaction mixtures with a higher concentration of VioB.

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.

**Figure 8.3** 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. A: fragment spectrum of violacein standard measured with direct infusion. B: fragment spectrum of deoxyviolacein standard measured with direct infusion. C: fragment spectrum of potential PDV measured with LC-MS; sample CS1 from prodeoxyviolacein *in vitro* assay .

Figure 8.3 shows the fragment spectra for violacein (A), deoxyviolacein (B) and PDV (C). 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 loose 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.
This measurements and analysis of the in vitro prodeoxyvioalcein assay prove the functionality of the enzymes VioA, VioB and VioE which are necessary to produce PDV from L-tryptophan (violacein pathway). The results therefore show, that the integration of the bacterial pathway into Saccharomyces cerevisiae has been successful.

Many thanks to Felix Büchel and the MS platform, Cologne for extraordinary support.

Outlook

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 in vitro strategy including the missing enzymes VioC and VioD and move on towards already developed in vivo 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.

**Figure 8.4** 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 (John E. Dueber *et al.*, 2015) .

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.

Pex5 import with LOV2

Our GFP-LOV-PTS1 construct was successfully cloned and transformed into S. cerevisiae. As part of our 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).

Pex7 import

All three constructs of our Split-TEV PTS2 subproject have been successfully cloned to level 1 in regards to the (Dueber Toolbox). A level 2 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.

Optogenetically controlled gene expression

The TetO-Pmin promoter construct has been brought to level 1 with mRuby and Pex11 as genes of interest. The TetR-PIF6 construct has also been brought to level 1. The PhyB-VP16 construct has not been successfully integrated into the Dueber toolbox.

Outlook

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, 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 Avena sativa does not correctly function in S. cerevisiae, possibly due to the different cytosolic conditions, such as pH, ion- or enzyme concentrations. Future tests would involve using the LOV2-variant from Arabidopsis thaliana. Another aspect we considered was the possibility of steric inhibition of the protein of interests function by the LOV2 attached to it. A future approach 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.
Due to a lack of time 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 S. cerevisiae.
The optogenetic control of our secretion mechanism 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. Another approach we have not pursued yet is attaching the vSNARE-proteins we are using to PIF6 and insert Phytochrome B into the peroxisomal membrane via our Pex26 anchor (see membrane integration). In theory, illumination with red light would then lead to instant secretion.

Difference between revisions of "Team:Cologne-Duesseldorf/jan"

