Question

Insertion of a foreign DNA at BamHI site in an E. coli cloning vector pBR322 results in the loss of antibiotic resistance towards:

• A. Ampicillin and tetracycline
• B. Tetracycline
• C. Ampicillin
• D. Gentamycin

Hint:

Logic Tip: Memorize the restriction sites for pBR322: {Bam}HI and {Sal}I sit inside the $tet^R$ gene, while {Pst}I and {Pvu}I sit inside the $amp^R$ gene. Slicing into a gene always destroys its function!

Answer 1

The Correct Option is B

Step 1 : Understanding the Question:
The question is about the principle of insertional inactivation in biotechnology. We need to determine how the antibiotic resistance of the pBR322 vector changes when a foreign gene is inserted at the BamHI restriction site.
Step 2 : Key Formulas and Approach:
The pBR322 vector contains two main selectable markers:
1. $amp^R$ (ampicillin resistance gene).
2. $tet^R$ (tetracycline resistance gene).
Restriction sites for specific enzymes are located within these genes. If an enzyme cuts within a gene and foreign DNA is inserted there, the gene is disrupted and its function is lost.
Step 3 : Detailed Explanation:

pBR322 Map: In the pBR322 plasmid, the BamHI and SalI recognition sites are specifically located within the coding sequence of the tetracycline resistance ($tet^R$) gene.

Insertional Inactivation: When foreign DNA is ligated into the vector at the BamHI site, the $tet^R$ gene becomes split or "interrupted." This disruption means the gene can no longer produce the protein required to provide resistance to tetracycline.

Phenotypic Change: Consequently, the bacteria carrying this recombinant plasmid will grow on medium containing ampicillin but will lose the ability to grow on medium containing tetracycline.

Ampicillin Status: The ampicillin resistance gene ($amp^R$) contains sites for PstI and PvuI. Since these were not used for the insertion, the ampicillin resistance remains completely intact.

Step 4 : Final Answer:
Insertion at the BamHI site specifically inactivates the gene for tetracycline resistance. Therefore, the recombinant colonies will show a loss of resistance towards tetracycline, as stated in option (B).