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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 chose to use this part, as it is Team NAU China 2017's improved version of the original part of Team Debrecen-Hungary 2010 (BBa_K364202).

We have characterized the apoptotic potential of Bax protein under the expression of pGAL1, when the BAX gene is integrated into the yeast genome, as compared to when it is expressed on a high copy plasmid. Furthermore, we have also characterized the neccesary concentration of inducer (galactose) needed in order for high enough expression of BAX to kill the cells.

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 900 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.

Figure 1: Yeast colony PCR of genomically integrated transformants | The positive colony of yeast is confirmed by the expected band size of around 800 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 of both plasmids. 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, as the GAL1 promoter was induced, expressing BAX, thus killing the yeast.

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

To analyse the effect of BAX on our yeast under different expression levels, we conducted a galactose induction assay using raff-U plates with five different galactose concentrations. Cultures of yeast containing pGAL1-BAX or empty vector were grown O/N and then diluted to an OD_600nm of 0.5. On plates with either 0%, 0.025%, 0.05%, 0.1% or 0.2% galactose, 10 µl of each culture were spotted in increasing dilutions (10^-1 to 10^-4; Figure 3 and 4). After three days of incubation at 30 °C, two observations were made. First off, the strain with the integrated pGAL1-BAX construct showed decreased growth compared to the control strain even when galactose was absent (Figure 3). This suggests that the galactose promoter is leaky and a low amount of BAX is produced at all times.

[Insert discussion of plates/results here]

Figure 3: Growth of pGAL1-BAX and the empty vector strain in the absence of galactose. The cultures were spotted in the dilutions 10^-1 to 10^-4 of an OD_600nm of 0.5 and incubated for three days at 30 °C.

Secondly, when comparing the spots of OV6 at a dilution of 10^-1 at different galactose concentrations, a clear inverse correlation of CFU/ml and percentage of galactose in the media can be observed (Figure 4). This suggests that successful induction of BAX leads to apoptosis in our yeast.

Figure 4: Growth comparison of pGAL1-BAX and control strain in the presence of varying galactose concentrations. | Shown are the CFUs at an OD_600nm of 0.05 after incubation at 30 °C for four days. The three yellow colonies seen at 0.2% galactose on the pGAL-BAX plate can be morphologically distinguished from the others, suggesting that they are contaminants.

As seen on figure 5, a clear reduction in growth was seen already at low galactose concentrations (0.05 %). In addition it appears BAX was expressed in sufficient concentrations to kill the yeast cells at a galactose concentrations of 0.3 %. Unfortunately our control strain was only plated on raff-U with 1 % galactose. However, no reduction in growth was seen at this concentration (compared to the empty vector grown in 0 % galactose media). . In addition to a reduction in the number of colonies formed between the two strains, there was also a clear difference on the size of the colonies. Only very small colonies were formed upon induction of BAX, this that the cells were killed before a normal sized colony could be formed. To test this, the pGAL1-BAX plate was incubated for an additional day at 30 °C and subsequently left at room temperature. No additional growth was observed. (Figure 5).

Quantitative galactose induction assay

To further analyze the effect of BAX on our yeast under different expression levels, we conducted a quantitative galactose induction assay. Here, an O/N culture of yeast containing pGAL1-BAX was diluted to an OD_600nm of 0.5. Subsequently, 100 µl of the culture in dilutions of 10^-3 and 10^-4 were spread on plates with 0%, 0.05%, 0.1%, 0.3% and 1% galactose, respectively. Each plate was made in duplicate. After incubation for three days at 30 °C, the CFU/ml were calculated and compared to the control (Figure 5).

[Discussion of below graph]

Figure 5: Gradient induction of BAX in yeast using the inducible GAL1 promoter. | If no errorbar is indicated, only one of the used duplets showed a quantifiable amount of colonies.

As seen on figure 6, a clear reduction in growth was seen already at low galactose concentrations (0.05 %). In addition it appears BAX was expressed in sufficient concentrations to kill the yeast cells at a galactose concentrations of 0.3 %. Unfortunately our control strain was only plated on raff-U with 1 % galactose. However, no reduction in growth was seen at this concentration (compared to pGAL1-BAX grown in 0 % galactose media). . In addition to a reduction in the number of colonies formed between the two strains, there was also a clear difference on the size of the colonies. Only very small colonies were formed upon induction of BAX, this that the cells were killed before a normal sized colony could be formed. To test this, the pGAL1-BAX plate was incubated for an additional day at 30 °C and subsequently left at room temperature. No additional growth was observed (Figure 6).

Figure 6: Comparison of colony sizes of pGAL1-BAX and the control containing the empty vector grown on raff-U agar with galactose. | Left: Colonies of the control strain (dilution 10^-3) in the presence of 1% galactose after incubation for three days at 30 °C. Right: Colonies of the pGAL1-BAX strain (dilution 10^-3) in the presence of 0.1% galactose after incubation for four days at 30 °C.

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