Part:BBa_K2220020:Experience

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Applications of BBa_K2220020

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_K2220020 expression vector.

We successfully transfected the correct carrier into S.cerevisiae EBY100 and induced expression.

Fig.2 The Western blot analysis carried out with an anti-V5 antibody.

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.

Fig.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–β tubulin;
D immunofluorescence micrograph of S. cerevisiae EBY100 cells harbouring pYD1–β tubulin.

We did the polymerization reaction with tubulins extracted from the brain tissue of Sus scrofa domesticus and yeasts displaying β-tubulin. We did microscopic exam with HRTEM. Polymerized microtubules were observed on the cell wall of the yeasts, of which the quantity and length were consistent with our model prediction. The followings are the results.

Fig.4 Microscopic images of polymerized microtubules on yeast cell wall. A Polymerization with tubulins extracted from the brain tissue of Sus scrofa domesticus and yeasts displaying β-tubulin. Considerable numbers of microtubule were displayed on the yeast cell wall; B Control: Yeasts displaying β-tubulin without free tubulins. There were no observable microtubules on the yeast cell wall.

Fig.5 Enlarged view of microtubules on the yeast surface in Fig. 11A The red arrows indicate the microtubules.

Fig.6 More enlarged view of microtubules on the yeast surface in Fig. 11A The red arrows indicate the microtubules.

Fig.7 Microscopic image of a free microtubule, which is obviously longer than those polymerized on yeast surface.

Here are the protocols we used.

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