Question

The pBR322 cloning vector has genes coding for tetracycline and ampicillin resistance. A foreign DNA to be cloned is inserted into the tetracycline resistance gene and the recombinant plasmid is then transformed into E. coli cells. Which one of the following choices is the most likely outcome of this cloning reaction?

• A. The cells with the recombinant plasmid can grow in the presence of ampicillin but not tetracycline
• B. The cells with the recombinant plasmid can grow in the presence of both ampicillin and tetracycline
• C. The cells with the non-recombinant plasmid can grow in the presence of ampicillin but not tetracycline
• D. The cells with the non-recombinant plasmid can grow in the presence of tetracycline but not ampicillin

Hint:

Insertional Inactivation Rule:
Whichever marker gene you "insert" into is "inactivated" (destroyed).
Inserted in $tet^R$ $\rightarrow$ Tetracycline resistance is lost.
Inserted in $amp^R$ $\rightarrow$ Ampicillin resistance is lost.
The other marker gene remains perfectly intact and serves for primary selection.

Answer 1

The Correct Option is A

Step 1: Understanding the Question:
This question is based on recombinant DNA selection using the plasmid vector pBR322. We need to determine the growth phenotypes of host bacterial cells containing either recombinant or non-recombinant plasmids.

Step 2: Key Formula or Approach:
Recall the concept of insertional inactivation.
When a foreign DNA sequence is ligated into a restriction site located within the coding sequence of an antibiotic resistance gene, it disrupts the open reading frame of that gene.
As a result, the gene becomes non-functional, and the host cell loses resistance to that specific antibiotic.

Step 3: Detailed Explanation:

• The plasmid pBR322 naturally carries two selectable marker genes:
1. $\text{amp}^R$ (conferring resistance to ampicillin)
2. $\text{tet}^R$ (conferring resistance to tetracycline)

• During the cloning experiment, the foreign gene of interest is inserted into a restriction site situated inside the $\text{tet}^R$ gene.

• This insertion disrupts the $\text{tet}^R$ gene, leading to its inactivation. Consequently, the recombinant plasmid loses the ability to express a functional protein that protects against tetracycline.

• However, the $\text{amp}^R$ gene remains completely intact and functional because no insertion occurred within its sequence.

• Therefore, host E. coli cells transformed with the recombinant plasmid will:
- Survive and grow on media containing ampicillin.
- Die on media containing tetracycline.

• In contrast, non-recombinant plasmids (which did not take up the insert) retain both fully functional $\text{amp}^R$ and $\text{tet}^R$ genes, allowing their transformants to grow on both antibiotics.

Step 4: Final Answer:
Recombinant plasmid-containing cells are ampicillin-resistant but tetracycline-sensitive, matching option (A).