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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 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 recombinant proteins are expressed by the engineered yeasts which contain pYCα-alpha, pYCα-beta, pYCα-mCherry and pYCα-mCherry-αtubulin, respectively. After 24h inducing，the recombinant proteins are extracted and analysed by Western blot.

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.The recombinant proteins are expressed by S.cerevisiae INVSCi pYCα-FilC(PETase), pYCα-FliC(XynA), respectively. After 24h inducing，the recombinant proteins are extracted and analysed by Western blot.

figure 6 The results of a Western blot analysis carried out with an anti-His antibody.