If cloning of a 0.8 kb DNA segment was performed at the PstI site of plasmid...

If cloning of a 0.8 kb DNA segment was performed at the PstI site of plasmid pBR322 and the E. coli strain HB-101 was transformed with the construct:
a) What is the selection criteria for E. Coli transformants with the recombinant plasmid ?:
-Ecoli growth in nutrient-rich culture medium____
-Ecoli growth in culture medium rich in nutrients plus ampicillim_____
- Growth of E. coli in a culture medium rich in nutrients plus tetracycline_____
-Ecoli growth in culture medium rich in nutrients plus ampicillim and no growth in tetracycline_____
-Ecoli growth in culture medium rich in nutrients plus tetracycline and no growth in ampicillim_____
b) How would it be verified that the selected transformant has the recombinant molecule, if it is known that the cloned DNA segment does not have restriction sites with PstI?

Expert Solution

pBR322 is a well-characterized plasmid of 4.36 kb and has antibiotic resistance genes for both Ampicillin and tetracycline resistance. Ampicillin resistance coding region has the recognition sites for Pst I and the tetracycline resistance coding region has the sites for cleavage by EcoR V, BamH I, and Sal I. So, if a gene is cloned into the plasmid using Pst I, the ampicillin resistance gene would be rendered inactive, so the transformed E. coli HB-101cells would not have ampicillin resistance but would have tetracycline resistance. So, the transformed cells would be able to grow on plates having tetracycline alone but would not grow in the presence of ampicillin.

b) Verification for the insert could be done by first isolating the plasmid from the transformed cells, and then digesting the isolated plasmid with PstI enzyme. since both ends of the insert are complementary to PstI cleaved ends of the vector, there would now be two PstI sites on the plasmid containing the insert, one at each end of the insert. So, this digestion with PstI would give two fragments, one of 4.36 kb and another of 0.8 kb, when the digest is run on an agarose gel along with a DNA ladder.

gladiator answered 2 years ago

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