Latest revision as of 02:20, 2 November 2017

Results

Introduction

Sub-projects

Pex5 Import

MD simulations

Pex5 variant R19

Conclusion

Violacein assay

Pex7 Import

Membrane Integration

Figure 1: PEX26 expression and integration

Figure 1: Bacteriorhodopsin expression and integration

Outlook

Size and Number

Heading

Secretion

Sensors

Sensors

Localization

pHluorin2

roGFP2

Outlook

Nootkatone

Outlook

Violacein

Violacein

Outlook

Optogenetic enhancements

Pex5 import with LOV2

Pex7 import

Optogenetically controlled gene expression

Outlook

@@ Line 1: / Line 1: @@
 {{Template:Cologne-Duesseldorf/css}}
 {{Template:Cologne-Duesseldorf/header}}
 <html>
 <body>
 <article>
-<h1>Project description</h1>
+<h1>Results</h1>
 <div id="ToC"></div>
+<h2>Introduction</h2>
+<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 paid off! Below you can find the results of our efforts.
+</p>
+<h2>Sub-projects</h2>
+<button class="accordion">
+  <h2 id="Pex5 Import">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">
-    <svg xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink" viewBox="0 0 1000 1000">
+<figure>
-    	<defs>
+<img src="https://static.igem.org/mediawiki/2017/b/b9/Artico_815.png">
-    		<style>
+<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.
-    			.cls-1 {
+</figcaption>
-    				fill-rule: evenodd;
+</figure>
-    				fill: url(#fill1);
+</div>
-    			}
-    			.cls-2,
-    			.cls-3 {
-    				fill: #11604d;
-    			}
-    			.cls-3 {
-    				opacity: 0.15;
-    			}
-    			.cls-4 {
-    				fill: #45b497;
-    			}
-    			.cls-5 {
-    				fill: #84c8bb;
-    			}
-    			.cls-6 {
-    				fill: #45b498;
-    			}
-    			.cls-10,
-    			.cls-7,
-    			.cls-8,
-    			.cls-9 {
-    				fill: none;
-    				stroke-miterlimit: 10;
-    			}
-    			.cls-7 {
-    				stroke: #45b497;
-    				stroke-width: 6.34px;
-    			}
-    			.cls-10,
-    			.cls-8,
-    			.cls-9 {
-    				stroke-width: 1.98px;
-    			}
-    			.cls-8 {
-    				stroke: url(#fill2);
-    			}
-    			.cls-9 {
-    				stroke: #11604d;
-    			}
-    			.cls-10 {
-    				stroke: url(#fill2-2);
-    			}
-    			g#svgNootkatone *{fill: rgb(162, 217, 203);transition: all 0.5s ease;}
+<p>
-    			g#svgNootkatone:hover *{fill: rgba(69, 180, 151, 1);opacity:1;}
+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.
+</p>
-    			g#svgProteinImport * {fill: rgb(162, 217, 203);transition: all 0.5s ease;}
+<h3>Pex5 variant R19</h3>
-    			g#svgProteinImport:hover * {fill: rgba(69, 180, 151, 1);}
-    			g#svgMembraneIntegration rect{fill: rgb(162, 217, 203);transition: all 0.5s ease;}
+<p>
-    			g#svgMembraneIntegration:hover rect{fill: rgba(69, 180, 151, 1);}
+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 <i>Pex5</i> deficient yeast strain.
-    			g#svgMembraneIntegration circle{fill: rgb(162, 217, 203);transition: all 0.5s ease;}
+</p>
-    			g#svgMembraneIntegration:hover circle{fill: rgba(69, 180, 151, 1);}
+<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
-    			g#svgMembraneIntegration path {stroke: rgb(162, 217, 203);transition: all 0.5s ease;}
+</figcaption>
-    			g#svgMembraneIntegration:hover path {stroke: rgba(69, 180, 151, 1);}
+</figure>
+</div>
+<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 a <i>Pex5</i> deficient yeast strain either carrying the wild type Pex5 receptor and the fluorescence protein mTurquoise tagged with our designed PTS1* or the modified R19 receptor and the fluorescence protein mTurquoise with the wild type PTS1 sequence. Both negative controls did not reveal any localized fluorescence signal, indicating that the wild type Pex5 receptor is not capable to recognize our modified PTS1* sequence and on the other hand our R19 receptor does not recognize the wild type PTS1 sequence either. Albeit, this experiment did not verify whether the localized fluorescence signal are indeed the peroxisomes. For that reason, we validated these results by coexpressing the 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.
-    			g#svgPeroxicretion rect {fill: rgb(162, 217, 203);transition: all 0.5s ease;}
+</figcaption>
-    			g#svgPeroxicretion:hover rect {fill: rgba(69, 180, 151, 1);}
+</div>
-    			g#svgPeroxicretion line {stroke: rgb(162, 217, 203);	transition: all 0.5s ease;}
+<div class="half-width">
-    			g#svgPeroxicretion:hover line {stroke: rgba(69, 180, 151, 1);}
+<figure>
+<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>
-    			g#svgPeroxicretion line.cls-8 {stroke: url(#fill21);transition: all 0.5s ease;}
-    			g#svgPeroxicretion:hover line.cls-8 {stroke: url(#fill22);}
-    			g#svgPeroxicretion line.cls-10 {stroke: url(#fill2_21);transition: all 0.5s ease;}
-    			g#svgPeroxicretion:hover line.cls-10 {stroke: url(#fill2_22);}
-    		</style>
-    <radialGradient id="fill1" cx="489.36" cy="506.65"
-        		r="370.29" gradientUnits="userSpaceOnUse">
-        			<stop offset="0.5" stop-color="#fff" stop-opacity="0.7" />
-        			<stop offset="1" stop-color="#ccc" /> </radialGradient>
-        		<linearGradient id="fill21" x1="656.42"
-        		y1="662.26" x2="709.23" y2="662.26" gradientTransform="matrix(-0.96, 0.28, 0.28, 0.96, 586.01, 54.84)"
-        		gradientUnits="userSpaceOnUse">
-        			<stop offset="0" stop-color="#a2d9cb" />
-        			<stop offset="1" stop-color="#fff" stop-opacity="0" /> </linearGradient>
-        		<linearGradient id="fill22" x1="656.42"
+<p>
-        		y1="662.26" x2="709.23" y2="662.26" gradientTransform="matrix(-0.96, 0.28, 0.28, 0.96, 586.01, 54.84)"
+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.
-        		gradientUnits="userSpaceOnUse">
+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.
-        			<stop offset="0" stop-color="#45b497" />
+<br>
-        			<stop offset="1" stop-color="#fff" stop-opacity="0" /> </linearGradient>
+Conclusively, all our negative controls were not able to import mTurquoise into the peroxisomes, confirming the orthogonality of our artificial protein import mechanism.
+<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>
-        		<linearGradient id="fill2_21" x1="1293.64"
-        		y1="481.7" x2="1346.45" y2="481.7" gradientTransform="translate(-248.31 54.84) rotate(16.13)"
-        		xlink:href="#fill21" />
-        		<linearGradient id="fill2_22" x1="1293.64" y1="481.7"
-        		x2="1346.45" y2="481.7" gradientTransform="translate(-248.31 54.84) rotate(16.13)"
-        		xlink:href="#fill22" />
-    <linearGradient id="fill2-2" x1="1293.64" y1="481.7" x2="1346.45" y2="481.7" gradientTransform="translate(-248.31 54.84) rotate(16.13)" xlink:href="#fill2"/>
-    	</defs>
+<h3>Conclusion</h3>
-    	<title>artico</title>
+<p>
-    	<g id="Ring">
+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.
-    		<path class="cls-1" d="M489.36,876.94a369.08,369.08,0,1,1,144.13-29.09A368,368,0,0,1,489.36,876.94Z"
+				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 enables 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 increases the yield and efficiency of rare biomolecule production <em>in vivo</em>.
-    		/>
+				Furthermore, we managed to make a big step 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.
-    		<path class="cls-2" d="M489.36,151.22A354.23,354.23,0,1,1,351,179.14a353.18,353.18,0,0,1,138.34-27.92m0-29.72c-212.71,0-385.15,172.44-385.15,385.15S276.65,891.8,489.36,891.8,874.51,719.37,874.51,506.65,702.07,121.5,489.36,121.5Z"
+				This is why our new import machinery shows the potential for biotechnology and real world applications.
-    		/>
+</p>
-    	</g>
+<div class="callout">
-    	<a href="#Nootkatone">
+<h3>Violacein assay</h3>
-    	<g id="svgNootkatone">
+<p>
-    		<path class="cls-3" d="M666.49,521.12c-1.29,17.06-4.76,33.69-11.31,49.47-25.31,61-70.33,98.21-135.22,111.09-6.17,1.22-12.47,1.81-18.71,2.68H477.72a1.24,1.24,0,0,1-.32,0,20.14,20.14,0,0,0-2.15-.53c-8.22-1.23-16.53-2-24.65-3.71-68.88-14.5-124.94-73.2-136.22-142.47-1-6-1.7-12.05-2.5-18.09a26.93,26.93,0,0,1-.24-3.57V498.09a24.21,24.21,0,0,1,.43-4.67c.06-.3.11-.6.17-.9,1.91-11,2.64-22.4,5.91-33,21.63-70.18,68.56-112.95,140.59-127.85,6.39-1.32,12.95-1.84,19.43-2.72h21.46a1.24,1.24,0,0,1,.28,0,16.35,16.35,0,0,0,2.54.54,164.66,164.66,0,0,1,62,16.18c54.66,26.84,88.11,70.18,99.87,130,1.28,6.51,1.88,13.16,2.79,19.75a1.18,1.18,0,0,1,0,.16M433.33,624.12a1.22,1.22,0,0,1-.32-1.48c4.65-9.39,9.4-18.3,13.46-27.52,4.55-10.34,8.47-21,12.54-31.5a4.77,4.77,0,0,0,.2-2,1.22,1.22,0,0,0-2.12-.78l-52,56.38a1.22,1.22,0,0,0,.14,1.78c50.47,39.51,134,40.81,187.38-17.05s45.56-140.95,1.89-188.21a1.22,1.22,0,0,0-1.79,0l-41.62,45.15,4.12-.39c10-1,20-2.51,30-2.68,7-.12,14.24,1.15,21.16,2.7,4.74,1.06,8.79,4.34,9.08,9.76s-3.64,8.81-8.26,10.41a103.7,103.7,0,0,1-19,4.92c-19.53,2.79-39.21,2.54-58.89,2a9.73,9.73,0,0,0-3.73,1.09,1.23,1.23,0,0,0-.51,1.87c11.52,15.28,22.41,29.74,33.35,44.16a8.86,8.86,0,0,0,3,2.31c12.31,6.41,24.65,12.76,37.21,19.25a1.23,1.23,0,0,1,.63,1.35c-.24,1.13-.59,1.13-2.39.77-11.35-2.29-22.49-5.27-33-10.36-6-2.92-11.24-6.69-15-12.46-2.7-4.16-5.94-8-8.95-11.93l-20.2-26.61a1.22,1.22,0,0,0-1.87-.09l-4.74,5.11a1.23,1.23,0,0,0-.08,1.57c5.56,7.36,10.91,14.62,16.51,21.69,1.74,2.19,1.89,4,.94,6.52-3.41,9.2-6.68,18.46-9.72,27.8a11.68,11.68,0,0,0-.28,7c3.41,9.55,6.93,19.1,11,28.37C536,603.19,541.11,613,546,622.92a1.19,1.19,0,0,1-.11,1.26c-1.28,1.63-2.65,1.26-3.93-.4-3.94-5.11-8.31-10-11.61-15.48-8.57-14.26-13.93-29.9-18.42-45.84a8.53,8.53,0,0,1,.34-4.67c3-9.22,6.23-18.37,9.27-27.59.35-1.07.54-2.72,0-3.49-4.48-6.18-9.14-12.23-13.77-18.33a1.22,1.22,0,0,0-1.77-.18c-5.37,4.67-8.89,9.64-8.51,17.45.33,6.68-1.37,13.52-2.59,20.2-.55,3-1.85,6.77-5.46,6.74s-4.64-3.82-5.49-6.79-1.33-5.74-2-8.87a1.22,1.22,0,0,0-2.1-.58c-3.47,3.84-6.71,7.65-10.36,11s-5.08,6.71-2.43,11.32a4.9,4.9,0,0,1,.31,3.58c-5.46,16.22-11.16,32.37-19.93,47.15-3.26,5.5-7.76,10.28-11.71,15.37l-.11.11a1.16,1.16,0,0,1-1.42-.09Zm123.51-235a1.22,1.22,0,0,1,.11,1.7c-5.31,6.22-10.27,12.29-15.58,18-3.44,3.71-5.57,7.65-6,12.78-.52,6.31-1.75,12.55-2.6,18.83l40.84-44.26a1.22,1.22,0,0,0-.14-1.78C523.55,355,439.39,353,385.57,412s-44.15,142.93-.53,188.4a2.11,2.11,0,0,0,.6-.32q31.07-33.66,62.07-67.39a3.66,3.66,0,0,0,.58-3.26c-1.23-2.54-.27-4.27,1.24-6.24q14.27-18.57,28.37-37.27c.17-.22.33-.45.5-.7a1.22,1.22,0,0,0-1.15-1.89c-1.65.19-3.18.38-4.68.44a10.76,10.76,0,0,0-8.17,4.32Q446,512.8,427.26,537.34a17.43,17.43,0,0,1-3.91,3.86c-12.68,8.68-27.18,12.28-41.89,15.26a1.22,1.22,0,0,1-1.4-.83c-.62-2-.35-2,1.36-2.8q18.51-9,37-18.17a10,10,0,0,0,3.25-3Q438,509.89,454.22,488c.2-.27.38-.54.57-.84a1.22,1.22,0,0,0-1.1-1.87c-1.11.07-2.08.13-3.06.16-21.52.76-43,.87-64.39-2.47-6.52-1-13-2.44-18.49-6.43-5.72-4.16-6.1-10.44-.58-14.84a24.84,24.84,0,0,1,8.92-4.32c10.1-2.68,20.52-2.54,30.68-1,19.05,2.93,38,6.62,56.95,10.13,4.88.9,7.14.34,9.82-3.53a1.22,1.22,0,0,0-.64-1.86,64.38,64.38,0,0,1-23.29-12.95,9.43,9.43,0,0,1-3-5.13c-1.23-8-1.93-16.07-3.14-24.06a13.9,13.9,0,0,0-2.75-6.42c-5.94-7.31-12.13-14.43-18.38-21.76a1.22,1.22,0,0,1,.11-1.7l.77-.68a1.22,1.22,0,0,1,1.62,0c8.33,7.49,16.52,14.8,24.59,22.22a6.33,6.33,0,0,1,1.61,3.63c1.09,8.25,1.94,16.53,3.1,24.77a7.51,7.51,0,0,0,2.32,4.34c6.42,5.43,14,8.5,22,10.93a1.22,1.22,0,0,0,1.33-.43l2.6-3.45a1.22,1.22,0,0,0-.63-1.9l-.59-.16c-6.44-1.54-8.59-6.79-4.51-12,1.87-2.36,4.82-3.84,7.11-5.91a6.11,6.11,0,0,0,2.1-3.37c.48-4.71.58-9.46.87-14.2.36-5.88.76-11.75,1.13-17.44a1.2,1.2,0,0,1,1-1.11c2.19-.33,2.46,1.06,2.62,2.83.92,9.78,2,19.56,2.92,29.34a6.33,6.33,0,0,0,3.06,5.32,19.43,19.43,0,0,1,6,6c2.75,4.6.58,9-4.62,10.41l-1.41.38a1.22,1.22,0,0,0-.66,1.91l2.56,3.41a1.22,1.22,0,0,0,1.35.43c7.55-2.44,15-5.09,21.33-10a7.9,7.9,0,0,0,2.65-4.49c1.56-9,2.81-18,4.26-27.05a5.34,5.34,0,0,1,1.38-3c8-7.17,16-14.25,24.22-21.46a1.22,1.22,0,0,1,1.62,0Zm-47.1,76.3,8-8.72-11.71,4.66a1.22,1.22,0,0,0-.37,2l2.33,2.1A1.22,1.22,0,0,0,509.74,465.45Z"
+ 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.
-    		/>
+</p>
-    		<path class="cls-4" d="M619.84,279.65a28.5,28.5,0,1,1-28.48,28.47A28.42,28.42,0,0,1,619.84,279.65Zm-27.06,28.5a27.08,27.08,0,1,0,27.12-27.07A27.12,27.12,0,0,0,592.78,308.15Z"
+</div>
-    		/>
-    		<path class="cls-4" d="M617,308.25l-4,1.35c-4.78,1.6-9.56,3.18-14.33,4.8a1.5,1.5,0,0,1-2.06-.64,10.4,10.4,0,0,1,0-11,2,2,0,0,1,1.68-.71c6.08,1.88,12.13,3.87,18.19,5.83A4,4,0,0,1,617,308.25Z"
-    		/>
-    		<path class="cls-4" d="M621,311.19a7.79,7.79,0,0,0,.48.84q5.42,7.3,10.86,14.58c.51.67.63,1.15,0,1.86-2.83,3.21-6.28,4.49-10.46,3.42a1.93,1.93,0,0,1-1.2-1.35c-.08-6.34-.05-12.68-.05-19Z"
-    		/>
-    		<path class="cls-4" d="M623.07,308l15.74-5.15,1.11-.35c2.89-.93,3.15-.84,4.2,2a9.94,9.94,0,0,1-1,9.22,1.85,1.85,0,0,1-1.55.74c-6.11-2-12.19-4-18.27-6.09C623.23,308.32,623.19,308.17,623.07,308Z"
-    		/>
-    		<path class="cls-4" d="M633,288.93l-12.29,16.63c-.05-.93-.1-1.4-.1-1.86,0-5.79,0-11.58-.1-17.36,0-1.49.51-1.88,1.87-2C626.72,283.85,630.34,285,633,288.93Z"
-    		/>
