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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 the 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). All parts have been validated by sequencing.

Figure 1 The electrophoresis image of 6 plasmids

Protein expression analysis- Fluorescence microscopy

These are the images recorded by the Fluorescence microscope. The engineered yeasts were induced at 30°C for 20 hours. From the image, we can see that our engineered yeasts have successfully expressed our recombinant proteins mCherry-α and 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 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 bright-field micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry;
F fluorescence micrograph of S. cerevisiae INVSc1 cells harbouring pYCα–mCherry

Recombinant S. cerevisiae EBY100 strain harboring the pYD1–1β plasmid was precultivated to mid-log growth phase and then induced for 24 h at 20℃. During the inducing period, cells equaling to 2 OD600 units were collected every two hours from 8 h to 24 h. To detect the displayed protein, immunofluorescence microscopy was performed, with mouse IgG against βI tubulin and donkey anti-mouse IgG conjugated with Cy3 as primary and second antibody respectively. Results showed that optimal detection of β-tubulin occurred at 12 h.

Figure 3 Induced 12h in SG-CAA medium；
A,B recipient strain with empty plasmid;
C bright-field micrograph of S. cerevisiae EBY100 cells harbouring pYD1–1β;
D immunofluorescence micrograph of S. cerevisiae EBY100 cells harbouring pYD1–1β

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

Figure 4 An obvious color of mCherry produced by our engineered yeast　with vector pYCα-mCherry α tubulin

Figure 5 The partly results of a Western blot analysis carried out with an anti-V5 antibody
A the purified supernatant of mα-engineered yeast culture, induced for 12 hours in SG-Ura.
B

Our engineered yeasts, containing pYD1-β tubulin vector, have been proved to have successfully expressed exogenous βtubulin by Western blot analysis.

Figure 5 The partly results of a Western blot analysis carried out with an anti-V5 antibody

From above results, we can validate our parts function and concluded that our proteins were all correctly

Flagellar Filament

Plasmid construction

We have successfully constructed the following 11 parts that have been described in detail in the 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. And we also constructed pYD1-FilC(eGFP) (BBa_K2220003) as our positive control. The length and sequence of each parts have been validated by sequencing. The length validation are presented on the part registry page.

In secretory module,we successfully constructed the following parts: pYCα-FliC-XynA (BBa_K2220011), 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 lengths and sequences of each part has been validated by sequencing. The length validations are presented on the part registry page.

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) and 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