Question

The bacterial one-hybrid assay holds E. coli cells hostage until our protein of interest can active transcription and translation of enzymes from the HIS3 and URA3 genes. These enzymes are needed to produce Histidine and Uridine, which are both needed for cells to grow and divide. Make an estimate, based on enzyme kinetics and any other information you may need, of how many HIS3 and URA3 enzymes will an E. coli cell need to grow and divide at a normal rate. How many are needed when we add 10 mM 3-amino-1,2,4-triazole (3AT) to the growth medium as a competitive inhibitor of HIS3?

Answer 1

Transcription factor has two domains. One is for DNA binding and another for VP16 activation.

One protein is fused to the DNA-binding domain of LexA repressor to form bait.

Another protein is fused to the VP16 activation domain of LexA repressor to form prey.

If these two proteins interact with each other inside the yeast cell, the activation of Lac Z gene transcription will take place.

VP-16-adenylyl cyclase interacts with LexA binding domain-Ras to act as positive control

VP-16-adenylyl cyclase interacts with LexA binding domain-Lamin to act as negative control

The plasmid constructs with these modifications can be put into yeast cells consisting of His3 gene and lacZ gene

Growth Plate that lacks histidine allows the growth of cells with His3 gene. The plate that have blue colonies indicate that the cell consists of beta-galactosidase gene that can convert XGAL as substrate.

Table 1:

Plasmid constructs		Growth on plates lacking histidine	Growth on plates with XGAL
Bait	Prey
LexA-Ras		-	white
LexA-lamin		-	white
	VP16	-	white
	VP16-CYR	-	white
LexA-Ras	VP16	+	blue
LexA-Ras	VP16-CYR	+	blue
LexA-lamin	VP16	+	blue
LexA-lamin	VP16-CYR	-	white