To use plasmid pBR322 for cloning, it is common to use a strategy such as this:

Question

To use plasmid pBR322 for cloning, it is common to use a strategy such as this: a 2.35 kb fragment with SalI overhangs at each end was mixed with pBR322 that had been digested to completion with SalI, then DNA ligase was added. This mixture was used to transform E. coli cells with selection for ampicillin resistance. Plasmid pBR322 also contains a gene encoding a resistance protein for tetracycline. Why weren’t colonies initially selected for resistance to tetracycline? Why would a scientist subsequently test the ability of the ampicillin-resistant transformants to grow in the presence of tetracycline?

pBR322 Plasmid diagram:https://www.neb.com/products/n3033-pbr322-vector

Explanation / Answer

The tetracycline resistant gene has low copy number compared to ampicillin resistant gene. That is why ampicillin resistance is incorporated into the plasmid. If only tetracycline is present , and selected for antibiotic resistant gene in tetracycline medium only few colonies are selected because incorporation of tetracycline resistant gene is low. More over ampicillin resistant gene incorporation is high and more colonies have this resistant gene. Environmental stress also affects incorporation of tetracycline into colonies. Hence ampicillin resistant gene is included in addition with tetracycline resistant gene. Hence more transformants have ampicillin resistant gene compared to tetracycline.

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