-    		<path class="cls-4" d="M617.71,309.7c-4.08,5.68-7.9,11-11.77,16.29a1.69,1.69,0,0,1-1.5.39,10.25,10.25,0,0,1-6.57-9.19,1.71,1.71,0,0,1,.91-1.27C604.91,313.86,611.06,311.87,617.71,309.7Z"
-    		/>
-    		<path class="cls-4" d="M622.42,309.81l16,5.16.63.22c3.27,1,3.42,1.34,2.36,4.67a9.79,9.79,0,0,1-5.88,6.41A1.68,1.68,0,0,1,634,326c-3.9-5.28-7.74-10.62-11.58-15.94C622.38,310,622.4,310,622.42,309.81Z"
-    		/>
-    		<path class="cls-4" d="M606.84,327.59,618.91,311c.1.76.19,1.15.19,1.54,0,5.9,0,11.8.11,17.69a1.5,1.5,0,0,1-1.39,1.79A10.55,10.55,0,0,1,606.84,327.59Z"
-    		/>
-    		<path class="cls-4" d="M617.17,306.55l-13.26-4.23c-1.64-.52-3.27-1.06-4.91-1.55-.87-.25-1.3-.66-1.17-1.65.57-4.12,2.56-7.18,6.48-8.78a1.81,1.81,0,0,1,1.66.3c3.85,5.17,7.62,10.4,11.41,15.61Z"
-    		/>
-    		<path class="cls-4" d="M622.21,306.25l9.85-13.42.5-.67c1.89-2.59,2.15-2.63,4.81-.82a10.16,10.16,0,0,1,4.47,7.81,1.74,1.74,0,0,1-.91,1.39c-6.14,2-12.31,4-18.47,5.93Z"
-    		/>
-    		<path class="cls-4" d="M618.7,305.16l-11.85-16.27c1.83-3.62,7.5-5.7,11.27-4.13a1.29,1.29,0,0,1,.73.88c.09,6.39.12,12.79.16,19.18C619,304.88,618.87,305,618.7,305.16Z"
-    		/>
-    		<path class="cls-4" d="M242.34,456.65a34,34,0,1,1-34,34A33.9,33.9,0,0,1,242.34,456.65Zm-32.28,34a32.3,32.3,0,1,0,32.35-32.3A32.35,32.35,0,0,0,210.06,490.65Z"
-    		/>
-    		<path class="cls-4" d="M238.94,490.76l-4.83,1.61c-5.7,1.91-11.41,3.79-17.1,5.73a1.79,1.79,0,0,1-2.46-.77,12.41,12.41,0,0,1,0-13.16,2.36,2.36,0,0,1,2-.85c7.25,2.25,14.47,4.61,21.7,7A4.71,4.71,0,0,1,238.94,490.76Z"
-    		/>
-    		<path class="cls-4" d="M243.77,494.27a9.3,9.3,0,0,0,.57,1q6.46,8.71,13,17.4c.6.8.75,1.38,0,2.22-3.38,3.82-7.49,5.35-12.48,4.08a2.31,2.31,0,0,1-1.43-1.61c-.1-7.56-.06-15.13-.06-22.69Z"
-    		/>
-    		<path class="cls-4" d="M246.18,490.43,265,484.29l1.32-.42c3.45-1.11,3.76-1,5,2.38a11.86,11.86,0,0,1-1.17,11,2.21,2.21,0,0,1-1.85.88c-7.29-2.35-14.54-4.81-21.79-7.26C246.38,490.85,246.34,490.68,246.18,490.43Z"
-    		/>
-    		<path class="cls-4" d="M258,467.73l-14.66,19.84c-.06-1.11-.12-1.66-.12-2.22,0-6.9,0-13.81-.12-20.71,0-1.77.6-2.24,2.23-2.41C250.54,461.66,254.86,463.05,258,467.73Z"
-    		/>
-    		<path class="cls-4" d="M239.8,492.5c-4.87,6.77-9.42,13.13-14,19.44a2,2,0,0,1-1.79.47c-4.3-1.37-8-6.51-7.84-11a2,2,0,0,1,1.09-1.51C224.52,497.46,231.87,495.09,239.8,492.5Z"
-    		/>
-    		<path class="cls-4" d="M245.42,492.63l19.07,6.15.75.26c3.9,1.25,4.08,1.6,2.81,5.57a11.68,11.68,0,0,1-7,7.65,2,2,0,0,1-1.82-.31c-4.66-6.3-9.23-12.66-13.82-19C245.36,492.91,245.39,492.84,245.42,492.63Z"
-    		/>
-    		<path class="cls-4" d="M226.83,513.85l14.39-19.76c.12.9.23,1.37.23,1.84,0,7,0,14.07.13,21.11a1.79,1.79,0,0,1-1.66,2.14A12.59,12.59,0,0,1,226.83,513.85Z"
-    		/>
-    		<path class="cls-4" d="M239.14,488.74l-15.82-5c-2-.62-3.9-1.27-5.86-1.85-1-.3-1.55-.79-1.39-2,.68-4.91,3-8.57,7.73-10.47a2.16,2.16,0,0,1,2,.35c4.59,6.17,9.09,12.4,13.61,18.63Z"
-    		/>
-    		<path class="cls-4" d="M245.16,488.38l11.75-16,.6-.79c2.26-3.09,2.57-3.14,5.74-1a12.12,12.12,0,0,1,5.33,9.32,2.08,2.08,0,0,1-1.08,1.65c-7.32,2.42-14.68,4.73-22,7.07Z"
-    		/>
-    		<path class="cls-4" d="M241,487.08l-14.14-19.41c2.19-4.32,8.95-6.81,13.44-4.93a1.54,1.54,0,0,1,.87,1c.1,7.63.15,15.25.19,22.88C241.34,486.75,241.18,486.85,241,487.08Z"
-    		/>
-    		<path class="cls-4" d="M387.84,238.65a34.5,34.5,0,1,1-34.48,34.47A34.4,34.4,0,0,1,387.84,238.65Zm-32.76,34.5a32.78,32.78,0,1,0,32.83-32.77A32.82,32.82,0,0,0,355.08,273.15Z"
-    		/>
-    		<path class="cls-4" d="M384.39,273.27l-4.9,1.64c-5.78,1.93-11.57,3.85-17.35,5.81a1.81,1.81,0,0,1-2.5-.78,12.6,12.6,0,0,1,0-13.35,2.4,2.4,0,0,1,2-.86c7.36,2.28,14.68,4.68,22,7.06A4.78,4.78,0,0,1,384.39,273.27Z"
-    		/>
-    		<path class="cls-4" d="M389.29,276.83a9.43,9.43,0,0,0,.58,1q6.56,8.84,13.14,17.65c.61.82.76,1.4,0,2.25-3.42,3.88-7.6,5.43-12.66,4.14a2.34,2.34,0,0,1-1.45-1.64c-.1-7.67-.06-15.35-.06-23Z"
-    		/>
-    		<path class="cls-4" d="M391.74,272.93l19-6.23,1.34-.42c3.5-1.13,3.81-1,5.08,2.42A12,12,0,0,1,416,279.86a2.24,2.24,0,0,1-1.87.89c-7.39-2.38-14.75-4.88-22.11-7.37C391.94,273.35,391.9,273.18,391.74,272.93Z"
-    		/>
-    		<path class="cls-4" d="M403.71,249.89,388.84,270c-.06-1.12-.12-1.69-.12-2.25,0-7,0-14-.13-21,0-1.8.61-2.27,2.26-2.45C396.16,243.73,400.54,245.15,403.71,249.89Z"
-    		/>
-    		<path class="cls-4" d="M385.26,275c-4.94,6.87-9.56,13.33-14.25,19.72a2.05,2.05,0,0,1-1.82.48c-4.36-1.39-8.1-6.61-8-11.13a2.07,2.07,0,0,1,1.1-1.53C369.76,280.06,377.21,277.65,385.26,275Z"
-    		/>
-    		<path class="cls-4" d="M391,275.16l19.35,6.24.76.26c4,1.27,4.14,1.62,2.86,5.65a11.86,11.86,0,0,1-7.12,7.77,2,2,0,0,1-1.85-.32c-4.72-6.39-9.37-12.85-14-19.3C390.91,275.44,390.94,275.37,391,275.16Z"
-    		/>
-    		<path class="cls-4" d="M372.1,296.69l14.61-20c.12.92.23,1.39.23,1.87,0,7.14,0,14.28.14,21.42a1.82,1.82,0,0,1-1.69,2.17A12.77,12.77,0,0,1,372.1,296.69Z"
-    		/>
-    		<path class="cls-4" d="M384.6,271.21l-16.05-5.12c-2-.63-4-1.29-5.95-1.87-1-.31-1.58-.8-1.41-2,.69-5,3.09-8.7,7.84-10.63a2.2,2.2,0,0,1,2,.36c4.66,6.26,9.22,12.59,13.81,18.9Z"
-    		/>
-    		<path class="cls-4" d="M390.7,270.85l11.93-16.24.61-.81c2.29-3.14,2.61-3.19,5.83-1a12.3,12.3,0,0,1,5.41,9.46,2.11,2.11,0,0,1-1.1,1.68c-7.43,2.45-14.9,4.8-22.36,7.17Z"
-    		/>
-    		<path class="cls-4" d="M386.45,269.53l-14.34-19.7c2.22-4.39,9.08-6.91,13.64-5a1.57,1.57,0,0,1,.88,1.06c.11,7.74.15,15.48.2,23.22C386.82,269.19,386.66,269.29,386.45,269.53Z"
-    		/>
-    		<path class="cls-4" d="M607.85,688.65a24.5,24.5,0,1,1-24.48,24.48A24.43,24.43,0,0,1,607.85,688.65Zm-23.26,24.5a23.28,23.28,0,1,0,23.31-23.27A23.31,23.31,0,0,0,584.58,713.15Z"
-    		/>
-    		<path class="cls-4" d="M605.39,713.23l-3.48,1.16c-4.11,1.37-8.22,2.73-12.32,4.13a1.29,1.29,0,0,1-1.77-.55,8.94,8.94,0,0,1,0-9.48,1.7,1.7,0,0,1,1.45-.61c5.23,1.62,10.43,3.32,15.63,5A3.39,3.39,0,0,1,605.39,713.23Z"
-    		/>
-    		<path class="cls-4" d="M608.87,715.76a6.7,6.7,0,0,0,.41.72q4.66,6.28,9.33,12.54c.44.58.54,1,0,1.6a8.47,8.47,0,0,1-9,2.94,1.66,1.66,0,0,1-1-1.16c-.07-5.45,0-10.9,0-16.35Z"
-    		/>
-    		<path class="cls-4" d="M610.62,713l13.53-4.42,1-.3c2.49-.8,2.71-.72,3.61,1.72a8.54,8.54,0,0,1-.84,7.93,1.59,1.59,0,0,1-1.33.63c-5.25-1.69-10.48-3.47-15.7-5.23C610.76,713.3,610.73,713.17,610.62,713Z"
-    		/>
-    		<path class="cls-4" d="M619.12,696.63l-10.56,14.3c0-.8-.09-1.2-.09-1.6,0-5,0-10-.09-14.92,0-1.28.43-1.61,1.61-1.74C613.75,692.26,616.87,693.27,619.12,696.63Z"
-    		/>
-    		<path class="cls-4" d="M606,714.48c-3.51,4.88-6.79,9.46-10.12,14a1.46,1.46,0,0,1-1.29.34,8.81,8.81,0,0,1-5.65-7.9,1.47,1.47,0,0,1,.78-1.09C595,718.06,600.3,716.35,606,714.48Z"
-    		/>
-    		<path class="cls-4" d="M610.06,714.58,623.8,719l.54.19c2.81.9,2.94,1.15,2,4a8.42,8.42,0,0,1-5.06,5.51,1.44,1.44,0,0,1-1.31-.23c-3.36-4.54-6.65-9.13-10-13.7C610,714.78,610,714.73,610.06,714.58Z"
-    		/>
-    		<path class="cls-4" d="M596.67,729.87,607,715.63c.08.65.16,1,.17,1.33,0,5.07,0,10.14.1,15.21a1.29,1.29,0,0,1-1.2,1.54A9.07,9.07,0,0,1,596.67,729.87Z"
-    		/>
-    		<path class="cls-4" d="M605.54,711.78l-11.4-3.63c-1.41-.45-2.81-.92-4.22-1.33-.74-.22-1.12-.57-1-1.42.49-3.54,2.2-6.18,5.57-7.55a1.56,1.56,0,0,1,1.42.25c3.31,4.44,6.55,8.94,9.8,13.42Z"
-    		/>
-    		<path class="cls-4" d="M609.88,711.52,618.35,700l.43-.57c1.63-2.23,1.85-2.26,4.14-.7a8.73,8.73,0,0,1,3.84,6.72,1.5,1.5,0,0,1-.78,1.19c-5.28,1.74-10.58,3.41-15.88,5.09Z"
-    		/>
-    		<path class="cls-4" d="M606.86,710.58l-10.19-14c1.58-3.12,6.45-4.9,9.69-3.55a1.11,1.11,0,0,1,.62.75c.08,5.5.11,11,.14,16.49C607.12,710.34,607,710.41,606.86,710.58Z"
-    		/>
-    		<path class="cls-4" d="M720.34,482.65a27,27,0,1,1-27,27A26.92,26.92,0,0,1,720.34,482.65Zm-25.64,27A25.65,25.65,0,1,0,720.4,484,25.69,25.69,0,0,0,694.71,509.65Z"
-    		/>
-    		<path class="cls-4" d="M717.64,509.74,713.81,511c-4.53,1.51-9.06,3-13.58,4.55a1.42,1.42,0,0,1-2-.61,9.86,9.86,0,0,1,0-10.45,1.88,1.88,0,0,1,1.6-.67c5.76,1.78,11.49,3.66,17.23,5.52A3.74,3.74,0,0,1,717.64,509.74Z"
-    		/>
-    		<path class="cls-4" d="M721.48,512.53a7.38,7.38,0,0,0,.45.8q5.13,6.92,10.29,13.81c.48.64.59,1.09,0,1.76-2.68,3-6,4.25-9.91,3.24a1.83,1.83,0,0,1-1.13-1.28c-.08-6-.05-12-.05-18Z"
-    		/>
-    		<path class="cls-4" d="M723.4,509.48l14.91-4.88,1-.33c2.74-.88,3-.79,4,1.89a9.42,9.42,0,0,1-.93,8.74,1.75,1.75,0,0,1-1.47.7c-5.79-1.87-11.55-3.82-17.31-5.77C723.56,509.81,723.52,509.67,723.4,509.48Z"
-    		/>
-    		<path class="cls-4" d="M732.77,491.45,721.12,507.2c0-.88-.09-1.32-.1-1.76,0-5.48,0-11-.1-16.45,0-1.41.48-1.78,1.77-1.92C726.86,486.63,730.29,487.74,732.77,491.45Z"
-    		/>
-    		<path class="cls-4" d="M718.33,511.12c-3.87,5.38-7.48,10.43-11.15,15.43a1.61,1.61,0,0,1-1.42.37,9.71,9.71,0,0,1-6.23-8.71,1.62,1.62,0,0,1,.86-1.2C706.19,515.06,712,513.17,718.33,511.12Z"
-    		/>
-    		<path class="cls-4" d="M722.79,511.23l15.14,4.88.6.21c3.1,1,3.24,1.27,2.23,4.43a9.28,9.28,0,0,1-5.57,6.08,1.59,1.59,0,0,1-1.45-.25c-3.7-5-7.33-10.06-11-15.1C722.74,511.44,722.77,511.39,722.79,511.23Z"
-    		/>
-    		<path class="cls-4" d="M708,528.07l11.43-15.69c.09.72.18,1.09.18,1.46,0,5.59,0,11.18.11,16.76a1.42,1.42,0,0,1-1.32,1.7A10,10,0,0,1,708,528.07Z"
-    		/>
-    		<path class="cls-4" d="M717.81,508.13l-12.56-4c-1.55-.49-3.09-1-4.66-1.47-.82-.24-1.23-.62-1.11-1.56.54-3.9,2.42-6.81,6.14-8.32a1.72,1.72,0,0,1,1.57.28c3.65,4.9,7.22,9.85,10.81,14.79Z"
-    		/>
-    		<path class="cls-4" d="M722.58,507.85l9.33-12.71.48-.63c1.79-2.46,2-2.5,4.56-.77a9.62,9.62,0,0,1,4.23,7.4,1.65,1.65,0,0,1-.86,1.31c-5.82,1.92-11.66,3.76-17.5,5.61Z"
-    		/>
-    		<path class="cls-4" d="M719.26,506.81,708,491.4c1.74-3.43,7.11-5.4,10.68-3.92a1.23,1.23,0,0,1,.69.83c.08,6.06.12,12.11.15,18.17C719.55,506.56,719.42,506.63,719.26,506.81Z"
-    		/>
-    		<path class="cls-4" d="M320.85,650.65a24.5,24.5,0,1,1-24.48,24.48A24.43,24.43,0,0,1,320.85,650.65Zm-23.26,24.5a23.28,23.28,0,1,0,23.31-23.27A23.31,23.31,0,0,0,297.58,675.15Z"
-    		/>
-    		<path class="cls-4" d="M318.39,675.23l-3.48,1.16c-4.11,1.37-8.22,2.73-12.32,4.13a1.29,1.29,0,0,1-1.77-.55,8.94,8.94,0,0,1,0-9.48,1.7,1.7,0,0,1,1.45-.61c5.23,1.62,10.43,3.32,15.63,5A3.39,3.39,0,0,1,318.39,675.23Z"
-    		/>
-    		<path class="cls-4" d="M321.87,677.76a6.7,6.7,0,0,0,.41.72q4.66,6.28,9.33,12.54c.44.58.54,1,0,1.6a8.47,8.47,0,0,1-9,2.94,1.66,1.66,0,0,1-1-1.16c-.07-5.45,0-10.9,0-16.35Z"
-    		/>
-    		<path class="cls-4" d="M323.62,675l13.53-4.42,1-.3c2.49-.8,2.71-.72,3.61,1.72a8.54,8.54,0,0,1-.84,7.93,1.59,1.59,0,0,1-1.33.63c-5.25-1.69-10.48-3.47-15.7-5.23C323.76,675.3,323.73,675.17,323.62,675Z"
-    		/>
-    		<path class="cls-4" d="M332.12,658.63l-10.56,14.3c0-.8-.09-1.2-.09-1.6,0-5,0-10-.09-14.92,0-1.28.43-1.61,1.61-1.74C326.75,654.26,329.87,655.27,332.12,658.63Z"
-    		/>
-    		<path class="cls-4" d="M319,676.48c-3.51,4.88-6.79,9.46-10.12,14a1.46,1.46,0,0,1-1.29.34,8.81,8.81,0,0,1-5.65-7.9,1.47,1.47,0,0,1,.78-1.09C308,680.06,313.3,678.35,319,676.48Z"
-    		/>
-    		<path class="cls-4" d="M323.06,676.58,336.8,681l.54.19c2.81.9,2.94,1.15,2,4a8.42,8.42,0,0,1-5.06,5.51,1.44,1.44,0,0,1-1.31-.23c-3.36-4.54-6.65-9.13-10-13.7C323,676.78,323,676.73,323.06,676.58Z"
-    		/>
-    		<path class="cls-4" d="M309.67,691.87,320,677.63c.08.65.16,1,.17,1.33,0,5.07,0,10.14.1,15.21a1.29,1.29,0,0,1-1.2,1.54A9.07,9.07,0,0,1,309.67,691.87Z"
-    		/>
-    		<path class="cls-4" d="M318.54,673.78l-11.4-3.63c-1.41-.45-2.81-.92-4.22-1.33-.74-.22-1.12-.57-1-1.42.49-3.54,2.2-6.18,5.57-7.55a1.56,1.56,0,0,1,1.42.25c3.31,4.44,6.55,8.94,9.8,13.42Z"
-    		/>
-    		<path class="cls-4" d="M322.88,673.52,331.35,662l.43-.57c1.63-2.23,1.85-2.26,4.14-.7a8.73,8.73,0,0,1,3.84,6.72,1.5,1.5,0,0,1-.78,1.19c-5.28,1.74-10.58,3.41-15.88,5.09Z"
-    		/>
-    		<path class="cls-4" d="M319.86,672.58l-10.19-14c1.58-3.12,6.45-4.9,9.69-3.55a1.11,1.11,0,0,1,.62.75c.08,5.5.11,11,.14,16.49C320.12,672.34,320,672.41,319.86,672.58Z"
-    		/>
-    	</g>
-    	</a>
-    	<a href="#ProteinImport">
-    	<g id="svgProteinImport">
-    		<path class="cls-4" d="M496.49,903.39a15.16,15.16,0,1,0,25-11.55V850.75A15.15,15.15,0,1,0,498.1,832.4a10.22,10.22,0,0,0-1.61,5.5v65c0,.08,0,.16,0,.25S496.49,903.3,496.49,903.39Z"
-    		/>
-    		<path class="cls-4" d="M485.79,839.19a15.16,15.16,0,1,0-25,11.55v41.09a15.15,15.15,0,1,0,23.36,18.34,10.22,10.22,0,0,0,1.61-5.5v-65c0-.08,0-.16,0-.25S485.79,839.28,485.79,839.19Z"
-    		/>
-    		<polygon class="cls-5" points="490.94 945.23 497.86 957.21 484.03 957.21 490.94 945.23"
-    		/>
-    		<polygon class="cls-5" points="490.94 811.5 497.86 823.47 484.03 823.47 490.94 811.5"
-    		/>
-    		<polygon class="cls-5" points="475.15 782.5 486.41 790.52 473.83 796.26 475.15 782.5"
-    		/>
-    		<polygon class="cls-5" points="517.85 760.46 517.19 774.27 505.56 766.79 517.85 760.46"
-    		/>
-    		<polygon class="cls-5" points="500.75 972.59 511.74 980.99 498.97 986.3 500.75 972.59"
-    		/>
-    		<polygon class="cls-5" points="473.84 986.28 475.55 1000 462.81 994.62 473.84 986.28"
-    		/>
-    		<path class="cls-4" d="M486.37,948.8l4.58-7.93,4.63,8a17.83,17.83,0,1,0-9.21-.09Z"
