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Applications of BBa_K2365048

Submission by Team UCopenhagen 2019:

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).

As part of our studies we both integrated BAX into the chromosome of Saccharomyces cerevisiae and expressed it on an episomal plasmid. In both cases we found that increasing galactose concentrations resulted in reduction in growth.

Yeast transformation

Initially, our yeast was transformed with episomal plasmids containing BAX under a galactose inducible promoter. For our control strain we used an empty vector. Following transformation, each culture were split into two. One half was plated on a plate containing raffinose selection media with 0.0 % galactose, and the other half on raffinose selection media containing 1.0 % galactose. In both strains a second empty vector was introduced, placing both strains under Uracil (U) and Tryptophan (W) selection.

As can be seen on Figure 2, no colonies were visible on the plate containing galactose, suggesting that the BAX protein was expressed in such a high concentration that all cells died. For our control strain an equal amount of cells were seen both with and without galactose.

For more stable expression, we also interated pGAL1-BAX into the chromosome of S. cerevisiae, using U selection. Here, positive transformants were selected for by performing yeast colony PCR (Figure 1). This strain was used for all further experiments.

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.

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 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) and incubated for three days at 30 °C. 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.

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.

Moreover, a clear inverse correlation of CFU/ml and percentage of galactose in the media can be observed (Figure 4) when comparing the spots of the pGAL1-BAX strain at a dilution of 10^-1 at different galactose concentrations. 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.

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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