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  • Microtubule

  • Flagellar Filament

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

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;

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

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)

The image shows the results of a Western blot analysis carried out with an anti-V5 antibody.

figure 6