In a gene cloning project, the goal is to produce a recombinant plasmid DNA that

Question

In a gene cloning project, the goal is to produce a recombinant plasmid DNA that harbors the red segment of the genomic DNA specified in the following figure. The genomic DNA is ~ 1600 bp, with multiple BamHI and EcoRI restriction sites.

Digesting this DNA with BamHI enzyme results in multiple cut fragments of 300 bp, 750 bp, 75 bp, and other fragments of unknown sizes at both tails. The plasmid DNA, on the other hand, is 2600 bp long and contains only one BamHI restriction site in the Tetracycline Resistant gene (TetR), such that insertion of any forign DNA in this site results in “insertional inactivation” of TetR.

In a test tube we digest the genomic DNA and plasmid DNA with restriction enzyme BamHI and then use the enzyme ligase with the hope to bring the red segment (750 bp) into the BamHI cut site of the plasmid DNA. Then, frozen E. coli will be heat shocked in the presence of this plasmid such that the bacteria is transformed with plasmid DNA which we hope contains the red-insert segment. We then plate the bacteria solution on to three types of plates A) without any antibiotic, B) with both ampicillin and tetracycline antibiotic, and C) with ampicillin only.

Assuming that a normal (untransformed) E. coli is not resistant to any antibiotic, what will happen if a ligation product that does not have any insert in it enters the bacteria through heat shock transformation? Will the transformed bacteria grow in the presence of ampicillin only, tetracyclin only, both antibiotics, or neither antibiotic?

Ampicillin only

Tetracycline only

Both ampicillin and tetracycline

Neither ampicillin or tetracycline

Explanation / Answer

If a foreign DNA ligates at the BamHI site of tetracyclin resistant gene in the vector, the recombinant plasmid loses the tetracyclin resistance due to insertion of foreign DNA.

The recombinants can grow in ampicillin containing medium but not on that containing tetracyclin whereas non recombinants can grow on the medium containing both the antibiotics.

So, I think the transformed bacteria will grow only in the medium containing ampicillin.

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