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<h2 id="PEX5Import">PEX5 Import</h2>
<h2 id="PEX5Import">PEX5 Import</h2>
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<h3>MD simulations</h3>
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Line 38: Our second approach for the modification of the importer PEX5 was our so called 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. In the following figure one can see the microscopy image of this import system variant cloned in PEX5 knock-out yeast.
Our second approach for the modification of the importer PEX5 was our so called 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. In the following figure one can see the microscopy image of this import system variant cloned in PEX5 knock-out yeast.
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+ PEX5 variant R19 with the PTS1* and the two negative controls consisting of
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Our first results show that coexpression of R19 with mTurquoise tagged to PTS1* leads to import of the flourescent reporter protein, visible as little dots. The negative control consisting of the wild-type strain carrying mTurquoise tagged with our PTS1* shows exactly the opposite: Flourescence was detected in the whole cell, indicating that R19 is not able to recognize and import the modified peroxisomal targeting signal with its cargo. Though, this figure does not prove that the reporter protein is located in the peroxisomes. Therefore we validated this results by coexpressing this import machinery with an peroxisomal marker protein as can be seen in the following.
Our first results show that coexpression of R19 with mTurquoise tagged to PTS1* leads to import of the flourescent reporter protein, visible as little dots. The negative control consisting of the wild-type strain carrying mTurquoise tagged with our PTS1* shows exactly the opposite: Flourescence was detected in the whole cell, indicating that R19 is not able to recognize and import the modified peroxisomal targeting signal with its cargo. Though, this figure does not prove that the reporter protein is located in the peroxisomes. Therefore we validated this results by coexpressing this import machinery with an peroxisomal marker protein as can be seen in the following.
Revision as of 02:13, 1 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 payed off! Below you can find the results of our efforts.
Sub-projects
PEX5 Import
The orthogonalization of the Pex5 import mechanism was an ambitious and challenging task − interested how we did? See our results!
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.
PEX5 variants obtained in the course of molecular dynamics simulations. Unfortunately, none of the constructs showed signs of import.
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 see import.
PEX5 variant R19
Our second approach for the modification of the importer PEX5 was our so called 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. In the following figure one can see the microscopy image of this import system variant cloned in PEX5 knock-out yeast.
PEX5 variant R19 with the PTS1* and the two negative controls consisting of
Our first results show that coexpression of R19 with mTurquoise tagged to PTS1* leads to import of the flourescent reporter protein, visible as little dots. The negative control consisting of the wild-type strain carrying mTurquoise tagged with our PTS1* shows exactly the opposite: Flourescence was detected in the whole cell, indicating that R19 is not able to recognize and import the modified peroxisomal targeting signal with its cargo. Though, this figure does not prove that the reporter protein is located in the peroxisomes. Therefore we validated this results by coexpressing this import machinery with an peroxisomal marker protein as can be seen in the following.
PEX5 variant R19 with the PTS1* and PTS*, co-transformed with the PEX13-mRuby construct − the localization of mTurquoise tagged with PTS1* P* is clear and due to that import was successfully validated whereas the control did not show any signs of import.
Wild type PEX5 with the PTS1* and PTS*.
PEX13, as an integral protein of the peroxisomal membrane, provides perfect features to mark the membrane to validate proper location. As described in our experimental design , we used Pex13's transmembrane domain and fused mRuby to it. The figure above shows clearly the location of the peroxisomes and the fluorescent signal of mTurquoise is definitely located in the peroxisomes., which proves that R19 transport our cargo into the peroxisomes. On the contrary, mTurquoise is located in the whole cytosol in the wild type strain with PTS1* and the R19 strain, meaning that there is no functional import.
On the other hand we observed similar fluorescent signals in wild type yeasts that possess the modified PTS tagged to mTurquoise, as can be seen in figure xx: In the same figure one can see the wild-type yeast expressing mTurquoise tagged to the natural PTS1. If one compares both pictures, it becomes directly clear that the wild-type receptor is not able to recognize our PTS1* and thus no import could be detected. So all our negative controls were not able to import mTurquoise into the peroxisome, which is exactly what we wanted to achieve.
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.
Conclusion
This subproject was a big challenge but also a big opportunity for our project. 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 yeast. We modified one of the most conserved import machineries within the domain of eukaryotes - no matter if it is plants, mammals or fungi. This opens up new possibilities for biotechnological applications since this import system can be used to shift toxic compound reactions into the natural stress-resistant peroxisomes and thereby it can increase the yield and efficiency of rare biomolecule production in vivo .
Furthermore, we managed to make a big step further towards a synthetic cell: While many research groups try to build up a synthetic cell from scratch, we decided to build it up from the inside by subverting its natural functional systems and making it fully customizable and controllable.
This is why our new import machinery shows the potential for biotechnology and real world applications in general.
Pex7 Import
Abstract
Heading
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 (2014) . Luciferase is highly efficient because almost all energy is converted into light, the protein is thus very sensitive Azad (2014) . 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 (2003) .
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 (2015) . 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 XXX Colonies of the PTS2 library show a colour range of white to green indicating 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, 2016 600 . A wide distribution of different values were observed indicating a broad variety of different PTS2 versions.
Figure XXX The fluorescence per OD600 show a broad range in the PTS2 library indicating varying import efficiencies
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 part for different import effiency.
Size and Number
Abstract
Secretion
Abstract
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 5). Shown in figure 4 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 5 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 4 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*needs to be change* . 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. decorated with snares*needs to be change* 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 6.)
Figure 6 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.
Membrane Integration
Abstract
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. Under the fluorescent microscope the colocalization of both, the green fluorescing GFP and the red fluorescing mRuby is clearly visible, showing that our anchors integrated into the peroxisomal membrane.
Finally we used the same approach to send 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.
Sensors
Abstract
Nootkatone
Abstract
Violacein
Abstract
