Question

1. Two CRISPR experiments (A and B) have been conducted in yeast cells to understand the effect of inducible promoters. The yeast cells used in both experiments contain 2 plasmids: one plasmid that has the Cas9 gene and one plasmid that has the CAN1.Y gRNA sequence. However, the two experiments differ in the promoter used in one of the plasmid.

The graph below shows the colony count from the canavanine selection plates (YNB + met + his + canavanine) that use either glucose or galactose as a carbon source.

Which experiment uses a galactose-inducible promoter, justify your answer? (1pt)

Which plasmid contains the galactose-inducible promoter, justify your answer? (2pt)

colonies HA YNB YNB GALACTOSE GLUCOSE

Answer 1

colonies HA YNB YNB GALACTOSE GLUCOSE

From the above graph, it can e interpreted that the number of colonies in the case of experiment B has remained the same, whereas the number of colonies has reduced drastically in the case of experiment A when galactose is absent in the medium. This clearly shows that experiment A uses a galactose inducible promoter, possibly Gal1, which is induced by galactose and repressed by glucose in the medium.

DiCarlo et al. in their 2013 publication on using CRISPR-Cas9 in yeast cells have used the GAL1 promoter in the plasmid with Cas9 gene in order to reduce or prevent the toxicity associated with the cas9 protein. Therefore, the plasmid with the cas9 gene contains the galactose inducible promoter.