Part:BBa_K2220021:Experience

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Applications of BBa_K2220021

We ligated this part to shuttle plasmid pYD1 and did the colony PCR to verify the efficiency(Fig.1). Meanwhile, the sequencing results further confirmed that we successfully cloned the expression vectors.

Fig.1 Electrophoresis result of BBa_K2220021 expression vector.

We successfully transfected the correct carrier into S.cerevisiae EBY100 and induced expression. The recombinant proteins were extracted and analysed by Western blot. The image shows the results of a Western blot analysis carried out with an anti-V5 antibody.

Fig.2 Western blot analysis of the supernatant from cell lysates of engineered yeasts mentioned above, carried out with an anti-V5 antibody.

Furthermore, it has been proven that our recombinant proteins can be secreted normally and worked as it expected. Firstly, the secretion function of part pYCα-mCherry have been proven by western blot analysis.

Fig.3 The results of Western blot analysis carried out with an anti-V5 antibody.
Lane A the purified supernatant of S.cerevisiae INVSc1 harboring pYCα-mCherry culture, induced for 12 hours in SG-Ura.
Lane B the supernatant from cell lysates of S.cerevisiae INVSc1 harboring pYCα-mCherry (without purified), induced for 12 hours in SG-Ura.

ig.4 Fluorescence image of mCherry expressed by the engineered yeast cell.

Here are the protocols we used.

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