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Applications of BBa_K2365048

Submission by Team UCopenhagen 2019:

pGAL1-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

We confirmed the sequences of the pGAL1-BAX constructs cloned in ~~E. coli~~, 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 the GAL1-BAX cloned in a high copy plasmid (200 copies/cell) with a URA marker, as well

picked the positive E. coli colonies of the pGAL1-BAX cloned in a high copy plasmid, and purified the DNA from these. After confirming the sequence, we transformed the construct into S. cerevisiae. To select for positive transformants, we also transformed an empty vector with a TRP selection marker, and grew the colonies on plates without both URA and TRP. As a control, we spread the cells on plates both with and without galactose. No growth was detected in the galactose containing plate, as seen below:

[Insert plate image of yeast 10] Figure legend: Transformant plates of dual plasmid transformed S. cerevisiae, transformed with pGAL1-BAX and an empty vector. Right: plate with 1 % galactose. Left: plate with no galactose.

Galactose induction assay To compare

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 <b> Yeast transformation </b>
-We confirmed the sequences of the pGAL1-BAX constructs, both genomically integrated, and plasmid expressed prior to transforming into <i>S. cerevisiae</i>.
+We confirmed the sequences of the pGAL1-BAX constructs cloned in <s>E. coli</s>, prior to transforming into <i>S. cerevisiae</i>.
 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.