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. Bacteria in blue colonies are those who do not have recombinant DNA. Those bacteria have Xgal enzyme because during insertion the enzyme producer, lacZa gene, did not remove from multiple cloning site.

2. White colonies are those who have recombinant DNA. Those bacteria do not contain Xgal enzyme because during insertion the enzyme producer, lacZa gene, was successfully remove from multiple cloning site.

3. We will choose white colonies for further analysis because those contain the recombinant DNA.

4. If we forgot to add Xgal, all colonies will appear same in the medium, in the white color. That is because no bacteria can change the Xgal into blue color product even they have Xgal enzyme.