Question

A plasmid vector contains a multiple cloning site (MCS) within the lac-Z gene. If foreign DNA is inserted into the MCS, what happens when competent cells are transformed with this plasmid and allowed to grow on a nutrient medium plate with X-gal and IPTG?

• A. Transformed cells with recombinant plasmids will appear blue.
• B. Transformed cells with recombinant plasmids will appear white.
• C. All transformed cells will appear blue.
• D. Non-transformed cells will appear white.

Hint:

Remember the key idea of blue-white screening:

\textbf{Blue} = Bad (for the experimenter). The plasmid is non-recombinant.
\textbf{White} = Wanted. The plasmid is recombinant, containing your gene of interest.
The insert inactivates the "blue-making" gene.

Answer 1

The Correct Option is B

Step 1: Understanding the Concept:
This question describes blue-white screening, a common technique used in molecular cloning to identify bacteria that have taken up a recombinant plasmid (a plasmid containing an inserted piece of foreign DNA). The technique relies on the principle of insertional inactivation.
Step 2: Detailed Explanation:
Let's break down the components:

lac-Z gene: This gene codes for the enzyme \(\beta\)-galactosidase.
X-gal: A colorless artificial substrate for \(\beta\)-galactosidase. When cleaved by the enzyme, it produces a blue-colored product.
IPTG: An inducer of the lac operon, which ensures that the lac-Z gene is expressed.
Multiple Cloning Site (MCS): A short region containing many unique restriction sites, placed intentionally inside the lac-Z gene.
Now let's consider the two possible outcomes for transformed cells (cells that have taken up a plasmid):

Non-recombinant plasmid: The plasmid has re-ligated without an insert. The lac-Z gene is intact. The bacteria will produce functional \(\beta\)-galactosidase. This enzyme will cleave X-gal, and the bacterial colony will turn blue.
Recombinant plasmid: A piece of foreign DNA has been successfully inserted into the MCS. This insertion disrupts the lac-Z gene (insertional inactivation). The bacteria cannot produce functional \(\beta\)-galactosidase. As a result, X-gal is not cleaved, and the bacterial colony remains its natural color, which is white.
Non-transformed cells (cells that did not take up any plasmid) will not grow at all if the plasmid also contains an antibiotic resistance gene and the medium contains that antibiotic (which is standard practice). If they were to grow, they would be white as they lack the lac-Z gene.
Step 3: Final Answer:
Transformed cells containing the recombinant plasmid have an inactivated lac-Z gene and therefore will appear white on the X-gal/IPTG medium.