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.

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 signify cells that never get exposed to sunlight.”

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

In this cloning experiment, white colonies signify cells that are transformed or recombinant plasmids, while blue colonies signify non-recombinant plasmids. This is because the gene lac-Z is interrupted to insert the desired gene called antithrombin. The lac-Z gene is responsible in production of -galactosidase enzyme. The -galactosidase is involved in breakdown of lactose into glucose and galactose.

The white colonies are picked up for further analysis as these may contain the desired gene.

The color variation in the colonies cannot be seen in the absence of X-gal to agar solution. Thus, it becomes difficult to identify the transformed cells.

The absence of ampicillin in the medium has no role in development of different colored colonies. This is because the color of colonies is decided by the presence of X-gal. hence, the different colored colonies on agar plate may be white and blue.

IPTG is the mimic of natural compound allolactose, which triggers the transcription of lac operon.

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