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D fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry-α; <br> | D fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry-α; <br> | ||
E a bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry;<br> | E a bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry;<br> | ||
− | F fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry;</h4> | + | F fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry;<br></h4> |
+ | |||
<h3>Protein expression analysis- Western blot</h3> | <h3>Protein expression analysis- Western blot</h3> | ||
− | <p | + | <p>The image shows the results of a Western blot analysis carried out with an anti-V5 antibody. |
The target proteins are expressed by the engineered yeasts which contain pYCα-alpha, pYCα-beta, pYCα-mCherry and pYCα-mCherry-αtubulin, respectively.</p> | The target proteins are expressed by the engineered yeasts which contain pYCα-alpha, pYCα-beta, pYCα-mCherry and pYCα-mCherry-αtubulin, respectively.</p> | ||
<img src="https://static.igem.org/mediawiki/2017/2/25/T-BNU-China-results3.png" alt="Sorry, the image is not supported by your browser."> | <img src="https://static.igem.org/mediawiki/2017/2/25/T-BNU-China-results3.png" alt="Sorry, the image is not supported by your browser."> | ||
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<img src="https://static.igem.org/mediawiki/2017/e/e7/T--BNU-China--results5.png" alt="Sorry, the image is not supported by your browser."> | <img src="https://static.igem.org/mediawiki/2017/e/e7/T--BNU-China--results5.png" alt="Sorry, the image is not supported by your browser."> | ||
<h4>Figure 5 Fluorescence image of recombinant FliC(eGFP) expressed by the engineered yeast cell.A,B recipient strain EBY100 harbouring pYCα;<br>C a bright-field micrograph of S. cerevisiae EBY100 cells harbouring pYCα–FliC(eGFP);<br> | <h4>Figure 5 Fluorescence image of recombinant FliC(eGFP) expressed by the engineered yeast cell.A,B recipient strain EBY100 harbouring pYCα;<br>C a bright-field micrograph of S. cerevisiae EBY100 cells harbouring pYCα–FliC(eGFP);<br> | ||
− | D fluorescence micrograph of S. cerevisiae EBY100 cells harbouring pYCα–FliC(eGFP)</h4> | + | D fluorescence micrograph of S. cerevisiae EBY100 cells harbouring pYCα–FliC(eGFP)<br></h4> |
+ | |||
<h3>Protein expression analysis- Western blot</h3> | <h3>Protein expression analysis- Western blot</h3> | ||
− | <p | + | <p>The image shows the results of a Western blot analysis carried out with an anti-His antibody.</p> |
<img src="https://static.igem.org/mediawiki/2017/f/fb/T-BNU-China-results6new.png" alt="Sorry, the image is not supported by your browser."> | <img src="https://static.igem.org/mediawiki/2017/f/fb/T-BNU-China-results6new.png" alt="Sorry, the image is not supported by your browser."> | ||
<h4>figure 6</h4> | <h4>figure 6</h4> |
Revision as of 03:12, 23 October 2017
Results
Microtubule
Plasmid construction
We have accomplished the construction of 6 main parts whose functions are described respectively in previous design page. (Microtubule module)
Figure 1 The electrophoresis image of these 6 plasmids.
Protein expression analysis- Fluorescence microscopy
These are images recorded by the Fluorescence microscope.The engineered yeasts were induced at 30°C for 20 hours. And from the image, we can notice that our engineered yeasts have successfully expressed our recombinant proteins mCherry-αand mCherry. Moreover, the expression rate of mCherry is almost up to 100%.
Figure 2 Induced 20h in SG-Ura medium;
A,B recipient strain with empty plasmid;
C a bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry-α;
D fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry-α;
E a bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry;
F fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry;
Protein expression analysis- Western blot
The image shows the results of a Western blot analysis carried out with an anti-V5 antibody. The target proteins are expressed by the engineered yeasts which contain pYCα-alpha, pYCα-beta, pYCα-mCherry and pYCα-mCherry-αtubulin, respectively.
Figure 3 The results of a Western blot analysis carried out with an anti-V5 antibody.
Our engineered yeasts, containing pYD1-β tubulin vector, have been proved successfully expressing exogenous βtubulin by Western blot analysis.
Figure 4 The partly results of a Western blot analysis carried out with an anti-V5 antibody.
Flagellar Filament
Plasmid construction
We have successfully constructed the following 11 parts that have been described in detail in previous design page. (Filagellar filament module)The parts are named: , , , ,
The electrophoresis image of our parts.
Protein expression analysis- Fluorescence microscopy
The below image shows the expression of the S. cerevisiae INVSc1 cells harbouring pYCα–FliC(eGFP). The strong fluorescence signal validates the expression of recombinant protein: FilC fused with eGFP.
Figure 5 Fluorescence image of recombinant FliC(eGFP) expressed by the engineered yeast cell.A,B recipient strain EBY100 harbouring pYCα;
C a bright-field micrograph of S. cerevisiae EBY100 cells harbouring pYCα–FliC(eGFP);
D fluorescence micrograph of S. cerevisiae EBY100 cells harbouring pYCα–FliC(eGFP)
Protein expression analysis- Western blot
The image shows the results of a Western blot analysis carried out with an anti-His antibody.
figure 6
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