-    		/>
-    		<path class="cls-6" d="M484.88,105.11a15.16,15.16,0,1,0-24.94,11.61L460,157.8a15.15,15.15,0,1,0,23.4,18.29,10.22,10.22,0,0,0,1.6-5.51l-.14-65c0-.08,0-.16,0-.25S484.88,105.19,484.88,105.11Z"
-    		/>
-    		<path class="cls-6" d="M495.71,169.27a15.16,15.16,0,1,0,24.94-11.61l-.09-41.09a15.15,15.15,0,1,0-23.4-18.29,10.22,10.22,0,0,0-1.6,5.51l.14,65c0,.08,0,.16,0,.25S495.71,169.19,495.71,169.27Z"
-    		/>
-    		<polygon class="cls-5" points="490.29 43.72 497.23 55.68 483.4 55.71 490.29 43.72"
-    		/>
-    		<polygon class="cls-5" points="480.04 0 490.53 9.02 477.48 13.59 480.04 0"
-    		/>
-    		<polygon class="cls-5" points="508.77 17.23 509.42 31.05 497.13 24.7 508.77 17.23"
-    		/>
-    		<polygon class="cls-5" points="490.58 177.84 497.52 189.81 483.69 189.84 490.58 177.84"
-    		/>
-    		<polygon class="cls-5" points="475.01 202.13 475.04 215.96 463.05 209.07 475.01 202.13"
-    		/>
-    		<polygon class="cls-5" points="492.07 213.51 499.56 225.14 485.75 225.81 492.07 213.51"
-    		/>
-    		<polygon class="cls-5" points="506.33 199.33 517.61 207.34 505.04 213.1 506.33 199.33"
-    		/>
-    		<polygon class="cls-5" points="522.04 223.67 533.32 231.67 520.75 237.44 522.04 223.67"
-    		/>
-    		<path class="cls-4" d="M494.44,60.14,497.25,65l-13.83,0,2.85-5a17.83,17.83,0,1,0,8.16.09Z"
-    		/>
-    	</g>
-    	</a>
-    	<a href="#MembraneIntegration">
-    	<g id="svgMembraneIntegration" data-name="Membrane Integration">
-    		<rect class="cls-4" x="134.65" y="617.64" width="25.23" height="71.72"
-    		rx="12.62" ry="12.62" transform="translate(679.83 247.14) rotate(65.35)"
-    		/>
-    		<path class="cls-7" d="M210,627c-7,9-18,10-28,10q-7.5,1.5-12,9" />
-    		<circle class="cls-4" cx="212.42" cy="627.79" r="12.28" transform="translate(-242.44 145.77) rotate(-24.65)"
-    		/>
-    		<rect class="cls-4" x="821.82" y="618.83" width="25.23" height="71.72"
-    		rx="12.62" ry="12.62" transform="translate(1777.45 169.27) rotate(114.65)"
-    		/>
-    		<path class="cls-7" d="M772,628c11-2,18,7,25,15,4,4,10,4,15,5" />
-    		<circle class="cls-4" cx="769.29" cy="628.98" r="12.28" transform="translate(-123.19 1065.91) rotate(-65.35)"
-    		/>
-    		<rect class="cls-4" x="728.57" y="198.63" width="25.23" height="71.72"
-    		rx="12.62" ry="12.62" transform="translate(352.82 -438.94) rotate(42.47)"
-    		/>
-    		<path class="cls-7" d="M697,286q4.5-12,18-18c6-2,12-7,13-15" />
-    		<circle class="cls-4" cx="696.76" cy="288.65" r="12.28" transform="translate(13.38 607.67) rotate(-47.53)"
-    		/>
-    		<rect class="cls-4" x="223.9" y="198.7" width="25.23" height="71.72"
-    		rx="12.62" ry="12.62" transform="translate(569.35 247.88) rotate(137.53)"
-    		/>
-    		<path class="cls-7" d="M281,286c-7-3-12-8-14-14-3-8-9-15-17-18" />
-    		<circle class="cls-4" cx="280.93" cy="288.72" r="12.28" transform="translate(-121.23 265.46) rotate(-42.47)"
-    		/>
-    		<rect class="cls-4" x="250.84" y="173.54" width="25.23" height="71.72"
-    		rx="12.62" ry="12.62" transform="translate(599.73 195.97) rotate(139.23)"
-    		/>
-    		<path class="cls-7" d="M218,159c4,3,10,5,12,10,3,10,5,21,16,25" />
-    		<circle class="cls-4" cx="214.91" cy="158.92" r="12.28" transform="translate(-51.63 178.89) rotate(-40.77)"
-    		/>
-    		<rect class="cls-4" x="704.79" y="173.38" width="25.23" height="71.72"
-    		rx="12.62" ry="12.62" transform="translate(310.69 -417.68) rotate(40.77)"
-    		/>
-    		<path class="cls-7" d="M763,159c-2,11-12,15-21,22-4,3-7,6-7,13" />
-    		<circle class="cls-4" cx="765.95" cy="158.75" r="12.28" transform="translate(145.57 635.21) rotate(-49.23)"
-    		/>
-    	</g>
-    	</a>
-    	<a href="#Secretion">
-    	<g id="svgPeroxicretion">
-    		<line class="cls-8" x1="93.32" y1="902.37" x2="134.75" y2="859.09" />
-    		<line class="cls-9" x1="193.97" y1="850.99" x2="235.4" y2="807.71" />
-    		<rect class="cls-4" x="249" y="753.88" width="15.71" height="62.83" rx="4"
-    		ry="4" transform="translate(-100.62 1530.19) rotate(-136.25)" />
-    		<rect class="cls-4" x="188.3" y="817" width="15.71" height="62.83" rx="4"
-    		ry="4" transform="translate(-248.82 1596.94) rotate(-136.25)" />
-    		<rect class="cls-4" x="145.42" y="808.34" width="15.71" height="62.83"
-    		rx="4" ry="4" transform="translate(-316.69 1552.37) rotate(-136.25)"
-    		/>
-    		<line class="cls-9" x1="800.88" y1="850.99" x2="759.45" y2="807.71" />
-    		<rect class="cls-4" x="730.14" y="753.88" width="15.71" height="62.83"
-    		rx="4" ry="4" transform="translate(-338.14 728.33) rotate(-43.75)" />
-    		<rect class="cls-4" x="790.84" y="817" width="15.71" height="62.83" rx="4"
-    		ry="4" transform="translate(-364.94 787.82) rotate(-43.75)" />
-    		<line class="cls-10" x1="906.68" y1="905.93" x2="865.25" y2="862.66"
-    		/>
-    		<rect class="cls-4" x="838.88" y="811.91" width="15.71" height="62.83"
-    		rx="4" ry="4" transform="translate(-348.08 819.62) rotate(-43.75)" />
-    	</g>
-    	</a>
-    </svg>
 </div>
+<button class="accordion">
+  <h2 id="Pex7Import">Pex7 Import</h2>
+  <p>Imagine you need different protein concentrations in your artificial compartment. What to do? Take our modified PTS2 sequences with varying import efficiencies.</p>
+  </button>
+<div class="panel">
+<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.
+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>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>
+<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>
+<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>
+<img src="https://static.igem.org/mediawiki/2017/9/9d/T--cologne-duesseldorf--Platte_Sternchen_3.jpg  ">
+<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>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
+<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.
+">(DeLoache)</abbr></a>
+ 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>
+<img src="https://static.igem.org/mediawiki/2017/1/17/T--cologne-duesseldorf--PDV_test1.jpg">
+<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.
+  </figcaption>
+<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>
+<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>
+</div>
-<h2>Introduction</h2>
-    <h3>Real world application</h3>
-    <p>As a proof of concept for our compartmentation strategy we intend to establish the Nootkatone pathway inside the peroxisome. Nootkatone is a natural compound found inside the peel of grapefruit, which gives it its characteristic taste and smell. In addition, Nootkatone is a natural repellent for mosquitoes and ticks that is already being commercially used and industrially manufactured. Unfortunately, the production costs are extremely high, because it has to either be extracted from the peel of millions of grapefruit or synthesized inside of yeast. The difficulties lie in the toxicity of the Nootkatone pathway towards yeast and the resulting low efficiency. Here our compartmentation comes into play: we plan to translocate the whole pathway into the modified peroxisome to prove that we have transformed the peroxisome into an independent compartment with all the features we require.</p>
 <button class="accordion">
-<h2 id="ProteinImport">Protein Import</h2>
+  <h2 id="MembraneIntegration">Membrane Integration</h2>
-<p>The peroxisome possesses two pathways for importing proteins with the main transport proteins being PEX5 and PEX7. We created an orthogonal PEX5 binding pocket and corresponding recognition peptide (PTS1) by structural modeling. We also created a library of PEX7 recognition sequences for import of proteins incompatible with the PTS1 peptide.</p>
+  <p>Not only can we completely control the import into our compartment we also managed to integrate two marker proteins, namely Pex3 from yeast and PEX26 from human cells into our membrane. Furthermore did we manage to integrate the proton pump bacteriorhodopsin that containing seven membrane domains and was a much bigger challenge.</p>
-</button>
+  </button>
 <div class="panel">
-<h3>PTS1 Import</h3>
-   <p>
+<h4>Figure 1: <span style="color: rgb(0, 0, 0);"><a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> expression and integration</span></h4>
-The vast majority of peroxisomal matrix proteins is imported by the PEX5 importer. PEX5 recognizes the C-terminal PTS1 peptide whose evolutionary conserved sequence is (S/A/C)-(K/R/H)-(L/M) (<a> Gould <i>et al.</i>, 1989 </a>).  PEX5 is a 612 amino acid protein which contains seven tetratrico peptide repeats (TPR). The TPR is a 34 amino acid motif which forms a structure of alpha-helices separated by one turn. A whole TPR domain consists of three of those structures (<a href="https://www.nature.com/nsmb/journal/v7/n12/full/nsb1200_1091.html"><abbr title="Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.">Gatto Jr. et al 2000</abbr></a>).
+<div class="flex-row-3">
-TPR domains are often involved in protein&minus;protein interactions. As can be seen in the following figure, the TPR regions mediate the binding of the peroxisomal targeting signal.
+<div><img src="https://static.igem.org/mediawiki/2017/a/a9/PMP_RESULT_PEX26-mRuby-sfGFP-PTS1_red_channel.png"></div>
-</p>
+<div><img src="https://static.igem.org/mediawiki/2017/f/f3/PEX26-mRuby-sfGFP-PTS1_merged.png"></div>
+<div><img src="https://static.igem.org/mediawiki/2017/f/f5/PMP_RESULTS_PEX26-mRuby-sfGFP-PTS1_green_channel.png"></div>
+</div>
+<figcaption><b>Fluorescent microscopy of bacteriorhodopsin coexpressed with sf-GFP:</b>.<br>
+<span style="color: rgb(0, 0, 0);">Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). The green fluorescent spots (on the right) shows a typical peroxisomal shape. The signal for membrane marker mRuby-PEX26 is shown in red (on the left). Both signals show colocalization when merged (middle), which indicates that the protein gets integrated into the membrane.</span></figcaption>
+<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>
+<h4>Figure 1: <span style="color: rgb(0, 0, 0);">Bacteriorhodopsin expression and integration</span></h4>
 <figure>
-<img src="https://static.igem.org/mediawiki/2017/b/b3/Artico_tpr.png">
+<div class="flex-row-3">
-<figcaption>
+<div><img src="https://static.igem.org/mediawiki/2017/5/55/PMP_BACR-mRuby_and_gfp-pts1_red_Channel.png"></div>
-<strong>Figure 1.1: </strong>TPR domain of the human PEX5, with a pentapeptide in its binding pocket (<a href="https://www.nature.com/nsmb/journal/v7/n12/full/nsb1200_1091.html"><abbr title="Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.">Gatto Jr. <i> et al. </i>, 2000</abbr></a>)
+<div><img src="https://static.igem.org/mediawiki/2017/1/1a/PMP_RESULTS_bacr-mRuby_and_GFP-PTS1_merged.png"></div>
-</figcaption>
+<div><img src="https://static.igem.org/mediawiki/2017/2/23/PMP_BACR-mRuby_and_GFP-PTS1_green_Channel.png"></div>
+</div>
+<figcaption><b>Fluorescent microscopy of bacteriorhodopsin coexpressed with sf-GFP:</b>.<br>
+<span style="color: rgb(0, 0, 0);">Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). The green fluorescent spots (on the right) shows a typical peroxisomal shape. The signal for the membrane marked bacteriorhodopsin is shown in red (on the left). Both signals show colocalization when merged (middle), which indicates that the protein gets integrated into the membrane.</span></figcaption>
 </figure>
 <p>
-The following figure depicts the import mechanism of PTS1 tagged proteins via PEX5.
+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>
-<figure>
+<h4>Outlook</h4>
-   <img src="https://static.igem.org/mediawiki/2017/e/e7/Artico_p5shuttle.jpeg">
-   <figcaption><strong>Figure 1.2: </strong>Pex5 import mechanism (<a href="https://www.nature.com/articles/nrm1710"><abbr title="Peroxisomal matrix protein import: the transient pore model">Erdmann et al., 2005</abbr></a>)
-<br>
-Upon recognition of the PTS1 in the cytosol, PEX5 binds to its cargo (i). It docks to the peroxisomal membrane complex, consisting of PEX13, PEX14 and PEX17 (ii). This docking complex is connected to the RING-finger complex, consisting of PEX2, PEX10 and PEX12, via PEX8. This multi-protein complex is known as the importomer. PEX5 and PEX14 form a pore in the membrane, through which the cargo is translocated (iii). Due to competitive binding of PEX8's PTS1 motif, the receptor–cargo complex dissociates at the matrix site of the membrane (iv). The integral PTS1-receptor is either monoubiquitinated by the E2-enzyme PEX4 or polyubiquitinated by Ubc4 or Ubc5. The AAA peroxins PEX1 and PEX6, which are anchored to the peroxisomal membrane by PEX15, dislocate the ubiquitinated PEX5 from the membrane back to the cytosol (v). The polyubiquitinated PTS1-receptors are degraded by the proteasome, whereas the monoubiquitinated receptors are recycled for further rounds of import.
-</figcaption>
-</figure>
-<p>
+<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.
