You are cloning bovine growth hormone BGH in the lab using a

You are cloning bovine growth hormone (BGH) in the lab using a plasmid, pJET, that contains an ampicillin resistance marker and also a lacZ gene interrupted by a multiple cloning site. You transformed the rDNA into competent E. coli cells by electroporation, then plated transformed E. coli on agar plates containing ampicillin and X-Gal and waited one day until colonies are visible. You then use blue/white screening to help you identify clones that carry BGH. You observe numerous blue and white colonies on the agar plate.

Answer the following questions in complete sentences and in paragraph format. Do not list the questions and answers separately. Instead combine the question content with your answer. For question 1, for example, you write, “In this cloning experiment, the white colonies represent bacterial cells that never get exposed to sunlight.”

1.What do blue colonies signify? Briefly explain your answer.

2.What do white colonies signify? Briefly explain your answer.

3.Which colonies (blue, white, or both) would you want to pick for further analysis to check for the successful cloning of the BGH gene? Briefly explain your answer.

4.If you forgot to add X-gal to the agar selection medium, how would the colonies that grow differ phenotypically from the ones that grow in plates with X-Gal? Briefly explain your answer.

5.If you forgot to add ampicillin to the agar selection medium, what other colonies would grow that won’t normally grow in plates with ampicillin? What color would those other colonies most likely be? Briefly explain your answer.

6.Research and describe the role of the compound, IPTG, in blue-white screening. What naturally occurring compound does it mimic, or replace?

Solution

1.Answer : The transformed cell which was plated on x - gal agar will yield blue colonies.These colonies grow because they contain plasmid .These plasmid are resistant to antibiotics in plate .The colonies are blue color because plasmid encodes alpha peptide of beta galactosidase.This peptide together with large fragments of enzyme encoded on bacterial genome form enzyme beta galactose which is responsible for blue color.

2Answer. Ampicillin resistant cell are incubated for long time grow white because enzyme beta lactamase that make cell resistant to ampicillin has diffused into agar broken ampicillin around main colony..if you transfer a small white colony to fresh amp plate it not grow and they are white.

3.Cells transformed with vector containing recombinant DNA will produce white colonies.

4.Answer: in absence of x gal colonies will not be blue

5.Answer: In absence of ampicillin there is