You are cloning human antithrombin in the lab using a plasmid, pBlueScript, that

Question

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

1) What do white colonies signify? Briefly explain your answer.

2) What do blue 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 antithrombin 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 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 synthetic compound, IPTG, in blue-white screening. What naturally occurring compound does it mimic, or replace?

Explanation / Answer

1) Colonies that lack lacZ gene (lacZ-) appear white in color as these cannot metabolize the X-gal product added in the medium. The colonies appeared are ampicillin resistant and hence can grow even in presence of ampicillin.

2) The blue colonies specify that they encode for lacZ gene (lacZ+ ) and these colonies can metabolize X-Gal product and form blue colored colonies and also the colonies are ampicillin resistant.

3) Both white and blue colored colonies are screened for the trnasformation of antithrombin gene. This is because the colonies that encode or lack (lacZ+ or lacZ-) can uptake the antithrombin gene through electroporation and grow on the medium when plated.

4) If X-Gal is not added to medium, the colonies appear white but can grow in presence of ampicillin which is an antibiotic marker depending on which the cloned cells can be screened.

5) If ampicillin is not added, then ampicillin susceptible cells can grow along with cloned cells that can be identified by blue colored colonies which are able to metabolize the X - Gal and hence these colonies encode for lacZ gene.

6) IPTG is known as -D-1-thiogalactopyranoside, it mimics a compound called allolactose which is a lactose metabolite that induces the lac operon to metabolize lactose. Thus, IPTG an inducer of lac operon due to which the X-Gal product is metabolized by lacZ encoding cells and these appear blue in color. The cells unable to metabolize X - Gal appear white since they lack lacZ gene.

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