-In this subproject we mutated the PEX5 receptor in a way that enables it to recognize a new signal peptide which does not occur in nature. As PEX5 is responsible for the import of most proteins , we have complete control over the peroxisomal content once we knock out the wild type receptor and replace it with our newly mutated one.
-Corresponding to the new receptor, a peroxisomal targeting signal that provides favorable interactions with the residues of the amino acids within the TPR needs to be designed.
-<br>
-Our first approach for the mutation deals with the introduction of site-directed mutagenesis in the TPR of PEX5 followed by computational simulation of the binding affinity between our new designed PEX5 receptor and several peptide variants via <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655909/">Molecular Dynamics</a>. In the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model">model section </a>we explain the molecular dynamics approach in more detail.
-<br>
-Our second approach relies on recently published literature. We designed a receptor similar to what <a href="https://www.nature.com/articles/s41467-017-00487-7">Baker <i>et al.</i> </a> did in the moss <i>Physcomitrella patens</i> in 2017. To understand how and where we set the mutations in the PEX5 receptor following this approach, please proceed with the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design">design section</a>.
 </p>
-<h3>PTS2 Import</h3>
+</div>
-<p>The peroxisomal import depends on two pathways. A vast majority of the proteins normally found in the peroxisome are imported via the <a href="#PTS1">Pex5 importer</a>.
-In <i>S. cerevisiae</i> only one protein, the 3-Oxoacyl-CoA thiolase <a href="http://onlinelibrary.wiley.com/doi/10.1002/yea.320100708/full"><abbr title="Erdmann, R. (1994). The peroxisomal targeting signal of 3‐oxoacyl‐CoA thiolase from Saccharomyces cerevisiae. Yeast, 10(7), 935-944."> (Erdmann)</abbr></a>, localized in the peroxisome, is imported by the receptor Pex7 and some coreceptors instead <a href="https://www.ncbi.nlm.nih.gov/pubmed/17445803"><abbr title="Platta, H. W., & Erdmann, R. (2007). The peroxisomal protein import machinery. FEBS letters, 581(15), 2811-2819.
-"> (Erdmann)</abbr></a>.</p>
-<p>The targeting signal for this pathway is localized near the N-terminus of each protein. Kunze and colleagues described the PTS2 consensus sequence (see figure 2.1)</p>
-<figure>
-    <img src="https://static.igem.org/mediawiki/2017/c/c3/T--cologne-duesseldorf--PTS_richtig.png  ">
-    <figcaption>Figure 2.1: The peroxisomal targeting signal type two consists of nine amino acids. Residue one contains Arginine or Lysine, residue two Leucine, Valine or Isoleucine. The amino acids three to seven are highly variable. Residue number eight consists of Histidine or Glutamine and the ninth is either Leucine or Alanine.
-<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247985/ ">
-<abbr title="2011, Kunze, M., Neuberger, G., Maurer-Stroh, S., Ma, J., Eck, T., Braverman, N., Schmid, J., Eisenhaber, F. & Berger, J. - Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7."> (Kunze)</abbr></a>
-</figcaption>
-</figure>
-<p>The five amino acids in the center are not conserved and highly variable. In yeast, among other organisms, the protein Pex7 works as a soluble chaperone, which recognizes PTS2 and directs the protein to the import pore at the peroxisomal membrane
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/17445803 "> <abbr title="2007, Platta, Harald W., and Ralf Erdmann - The peroxisomal protein import machinery">(Erdmann)</abbr></a>.</p>
-<p>Towards the aim of implementing a valuable import device for our toolbox we created a library of different PTS2 versions showing variable import efficiencies. Subsequently, one can ensure customizable concentrations of different pathway parts in the peroxisome. Moreover, proteins which require an unmodified C-terminus can be imported via PTS2 since this sequence is located on the N-terminus of the protein (<a href="#PTS1">PTS1 import</a>).</p>
-<p>Kunze <i>et al.</i> performed a mutational analysis for the PTS2 containing human thiolase, specifically for the five variable residues in the core region. The wild type sequence of those residues was defined as glutamine, valine, valine, leucine and glycine. These amino acids were substituted by specific amino acids to be able to evaluate the effect of distinct types in the above stated positions within the sequence. The selected amino acids represent different groups to investigate the biochemical effects of different side chains or other factors: aspartate as a negatively charged, tryptophan as an aromatic, arginine as a basic, leucine as a bulky and lysine as a positively charged amino acid. The thiolase import was subsequently measured with immunofluorescence microscopy. The recognition and import of the PTS2 harboring protein of interest by Pex7 worked out with aspartate at position X1, but not on X2 or X3. Lysine on residue X3 lead to a strong decrease of import activity. Kunze et al. concluded that the import of a given protein relies highly on the amino acid groups in the core region of the PTS2 <a href="https://www.ncbi.nlm.nih.gov/pubmed/22057399 ">       <abbr title="2011, Kunze, M., Neuberger, G., Maurer-Stroh, S., Ma, J., Eck, T., Braverman, N., Schmid, J., Eisenhaber, F. & Berger, J. - Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7.">(Kunze) </abbr></a>.</p>
-<p>Besides a biased approach, which relies on substitution of single residues in the amino acid sequence of the PTS2, in a second approach we aim to randomly change the sequence to characterize a huge library of different sequence compositions.</p>
-</div>
 <button class="accordion">
-<h2  id="MembraneIntegration">Membrane Integration</h2>
+  <h2 id="SizeAndNumber">Size and Number</h2>
-<p>To optimize the luminal conditions of our compartment we focused on integrating new proteins to its membrane. This way we can alter specific properties or supply reactions inside with necessary co-factors. To test the import and integration mechanism, we fused our designed membrane anchors to fluorescent marker proteins and finally integrated the protein pump bacteriorhodopsin into the membrane to acidify our compartment</p>
+  <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>
-</button>
+  </button>
 <div class="panel">
-<h3>Introduction</h3>
+  <h3>Heading</h3>
+    <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 membrane 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>
+  </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>
+<figure>
+        <img src="https://static.igem.org/mediawiki/2017/a/a4/T--Cologne-Duesseldorf--Pex15_mVenus.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>
-<p>Many reactions rely on optimal conditions like pH and co-factors. Thus, this subproject aims at the optimization of those circumstances through the integration of new membrane proteins, which alter specific properties of the peroxisomal lumen. Such an approach promises to be very useful for metabolic engineering projects as it can help to adjust the pH, provide cofactors to enzymes or increase/decrease the concentrations of metabolites inside to peroxisome. In nature two distinct mechanisms exist, which are used for the integration of membrane proteins into the peroxisomal membrane – a Pex19-<a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> dependent and an ER-dependent one  <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255988"> <abbr title="I.A. Sparkes, C. Hawes, A. Baker, AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes, Plant Physiol. 139 (2005) 690–700"> (2005, Sparkes <i>et al.</i>) </abbr> </a>.</p>
-<img src="https://static.igem.org/mediawiki/2017/8/8c/PMP_pH_dependent_enzymes.png">
+</figure>
+<br>
+<br>
+<figure>
-<p>They rely on a so called mPTS sequence, that is used to mark the proteins for transport to and integration in the peroxisomal membrane  <a href="https://www.ncbi.nlm.nih.gov/pubmed/12839494"> <abbr title="H.F. Tabak, J.L. Murk, I. Braakman, H.J. Geuze, Peroxisomes start their life in the endoplasmic reticulum, Traffic 4 (2003) 512–518"> (2003, H.F. Tabak <i>et al.</i>)</abbr> </a>. We will try to utilize the capability of both mechanisms to incorporate new proteins into the peroxisomal membrane.
-However, to test whether yeast can integrate and use the foreign proteins in its peroxisomal membrane, we will design three different constructs, which will hopefully give us insights into the mechanisms and its efficiency to incorporate new proteins into the peroxisomal membrane.</p>
+        <img src="https://static.igem.org/mediawiki/2017/3/3d/T--Cologne-Duesseldorf--Pex26_overlay.png">
-<img src="https://static.igem.org/mediawiki/2017/7/7b/PMP_Import_ways.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>
-<p>As a proof of concept, we will incorporate three proteins through three different approaches into the peroxisomal membrane: (i) mRuby2-<a href="http://www.uniprot.org/uniprot/Q7Z412"><a href="http://www.uniprot.org/uniprot/Q7Z412" style="color:#DB8321">PEX26</a></a> as a proof for the Pex19-dependent mechanism, (ii)  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>-mRuby2 itself to showcase the ER-dependent mechanism and (iii) <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a>, a unidirectional proton pump, fused to the N-terminal anchor of  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>. </p>
+</figure>
-<h4>Pex19-dependent Mechanism</h4>
+<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>
-<p>The exact mechanisms of mPTS binding,  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>/Pex19 disassembly, mPTS-PMP binding, and release from the  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>/Pex19 mediated mPTS-PMP docking to the full integration into the membrane are yet unknown <a href="https://www.ncbi.nlm.nih.gov/pubmed/20531392"> <abbr title="2010, Schueller - The peroxisomal receptor Pex19p forms a helical mPTS recognition domain"> (2010, Schueller <i>et al.</i>)</abbr> </a>. However, general principles of the integration of a new peroxisomal membrane protein (PMP) through Pex19 and  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> are studied. Most PMPs feature a membrane targeting signal (mPTS), multiple binding sites for Pex19p, and at least one transmembrane domain (TMD). The mPTS can appear in two different ways, either located in the middle of the primary amino acid sequence, which is the rather complex form, or it can be found at the N-terminal part of the PMP as in Pex25. Pex19p is a cytosolic protein, which recognizes the mPTS of the PMP to be incorporated. In the first step Pex19p attaches to the PMP by binding to the mPTS and acts like a chaperone, guiding it to the peroxisome. Next, Pex19p binds N-terminally to the peroxisomal membrane protein  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>p, which is attached to the peroxisomal membrane through an N-terminal membrane anchor. This will bring the PMP in close proximity to the peroxisomal membrane. Last, Pex19p initiates the membrane integration of the PMP.  <a href="https://www.ncbi.nlm.nih.gov/pubmed/26777132">
+<div class="half-width">
-   <abbr title="(2016, Liu et al)">.</a></p>
+<figure>
+<img src="https://static.igem.org/mediawiki/2017/e/e1/T--Cologne-Duesseldorf--Gus_Assay_Secret2.jpg">
-<h4>Additional Sources/References</h4>
-<p>2001, Jones - Multiple Distinct Targeting Signals in Integral Peroxisomal Membrane Proteins</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.
-<p>2004, Jones - PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins</p>
+</figcaption>
-<p>2004, Rottensteiner - Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals</p>
+</figure>
-<p>2016, Mayerhofer - Targeting and insertion of peroxisomal membrane proteins ER trafficking versus direct delivery to peroxisomes</p>
+</div>
-<p>2016, Hua - Multiple paths to peroxisomes Mechanism of peroxisome maintenance in mammals</p>
+<!--
-<p>2016, Giannopoulou - Towards the molecular mechanism of the integration of peroxisomal membrane proteins</p>
+<figure>
+        <img src="">
+        <figcaption>  </figcaption>
+</figure>
+-->
 </div>
@@ Line 482: / Line 268: @@
 <button class="accordion">
-<h2 id="Secretion">Secretion</h2>
+  <h2 id="Sensors">Sensors</h2>
-<p>Downstream processing is not only time consuming but also cost and energy intensive. Therefore, we aim to simplify the purification of compounds produced in our artificial compartment. We used a concept based on the peroxicretion described by Sagt and colleagues <a href=" https://www.ncbi.nlm.nih.gov/pubmed/19457257">
+  <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>
-  <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.">
+  </button>
-      (Sagt <i> et al</i>, 2009)
-  </abbr>
-</a>. </p>
-</button>
 <div class="panel">
-<h3>Introduction </h3>
+    <p>
