Question

Arrange the following steps involved in the transformation of bacteria in a sequence from initiation to end.
(A) Incubation of rDNA with bacterial cell on ice
(B) Treatment with divalent cations
(C) Heat shock treatment
(D) Selection on antibiotic containing agar plate
(E) Placed them again on ice
Choose the correct answer from the options given below:

• A. (A), (B), (D), (C), (E)
• B. (B), (A), (C), (E), (D)
• C. (B), (C), (D), (A), (E)
• D. (A), (C), (B), (D), (E)

Answer 1

The Correct Option is B

The transformation of bacteria using recombinant DNA (rDNA) involves several key steps, each critical for the successful uptake and expression of new genetic material. Here's a detailed breakdown of the correct sequence: 

1. Treatment with divalent cations (B): Initially, bacteria are treated with divalent cations like calcium or magnesium. This treatment increases the permeability of the bacterial cell membrane, making it more receptive to the acceptance of foreign DNA.
2. Incubation of rDNA with bacterial cell on ice (A): The rDNA is mixed with the treated bacterial cells and incubated on ice. This step allows the DNA to approach the bacterial cells in a less active environment, facilitating interaction.
3. Heat shock treatment (C): The mixture is then subjected to a heat shock, a rapid increase in temperature which temporarily opens up the pores in the bacterial membrane, allowing the rDNA to enter the cell.
4. Placed them again on ice (E): Post heat shock, the cells are placed back on ice to stabilize the membrane and enable the cells to recover, now housing the rDNA.
5. Selection on antibiotic containing agar plate (D): Finally, the transformed bacteria are spread onto agar plates containing an antibiotic. Only bacteria that have successfully incorporated the rDNA, which includes a resistance gene, will survive and grow in this medium, allowing for selection of transformed colonies.

Thus, the correct sequence is: (B), (A), (C), (E), (D).

Answer 2

Correct Sequence of Bacterial Transformation 

Bacterial transformation is a crucial technique in genetic engineering, allowing the introduction of foreign DNA into bacterial cells. The process involves several key steps to ensure the efficient uptake of genetic material.

1. Treatment with Divalent Cations

Bacteria are first treated with divalent cations such as calcium chloride (CaCl₂). This step:

• Neutralizes the negative charge on the bacterial cell membrane.
• Facilitates the binding of plasmid DNA to the cell surface.

2. Incubation on Ice

After cation treatment, the bacterial cells are incubated on ice for approximately 15–30 minutes. This:

• Stabilizes the cell membrane.
• Prepares cells for DNA uptake.

3. Heat Shock

The bacterial suspension is subjected to a heat shock (typically at \(42^\circ C\) for 30–60 seconds). This step:

• Creates a temperature gradient, inducing the uptake of plasmid DNA.
• Temporarily increases membrane permeability.

4. Recovery in Nutrient Medium

After heat shock, bacteria are transferred to a nutrient-rich medium (such as LB broth) and incubated at \(37^\circ C\) for about an hour. This allows:

• Cells to express antibiotic resistance genes (if present in the plasmid).
• Recovery from heat shock stress.

5. Selection on Antibiotic Plate

Finally, bacteria are plated on an antibiotic-containing agar plate. Only transformed cells containing the plasmid (with the antibiotic resistance gene) will survive, leading to:

• Positive selection of transformed bacteria.
• Formation of colonies that can be further studied.

Conclusion

The correct sequence of bacterial transformation involves:

1. Treating bacteria with divalent cations (e.g., CaCl₂).
2. Incubating on ice to stabilize the membrane.
3. Applying a heat shock to induce DNA uptake.
4. Recovering bacteria in a nutrient medium.
5. Selecting on an antibiotic plate to identify transformed cells.

This process ensures successful transformation, enabling genetic modifications in bacterial cells for research and biotechnology applications.