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

blue colony signify the nonrecombinant colony and white represent the recombinant colony having ligated insert. therefore white colony we need to pick for futher screening of the clone. X-gal is a chromogenic substrate added to the agar plate. If -galactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-indigo. The colonies formed by non-recombinant cells, therefore appear blue in color while the recombinant ones appear white. if X-gal is not there all colonies will be white. if we forget to add amplicillin then there will be no antibiotic selection means colony which having amplicillin resistance as well as non resistance one. iamplicillin addition we not alter the colour of the colony.IPTG is a non-metabolizable analog of galactose that induces the expression of lacZ gene. It should be noted that IPTG is not a substrate for -galactosidase but only an inducer.