-<p>Downstream processing is a very important part of industrial biological compound production. For most biotechnological produced compounds, it is the most expensive part of the production <a href=" https://www.ncbi.nlm.nih.gov/pubmed/11602307 ">
+<h3>Sensors</h3>
-  <abbr title=" Keller, K., Friedmann, T., & Boxman, A. (2001). The bioseparation needs for tomorrow. TRENDS in Biotechnology, 19(11), 438-441.">
-      (Keller <i> et al</i>, 2001)
+<h4>Localization</h4>
-  </abbr>
+<p>pHlourin2 and roGFP2 was detectable by fluorescence microscopy and showed a 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>
-</a>
+<div class="flex-row-2">
-. One step to decrease the costs is to secrete the products into the supernatant <a href=" https://www.ncbi.nlm.nih.gov/pubmed/23385853">
+	<div>
-  <abbr title=" Berlec, A., & Štrukelj, B. (2013). Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells. Journal of industrial microbiology & biotechnology, 40(3-4), 257-274.
+  	<img src="https://static.igem.org/mediawiki/2017/0/04/L1_ruc%2B_gfp-mcherry_200ms_str8green.png">
-">
+	</div>
-      (Berlec <i> et al</i>, 2013)
+	<div>
-  </abbr>
+  	<img src="https://static.igem.org/mediawiki/2017/e/e3/WT_GFP_mCherry_WT_GFP-mCherry_500ms_str4green.png">
-</a>. After secretion, it is possible to remove most cellular compounds from valuable products with one simple centrifugation step. Due to this, secretion is not only a great tool for a compartment toolbox but also has an economic value. <br>
+	</div>
-In regards to the whole project, this is an important part for making the compartment more applicable. Through it we go a step further by thinking about the extraction of products after production.<br>
-At the end of this sub project it should be possible to secrete every compound produced in the modified compartment to the supernatant. This is not trivial because peroxisomes, which are the basis of our compartment do not possess a known natural secretion mechanism. <br>
-We overcome this problem by using the "peroxicretion" concept of Sagt and colleagues <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>. They used a v-SNARE (<b>v</b>esicle- synaptosome-associated-<b>S</b>oluble <b>N</b>-ethylmaleimide-sensitive-factor <b>A</b>ttachment <b>RE</b>ceptorprotein) fused to a peroxisomal membrane-protein to secrete the content of peroxisomes. V-SNAREs interact with the t-SNARE (<b>t</b>arget synaptosome-associated-SNARE) at the cell membrane, which leads to an fusion of the vesicle with the membrane <a href=" https://www.nature.com/articles/35052017">
-  <abbr title=" Chen, Y. A., & Scheller, R. H. (2001). SNARE-mediated membrane fusion. Nature reviews Molecular cell biology, 2(2), 98-106.">
-      (Chen <i> et al</i>, 2001)
-  </abbr>
-</a>.
-</p>
 </div>
+<figcaption>
-<button class="accordion" >
-<h2 id="SizeAndNumber">Size and Number</h2>
 <p>
-Peroxisomes are eukaryotic organelles that are involved in a vast variety of metabolic processes, most notably the metabolism of lipids as well as various oxidative reactions [1, 6]. They are remarkably versatile when it comes to adapting to both, intra- and extracellular cues and changes, responding to environmental variation by changing their size, shape and content accordingly. These exceptional abilities of peroxisomes do not only provide cells with a microenvironment for metabolic reactions but also enable them to respond to metabolic or environmental stress as well as cope with needs of cell division [6]. Controlling the size and shape of peroxisomes is therefore not only crucial to understanding peroxisome dynamics and utility but also provides a promising opportunity to influence bioengineering pathways within cells. Many studies that have been conducted in order to investigate the complexity of the shape, size and number of peroxisomes have led to the recognition of several PEX genes involved in peroxisome biogenesis and proliferation [3]. In order to control the size and number of peroxisomes as part of our toolbox, we further investigated the Pex11, Pex31,32, and Pex34 genes.
+<font size="3"><strong>Figure5.1 </strong>Left: cytosolic pHLuorin2 expression. Right: wild type, without any fluorescence.
 </p>
-</button>
+</font>
-<div class="panel">
+</figcaption>
-<h3>Introduction</h3>
-<h4>Peroxisome Biogenesis and Proliferation</h4>
-<p>
-Peroxisomes can be generated in different ways and their size and abundance is controlled by a number of pathways [8]. In yeast, peroxisomes can be generated de novo by budding from the endoplasmatic reticulum (ER) or through division from pre-existing peroxisomes using new proteins and lipids supplied from the ER in the form of vesicles [1]. Both pathways are still being investigated and to date haven’t been fully understood.
-</p>
-<figure>
-    <img class="half-width" src="https://static.igem.org/mediawiki/2017/4/44/T--Cologne-Duesseldorf--Peroxisomes-can-form-through-two-pathways.jpg">
-    <figcaption>Fig.1: Peroxisomes can form through two pathways Nat Rev Mol Cell Biol. 2013 Dec; 14(12): 803–817.</figcaption>
-</figure>
 <p>
-Peroxisomes are extremely sensitive to environmental cues and are able to proliferate or be degraded accordingly [3]. Depending on the growth medium and their extracellular environment, peroxisomes are able to divide and multiply separately from cell division [3]. Their size and number is directly influenced by the presence of e.g. fatty acids, which lead to an increase in both size and number. Furthermore, peroxisome population is regulated by different peroxisomal integral membrane proteins, so called peroxins [2].
+Carboxyl terminal fusion of the peroxisomal targeting signal 1 resulted in small areas with high intensity 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>
+  	<img src="https://static.igem.org/mediawiki/2017/e/ef/Pup%2B%2B_p13_gfp-mcherry_500ms_str4_m2green.png">
+	</div>
+	<div>
+  	<img src="https://static.igem.org/mediawiki/2017/c/cb/L1_rup-_gfp-mcherry_200ms_str8green.png">
+	</div>
+</div>
+<figcaption>
+<p>
+<font size="3"><strong>Figure5.2 </strong>Left: pHLuorin2 localized in the peroxisome. Right: roGFP2 localized to the peroxisome
 </p>
-<h4>Peroxins</h4>
+</font>
+</figcaption>
 <p>
-The formation of peroxisomes, both by de novo generation as well as growth and fission, is a highly controlled mechanism. Multiple studies have shown that the growth and division of peroxisomes are regulated by protein families specific to peroxisomes, so called peroxins [2]. Since <I>S. cerevisiae</I> naturally contains a very small amount of peroxisomes when growing under glucose-rich conditions, biosynthesis and target yield can be increased by altering peroxisome size and number. A short introduction to the peroxins used in this part of the project is given hereafter.
+<br>
+peroxin13 mRuby construct showed concentrated localization inside cellular areas too. In comparison to our tested sensors with the comparable promoter parts <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design">016 and 017</a> peroxin13 mRuby 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 level 2 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>
+<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>
-<h5>PEX11</h5>
+</font>
+</figcaption>
+<div class="flex-row-2">
+	<div>
+  	<img src="https://static.igem.org/mediawiki/2017/9/94/Puc%2B%2B_p13_gfp-mcherry_500ms_str4_m2green.png">
+	</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>
-Due to its unique ability to promote peroxisome division and its role in peroxisome biogenesis [5], the first peroxin we chose for our purpose is Pex11, which is located in the inner surface of the peroxisomal membrane [9]. Erdmann and Blobel have shown that the deletion of the Pex11 gene in S. cerevisiae results in cells with fewer, larger peroxisomes, whereas overexpression results in cells with a higher quantity of smaller peroxisomes [2]. Studies conducted by Smith and Aitchison confirmed that Pex11p-deficient cells growing on fatty acids failed to increase the amount of peroxisomes and instead the accumulation of a few giant peroxisomes was observed [1]. Other media, like oleate-containing ones, cause an induction of peroxisomal proliferation, which is due to an oleate responsive element of the Pex11 promoter [2].
+<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.
-<br>
-In order to find out more about the complexity of peroxisome biogenesis and proliferation and also get constructive feedback on our work, we consulted with Florian David from Biopetrolia in Sweden. Their company specializes in yeast engineering in order to improve production titers, yields and rates for the production of biofuels, pharmaceuticals and other products. He suggested to expand our project to working not only with Pex11, but Pex31,32 and Pex34 as well.
 </p>
+</font>
+</figcaption>
+<h4>pHluorin2</h4>
+<p> Initially we desired an <i>in vivo</i> calibration with pH values ranging from pH 5.8 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. The ionophore 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 .
+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>
+<figure>
+	<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>
-<h5>PEX30-32</h5>
-<p>
-According to Zhou et al., the genes of the Pex30 – 32 family have been shown to influence peroxisome proliferation [2]. Their deletion resulted in the production of a higher quantity of large peroxisomes. Zhou et al. further investigated the effect of a Pex31,32 knockout, showing both number and size increase, also leading to a higher metabolic yield. However, a Pex31,32 knockout has been proven to attribute to a change in the membrane structure, resulting in higher permeability of the peroxisome membrane for fatty aldehydes and other intermediates and byproducts [2]. Due to these side effects we decided to discard working with a Pex31,32 knockout for now.
-<p/>
+<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 ODs<sub>600</sub>. 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>
-<h5>PEX34</h5>
+<br>
-<p>
+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>.
-Similar to Pex11, Pex34p is another peroxisomal integral membrane protein that can act both, independently and in combination with Pex11p, Pex25p, and Pex27p to control the peroxisome morphology and population. Pex34p is suggested to directly influence peroxisome proliferation as well as constitutive peroxisome division. Specifically, Pex34p overexpression positively affects peroxisome numbers in wild type and pex34 cells, whereas Pex34 deletion results in cells with fewer peroxisomes [6, 2]. In their studies Zhou et al. targeted synthetic pathways to peroxisomes in order to increase the production of fatty-acid-derived fatty alcohols, alkanes and olefins. By harnessing peroxisomes to produce fatty-acid-derived chemicals and biofuels they were able to show that peroxisome increases the production of target molecules while decreasing byproduct formation. Additionally, analyzing the effect of peroxin knockouts and overexpression, their research revealed that Pex34 overexpression significantly increased their yield [2].  The main advantage of working with Pex34p over Pex31,32 is the effect on the peroxisomal membrane. While Pex31,32 significantly increases membrane permeability, Pex34 has less effects on the membrane structure [2].
+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>
-</p>
+<h3>roGFP2</h3>
+<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>
 <figure>
-    <img src="https://static.igem.org/mediawiki/2017/5/52/T--Cologne-Duesseldorf--peroxisome-quantity-and-morphology.png">
+	<img src="https://static.igem.org/mediawiki/2017/a/a2/Artico_rogfp_verlauf.svg">
-    <figcaption>Fig. 2: Regulation of peroxisome quantity and morphology by different peroxins
+   <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
+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>
-<h4>Our Project</h4>
+<figure>
+	<img src="https://static.igem.org/mediawiki/2017/f/f9/Artico_oxidized_rogfp.svg">
+   <figcaption><font size="3"><strong>Figure5.8 </strong>405/485nm excitation ratio plotted against the oxidized proportion of roGFP2 </font
+</figcaption>
+</figure>
 <p>
-One step towards achieving the creation of a fully controllable artificial compartment is the regulation of and control over its morphology. In our case we are aiming at achieving the exact regulation of the size and number of the peroxisome. As a first approach we have chosen to control the Pex11 concentration in the cell. Furthermore Pex11 is to be designed as a 3b toolbox part so it can be combined and its effects tested with different promoters. For that purpose we are working with two constitutive promoters of varying strength as well as two inducible promoters which increase gene expression when grown in varying concentrations of galactose or copper sulfate. To control the range from a few giant peroxisomes to a high quantity of small ones we are working in a pex11D knockout strain. Secondly, following the advice of Florian David from Biopetrolia, we intend to increase both, the size and quantity of peroxisomes in the cell via a Pex34 overexpression. By working with Pex34 we will not only be able to control the peroxisome morphology, but also positively influence production yields.
