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

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  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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<figcaption> 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.  </figcaption>
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<figcaption>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.  </figcaption>
  
  
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  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>
 
  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>
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<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>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 part for different import effiency. </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 part for different import effiency. </p>
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<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 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</p>
 
<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 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</p>
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         <figcaption><b> Figure 5 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>
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         <figcaption><b> Figure 5 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>
  
  
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         <figcaption><b>Figure 4 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>
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         <figcaption><b>Figure 4 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>
  
  
 
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<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"> GUS-assay*XXneeds to be change* </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>
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The fluorescence increase over time of the samples which are <a href="https://2017.igem.org/Team:Cologne-Duesseldorf"> decorated with snares*XXneeds to be change* </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 6.) </p>
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<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"> GUS-assay*needs to be change* </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>
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The fluorescence increase over time of the samples which are <a href="https://2017.igem.org/Team:Cologne-Duesseldorf"> decorated with snares*needs to be change* </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 6.) </p>
  
 
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<div><img src="https://static.igem.org/mediawiki/2017/d/d9/C2-PEX26-mRuby_red_Channel.jpeg"></div>
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<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. 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.</p>
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Finally we used the same approach to send 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.
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<h4>Outlook</h4>
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<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.
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Revision as of 00:43, 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. Months of lab work payed off! These are our results.

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.

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.