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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) They are pYD1-α tubulin (BBa_K2220019), pYD1-β tubulin (BBa_K2220020), pYCα-α tubulin (BBa_K2220022), pYCα-β tubulin (BBa_K2220023), pYCα-mCherry-α tubulin (BBa_K2220024), pYCα-β-tubulin-mGFP (BBa_K2220025) and pYCα-mCherry (BBa_K2220021).

Figure 1 The electrophoresis image of 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. 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. (Flagellar filament module)
In display module, we constructed and validated the following 6 parts. They are pYD1-FliC (BBa_K2220002), pYD1-XynA(BBa_K2220004), pYD1-PETase(BBa_K2220005), pYD1-BG(BBa_K2220007), pYD1-EG(BBa_K2220006), pYD1-CBH(BBa_K2220008), which means to fuse the target gene sequences with AGA2 gene respectively. The length and sequence of each parts are validated through sequencing. The length validation are presented as below.

Figure 5 The electrophoresis images of 6 parts mentioned above.

In secretory module,we successfully constructed the following parts: pYCα-FliC-XynA (BBa_K22200011), pYCα-FliC-BG (BBa_K2220014), pYCα-FliC-EG (BBa_K2220013), pYCα-FliC-CBH (BBa_K2220015)，and pYCα-FliC-eGFP (BBa_K2220003) as positive control. The length and sequence of each parts are validated through sequencing. The length validation are presented as below.

Figure 6 The electrophoresis images of 4 parts mentioned above.

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 INVSC1 pYCα-FilC(PETase), pYCα-FliC(XynA), respectively. After 24h inducing，the recombinant proteins are extracted and analysed by Western blot.

figure 7 The results of a Western blot analysis carried out with an anti-His antibody.