+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">
+   <figcaption><font size="3"><strong>Figure5.9 </strong>Oxidized roGFP2 proportion plotted against the redox potential of glutathione (mV)</font
+</figcaption>
+</figure>.
-<h4>How does it integrate into the overall project?</h4>
 <p>
-Controlling the size and number of peroxisomes is one of the multiple functions we plan to integrate into our artificial compartment toolbox so that it can be utilized for various projects. However, even though the exact control of proliferation can help understand the complex matter of peroxisome dynamics, the advantages of these findings exceed mere foundational research.
+Using our calibrated sensor we could compare the redox states within strains which differ in metabolic physiology.
-Integrating synthetic pathways into cells is often impeded by competing pathways and accruing intermediates or undesired byproducts that negatively influence biosynthesis. In order to achieve feasible results from microbial production, respective pathways need to be isolated into a suitable environment. Compartmentation provides microenvironments for metabolic functions of cells shielding them from the interference of simultaneously occurring reactions and therefore favoring biosynthesis. Going one step further, establishing synthetic pathways into a fully controlled compartment has the potential to increase the efficiency and productivity of these pathways resulting in higher yields of target products [2,6]. In our case, we change the peroxisome’s morphology by knocking out or overexpressing Pex11 and Pex34 to obtain either a large amount of smaller peroxisomes or a high amount of enlarged ones. Especially the overexpression of Pex34 which results in a high quantity of large peroxisomes has been shown to actively regulate metabolic processes [1] and increase the production of target molecules while decreasing byproduct formation [2]. Furthermore, evidence indicates that changing the morphology of a compartment, including both, its shape and size, influences the amount of chemical reactions embedded in that compartment [1], a trait that can be used to increase the yield of otherwise inefficient reactions.  Ultimately, even though we decided to discard our work on a Pex31,32 knockout, the effects this knockout has on membrane permeability and structure could potentially be used for further pathways within the peroxisome. </p>
+</p>
+<figure>
+	<img src=" https://static.igem.org/mediawiki/2017/a/af/Artico_rogfp_living_cells.svg">
+   <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>
+</figure>
+<p>We conducted two Mann-Whitney-Wilcoxen test on the different targets and on different promoters strength used(n<sub>1</sub>=6, n<sub>2</sub>=6) resulting in a p value of 0,69 and 0,82. Neither  comparisons of cytosolic and peroxisomal targeting nor comparison of the different promoters showed significant differences in glutathione redox states. 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>Overall goal of this subproject</h4>
+<h4>Outlook</h4>
-<p>
+<p>We planned to calibrate our sensors <i>in vivo</i> as well and wanted to follow up changes induced by <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Violacein">violacein</a> and <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-2">nootkatone</a> pathways. Furthermore our objective was testing the expected acidification upon induced expression and integration of <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Membrane_Integration">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
-In our subproject we want to achieve full control over peroxin concentrations in the yeast cell, in order to establish a simple method to regulate the peroxisome morphology and quantity.</p>
+Protein Sensors"> Schwarzländer <i>et al.</i> (2015)</abbr></a>.This property makes it interesting to further usage for the iGEM community.
-</div>
+<br>
+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>.
+<br>
+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>
+    </p>
+</div>
 <button class="accordion">
-<h2 id="Sensors"><i>In Vivo</i> Sensors</h2>
-<p>Designing new pathways or transferring pathways into cellular compartments requires a sound understanding of the present conditions and content, like cofactors in the peroxisomes.
+  <h2 id="Nootkatone">Nootkatone</h2>
-We aimed  measuring the peroxisomal pH, cofactors like NADP<sup>+</sup> and ATP in wild type yeast and our designed mutants over different time periods as well as in response to changing physiological conditions. Therefore, we used ratiometric fluorescent biosensors which we genetically attached to a peroxisomal targeting signal.
+  <p>After successfully integrating all our plasmids into yeast, we were able to verify the expression of each enzyme of the nootkatone pathway. </p>
-These measurements provide important insights into possible issues which may occur if non-peroxisomal pathways are transferred into the peroxisome and furthermore enable more precise predictions and modelling.</p>
+  </button>
-</button>
 <div class="panel">
+<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>
-<h4> pH Sensor </h4>
-<p>The activity of enzymatic Proteins is mostly pH-dependent. Therefore, it is of high interest to understand the pH-regulating mechanism of the peroxisome and the effects on the imported pathways. Literature has not agreed whether there is a common peroxisomal pH nor whether there is a regulating mechanism. For our measurements, we use pH Lourin2, a GFP variant with a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidification, the excitation spectrum shifts from 395 to 475 nm <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>
-</p>
 <figure>
-	<img src="https://static.igem.org/mediawiki/2017/9/93/Artico_pHLuorin2_Verlauf.png">
+     	<img src="https://static.igem.org/mediawiki/2017/2/21/T--Cologne-Duesseldorf--western-blot-nootkatone.png" ;="" style="width: 70%; height: 70%">
-	<figcaption><font size="3"> <strong>Figure5.1</strong>
+      	<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>
-pHLuorin2 emission at 509 nm, excited at wavelengths between 350 nm and 500 nm . Five different pH values, ranging from 5.8 to 7.8 are shown <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"><font size="3"> Mahon <i>et al.</i> (2011)</font></abbr></a>.</font> </figcaption>
+  	</figure>
-</figure>
-<h4> roGFP2 Sensor </h4>
-<p>To maintain thermodynamic driving forces and electron fluxes which are needed at steady state, the intact chemeostasis of the redox machinery is of high importance<a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539"> <abbr title="2016, Schwarzländer, et al. - Dissecting Redox Biology Using Fluorescent Protein Sensors"> (2016, Schwarzländer)</abbr></a>. Glutathione is considered to be inside the peroxisomal lumen <a href="https://www.ncbi.nlm.nih.gov/pubmed/25867539"> <abbr title="2014, Elbaz-Alon, Y., et al. -The Yeast Oligopeptide Transporter Opt2 Is Localized to Peroxisomes and Affects Glutathione Redox Homeostasis">(Elbaz-Alon, Y., et al. 2014)</abbr></a>. We therefore wanted to monitor glutathione redox potentials inside the peroxisomal lumen using the  GFP variant roGFP2, which is able to precisely detect redox changes of glutathione. Two cysteines in the beta barrel structure can either form two thiols or one disulfide bondage dependent on whether they are reduced or oxidized. This influences the proton transfer of the chromophore and ultimately leads to a ratiometric shift in excitation. Excitation at 485 nm of the reduced roGFP2 exceeds the excitation of oxidized roGFP2 at 485 whereas excitation at 405 nm  of oxidized roGFP2 exceeds excitation of reduced roGFP2<a href="https://link.springer.com/article/10.1007/s12268-016-0683-2"> <abbr title="Morgan, B. and M. Schwarzländer 2016 et al.- The Yeast Oligopeptide Transporter Opt2 Is Localized to Peroxisomes and Affects Glutathione Redox Homeostasis">(Morgan, B. and M. Schwarzländer 2016)</abbr></a>. </p>
+<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.
+<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>
+<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>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>
 <button class="accordion">
-<h2 id="Nootkatone">Nootkatone</h2>
+  <h2 id="Violacein">Violacein</h2>
-<p>As a proof of concept for our compartment toolbox we decided to shift the metabolic pathway of nootkatone into the peroxisome. With this transfer we want to overcome the obstacle of intermediate toxicity for the yeast cell. A working metabolism will pave the way for an efficient, safe and favorable solution of producing and providing an effective insect repellent. </p>
+  <p>
-</button>
+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.
+</p>
+  </button>
 <div class="panel">
+  <h3>Violacein</h3>
+<p>
+The experiments were implemented following the protocols for
+<a href="https://static.igem.org/mediawiki/2017/8/8a/T--Cologne-Duesseldorf--western-blot-protocol.pdf">western blot</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
+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.
+</figcaption>
+</figure>
-<p>Nootkatone is an oxidized sesquiterpene, which is highly valuable for industrial and pharmaceutical application. We will focus on its repellent effect towards insects
+<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
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/11441443">
+<a href="https://advansta.com/wikis/multiple-bands-in-western-blots-causes-and-solutions-2/">
-<abbr title="2001, Zhu et al. - Nootkatone is a repellent for Formosan subterranean termite (Coptotermes formosanus)">
+<abbr title="Multiple Bands in Western Blots – Causes and Solutions">
-    	Zhu <i>et al.</i> (2001)
+(Hurley A, 2017)
-	</abbr>
+</abbr>
 </a>.
+<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.
-Also, therapeutic activities of nootkatone have been reported, such as anti-platelet effects in rats
+To decrease protein degradation in the future, protease inhibitors should be added to the lysis solution and all samples should be kept on ice.
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/21354294">
+<br> For further analysis of the enzyme functionality an assay followed by HPLC-MS analysis was implemented.
-<abbr title="2011, Seo et al. - Antiplatelet effects of Cyperus rotundus and its component (+)-Nootkatone">
+</p>
-    	Seo <i>et al.</i> (2011)</abbr>
-</a>,
- anti-proliferative activity towards cancer cell lines
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/21377882">
-<abbr title="2011, Gliszczyńska et al. - Microbial Transformation of (+)-Nootkatone and the Antiproliferative Activity of Its Metabolites">
-   	Gliszczyńska <i>et al.</i> (2011)
-	</abbr>
-</a>
- and enhancement of energy metabolism through AMP-activated protein kinase activation in skeletal muscle and liver
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/24624065">
-<abbr title="2010, Murase et al. - Habituation of the responsiveness of mesolimbic and mesocortical dopamine transmission to taste stimuli
-">
-   	Murase <i>et al.</i> (2010)
-	</abbr>
-</a>.</p>
+<figure>
-<p>Nootkatone can be extracted from grapefruits, but the organic material is limited and the yield is very low. So far, industrial production of nootkatone requires toxic substances such as heavy metals and strong oxidants like tert-butyl hydroperoxide which is known to be carcinogenic
+<img
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/21115006">
+src="https://static.igem.org/mediawiki/2017/2/2c/Artico_PDV_MS.png">
-<abbr title="2010, Cankar et al. -  A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene
+<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>.
-   	Cankar <i>et al.</i> (2010)
+</figcaption>
-	</abbr>
+</figure>
-</a>.</p>
+<p>
+Figure 8.2 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).
+<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 catalyze 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
+<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>
+therefore we did the second reaction mixtures with a higher concentration of VioB.
+</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.
+</p>
+<div class="max-width">
 <figure>
-     	<img src="https://static.igem.org/mediawiki/2017/d/d9/Valencene_Nootkatol_Nootkatone.jpeg">
+<img
-      	<figcaption><strong>Figure 7.1</strong> Conversion of valencene  to Nootkatol and Nootkatone </figcaption>
+src="https://static.igem.org/mediawiki/2017/e/ef/T--Cologne-Duesseldorf--PDV_MSMS.jpg">
-  	</figure>
