The bacterial one-hybrid assay holds E. coli cells hostage until our protein of
ID: 280717 • Letter: T
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? See Cell Biology By The Numbers and Kinetics of His3 Inhibition by 3-AT on Canvas for relevant numbers.
Report you strategy by first listing all variables/ratios/numbers (with units) to be used and informally recognizing sources (copy-paste a link or “Table 4-1 in textbook” or “random number generator”). State all assumptions. For example, when estimating the amount of ATP used to do the world record bench press, we assumed that 100% of the energy from ATP hydrolysis went into the lift. It may have been more appropriate to assume perhaps 50% (intuitive guess).
Group variables used for unit conversions together and state starting and ending values. (Example: “Variables in List 1 were used to convert 3,600 Joules to 44 g of ATP”)
For equations, list forms from textbook and state how they were manipulated but don’t show steps. Use Insert Equation. Example: Solved y = mx + b for x to get x = (y-b) / m
Explanation / Answer
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
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
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