Part:BBa_K2365048:Experience
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Applications of BBa_K2365048
Submission by Team UCopenhagen 2019:
Characterization of BAX: Genomic integration vs. plasmid expression
We further characterized the Bax protein under the expression of an inducible promoter, pGAL1 (BBa_K3190050). We have characterized the apoptotic potential of Bax protein, when the BAX gene is integrated into the yeast genome, as compared to when it is expressed on a high copy plasmid.
Using USER ligation, we assembled the BAX gene with pGAL1 on a plasmid backbone compatible with multiplex integration cassette. The backbone used contains a URA selection marker, and will integrate the construct in the yeast genome at chromosome 11, site 2.
Furthermore, we also transformed yeast with a dual plasmid system. Using USER ligation, we assembled pGAL1 and BAX on a high copy plasmid backbone (200 copies/cell) containing a URA selection marker.
Yeast transformation
After cloning and purifying the pGAL1-BAX constructs in E. coli, we confirmed the sequences of these, prior to transforming into S. cerevisiae.
For the genomically integrated strain, the positive transformants were confirmed by performing yeast colony PCR. We used 2 primers, one in the forward direction for the backbone and one in the reverse direction for the yeast chromosome 11. In the presence of our construct, we expect to see a band at around 1000 bp as, that is the size of the fragment between the two primer regions. In the absence of the constructs, we expect to see the bands at around 1500 bp, as this is the size of site 2 of chromosome 11.
[INSERT GEL IMAGE of yeast colony PCR]
Figure 1: Yeast colony PCR | The positive colony of yeast is confirmed by the expected band size of around 1000 bp.
For the plasmid expressing strain, we transformed the yeast with two plasmid: the GAL1-BAX cloned in a high copy plasmid (200 copies/cell) with a URA marker, and an empty vector plasmid with a TRP marker. After transforming the yeast, we grew the colonies on plates without URA and TRP, thus selecting for positive transformants. To further confirm the functionality of the GAL1 promoter, we also plated the transformed yeast on a plate with 1 % galactose. As expected, we did not see any growth on the galactose positive plate.
Figure 2: Transformant plates of dual plasmid transformed S. cerevisiae | Both transformed with pGAL1-BAX (URA marker) and an empty vector (TRP marker). A: plate with no galactose. B: plate with 1 % galactose.
Galactose induction assay
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