+<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><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 loose 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.
+<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.
+<br>
+<br>
+<font size="2">
+Many thanks to Felix Büchel and the MS platform, Cologne for extraordinary support.</font>
+</p>
+<br>
-<p>The synthesis of nootkatone starts from the precursor farnesyl pyrophosphate (FPP) and requires at least two enzymes. The initial step is the formation of valencene from FPP by a valencene synthase (ValS) followed by the production of nootkatol, nootkatone and other by-products by a P450 BM3 monooxygenase (BM3). The co-expression of an alcohol dehydrogenase (ADH) with ValS improves nootkatone production by favoring the conversion from nootkatol into nootkatone
+<h4>Outlook </h4>
- <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>
+<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.
+</p>
+<figure>
+<img
-<p>Previous approaches of nootkatone synthesis in yeast often failed due to toxic intermediates. A specific problem is the toxicity of beta-nootkatol and nootkatone itself for <i>Saccharomyces cerevisiae</i> at concentration higher than 100 mg/L
+src="https://static.igem.org/mediawiki/parts/b/bc/T--Cologne-Duesseldorf--Violacein-Level2Kombis.png">
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/23518241">
+<figcaption>
-<abbr title="2013, Gavira et al. - Challenges and pitfalls of P450-dependent (þ)-valencene bioconversion by Saccharomyces cerevisiae">
+<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
-   	Gavira <i>et al.</i> (2013)
+<a href="http://www.nature.com/articles/ncomms11152">
-	</abbr>
+<abbr title="Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways">
-</a>.
+(John E. Dueber <i>et al.</i>, 2015)
-For an efficient industrial production, concentrations need to be in the range of g/L, which is lethal for yeast cells. Beta-nootkatol seems to accumulate in membranes because of its hydrophobic characteristics, resulting in changes of the membrane permeability, integrity and the function of membrane proteins
+</abbr>
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/23518241">
-<abbr title="2013, Gavira et al. - Challenges and pitfalls of P450-dependent (þ)-valencene bioconversion by Saccharomyces cerevisiae">
-   	Gavira <i>et al.</i> (2013)
-	</abbr>
-</a>.
-It is presumed that the toxicity is partly caused by this effect. As one of the original purposes of the peroxisome is to reduce hydrogen peroxide, which is harmful to the cell and also alters the membrane composition
-<a href="https:/https://www.ncbi.nlm.nih.gov/books/NBK9930/">
-<abbr title="2000, Cooper et al. - The Cell: A Molecular Approach. 2nd edition">
-   	Cooper <i>et al.</i> (2000)
-	</abbr>
-</a>
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/1902481">
-<abbr title="1991, Block et al. - Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells">
-   	Block <i>et al.</i> (1991)
-	</abbr>
 </a>
-, we assume that beta-nootkatol does not affect the peroxisomal membrane either. But to be fully sure if this hypothesis is true, we have to collect and evaluate our own data on how beta-nootkatol affects the peroxisome membrane and thus the yield of nootkatone.</p>
+.
+</figcaption>
+</figure>
+<p>
-<div class="half-width">
+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.
-<figure>
+</p>
-     	<img src="https://static.igem.org/mediawiki/2017/3/3c/Graph1.png">
-      	<figcaption><strong>Figure 7.2</strong> Yeast viability after 24 h in the presence of (+)-valencene, beta-Nootkatol or nootkatone in different concentrations <a href="https://www.ncbi.nlm.nih.gov/pubmed/23518241">
-<abbr title="2013, Gavira et al. - Challenges and pitfalls of P450-dependent (þ)-valencene bioconversion by Saccharomyces cerevisiae">
-   	Gavira <i>et al.</i> (2013)
-	</abbr>
-</a> </figcaption>
-  	</figure>
 </div>
-<p>Our goal is the successful integration of the nootkatone pathway into our compartment and to bypass the problem of high concentration toxicity of beta-nootkatol and nootkatone for the yeast cell. This would not only be a more efficient but also a more environmentally friendly method to satisfy the great industrial demand of this sesquiterpene. It would also facilitate the access to a high performing insect repellent in less developed regions of the world and therefore decrease the spread of diseases like malaria, dengue or the Zika virus.</p>
-</div>
+<button class="accordion"> <h3 id="optogenetics">Optogenetic enhancements</h3>
+<p>
+Here you can find our results and discussion regarding the optogenetics subtopic
+</p>
+ </button>
+<div class="panel">
-<button class="accordion">
+<h3>Pex5 import with LOV2</h3>
-<h2 id="Violacein">Violacein</h2>
-<p> Using the tools of synthetic biology in metabolic engineering unleashes the full potential of biofactories. Natural systems use compartmentalization to improve biochemical reactions. Here we present the use of peroxisomal import tags to engineer an artificial compartment in <i>Saccharomyces cerevisiae</i> cells to be further used in metabolic engineering approaches. As an application and proof of concept we are using the well studied biosynthetic pathway of violacein. By designing an import library for the different enzymes we aim to understand basic design principles that can guide future design of compartmentalization for metabolic engineering. We chose violacein, not only because of its wide range of biological benefits but also as a solid foundation to proof a sophisticated import machinery. </p>
-</button>
-<div class="panel">
-<figure class="floatleft">
-<img class="half-width" src="https://static.igem.org/mediawiki/parts/1/17/T--Cologne-Duesseldorf--Violacein_Struktur.png">
-<figcaption>
-<strong>Figure 8.1</strong> Structural formula of violacein.
-</figcaption>
-</figure>
 <p>
-Violacein (C<sub>20</sub>H<sub>13</sub>N<sub>3</sub>O<sub>3</sub>), a bisindole, is a violet pigment, formed by condensation of two tryptophan molecules. It can naturally be found in numerous bacterial strains, for example in the gram-negative <i> Chromobacterium violaceum</i>. Due to its wide range of biological properties, violacein is useful for various industrial applications in pharmaceuticals and cosmetics.
+Our GFP-LOV-PTS1 construct was successfully cloned and transformed into <i>S. cerevisiae</i>. As part of our 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).
-<br>
-Violacein is known to have a variety of different biological activities, including an antitumor
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/20416285">
-<abbr title="Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor">
-(Bromberg N<i> et al</i>, 2010)
-</abbr>
-</a>, antifungal
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/18949519">
-<abbr title="Amphibian chemical defense: antifungal metabolites of the microsymbiont Janthinobacterium lividum on the salamander Plethodon cinereus">
-(Brucker RM <i>et al.</i>, 2008)
-</abbr>
-</a>
-and antiviral
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/14595466">
-<abbr title="Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum">
-(Andrighetti-Fröhner CR <i> et al.</i>, 2003)
-</abbr>
-</a>
-function. Furthermore, it has been shown that violacein enhances the effect of most commercial antibiotics by working synergistically with them
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/24073823">
-<abbr title="Synergistic antimicrobial profiling of violacein with commercial antibiotics against pathogenic micro-organisms">
-(Subramaniam S <i> et al.</i>, 2014)
-</abbr>
-</a>. This is especially of high interest in the fight against recent antibiotic-resistant strains of pathogenic bacteria such as MRSA (multi resistant <i>Staphylococcus aureus</i>). Violacein’s antibacterial action against <i>S. aureus</i> has been proven by
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/21364597">
-<abbr title="Antibacterial activity of violacein against Staphylococcus aureus isolated from bovine mastitis">
-Cazoto LL <i>et al.</i> (2011)
-</abbr>
-</a>.
-<br>It is of high medical interest that toxic effects of Violacein on cultured cancer cells were shown within <i>in vitro</i> tests. Furthermore, the Ehrlich ascites tumor (EAT) mouse model gives the prove as an <i>in vivo</i> test: daily injection of violacein ($0.1\,\mu g/kg$ up to $1\,mg/kg$) led to a significant increased survival rate of the mice
-<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538413/">
-<abbr title="Violacein: Properties and Production of a Versatile Bacterial Pigment">
-(Seong Yeol Choi <i>et al.</i>, 2015)
-</abbr>
-</a>. The ability to weaken cancer growth draws more attention to violacein as a possible cancer therapeutic.
-<a href="https://www.ncbi.nlm.nih.gov/pubmed/16889929">
-<abbr title="Cytotoxic activity of violacein in human colon cancer cells">
-de Carvalho DD <i>et al.</i> (2006)
-</abbr>
-</a>showed that violacein is capable to induce apoptosis in various cancer cells by inducing the production of oxygen radicals.
-<br> A main focus also lies in violacein’s antimalarial activity, which was tested <i>in vitro</i>  and <i>in vivo</i> on human and murine blood stage forms of <i>Plasmodium</i> parasites
-<a href="http://aac.asm.org/content/53/5/2149.full">
-<abbr title="Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo">
-(Stefanie C. P. Lopes <i> et al.</i>, 2009)
-</abbr>
-</a>. <i>P. falciparum</i> is known to be the deadliest <i>Plasmodium</i> species that causes malaria in humans
-<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720412/">
-<abbr title="The origin of malignant malaria">
-(Stephen M. Rich <i>et al.</i>, 2009)
-</abbr>
-</a>. Violacein acted effectively against diseases caused by both, young and mature parasite strains, of <i>P. falciparum</i>, and parasite growth was reduced significantly compared to non-treated animals. Moreover, it has a protective effect as mice infected with a lethal strain (<i>P. chabaudi chabaudi</i>) died within 10 days, whereas the majority (80 %) treated with violacein survived the infection
-<a href="http://aac.asm.org/content/53/5/2149.full">
-<abbr title="Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo">
-(Stefanie C. P. Lopes <i> et al.</i>, 2009)
-</abbr>
-</a>. Not at least because the emerge resistance to plant-based malaria drugs becomes more frequent, it is time to look out for further possibilities in the worldwide battle against malaria <a href="https://www.ncbi.nlm.nih.gov/pubmed/26911755">
-<abbr title="Synthetic biology's first malaria drug meets market resistance">
-(Peplow M, 2016)
-</abbr>
-</a>.
 </p>
-<p> As the commercial production of violacein is rather difficult and limited for low productivity
+<h3>Pex7 import</h3>
-<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997675/">
+<p>
-<abbr title="Engineering Corynebacterium glutamicum for violacein hyper production">
+All three constructs of our Split-TEV PTS2 subproject have been successfully cloned to level 1 in regards to the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#yeast-toolbox">(Dueber Toolbox)</a>. A level 2 plasmid containing the PhyB-TEV2 and TEV1-PIF6 constructs is required in order to transform all constructs into <i>S. cerevisiae</i>. This has not been created so far.
-(Hongnian Sun <i>et al.</i>, 2016)
+</p>
-</abbr>
+<h3>Optogenetically controlled gene expression</h3>
-</a>, researchers are working on improving the fermentative titers by metabolic engineering.
+<p>
-<br>Here we want to make use of the existing potential violacein has and even try to promote it. With the great advantages a peroxisomal import has to offer, we want to develop a solid mechanism to not only prove the concept of our project, but also take advantage of violacein’s biological opportunities. By relocalization of the violacein pathway into yeast peroxisomes we want to create a space with optimized working conditions for the production of violacein to achieve a high yield of the bisindole.
+The TetO-Pmin promoter construct has been brought to level 1 with <I>mRuby</I> and <I>Pex11</I> as genes of interest. The TetR-PIF6 construct has also been brought to level 1. The PhyB-VP16 construct has not been successfully integrated into the Dueber toolbox.
+</p>
+<h3>Outlook</h3>
+<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, 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 approach 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>
+Due to a lack of time 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>.
+<br>
+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.
+Another approach we have not pursued yet is attaching the vSNARE-proteins we are using to PIF6 and insert Phytochrome B into the peroxisomal membrane via our Pex26 anchor (see <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results#MembraneIntegration">membrane integration</a>). In theory, illumination with red light would then lead to instant secretion.
 </p>
 </div>