Question 1

How does a bacteriophage transfer DNA between bacterial cells?

Accepted Answer

A bacteriophage attaches to a bacterial cell surface and injects its DNA, then replicates its genome while hijacking the host's machinery. Due to low-fidelity DNA packaging, the phage occasionally packages bacterial DNA fragments into its capsid. When this phage infects another cell, it transfers the bacterial DNA, which can recombine and integrate into the new host's chromosome, mediating gene transfer between bacteria.

Question 2

What are the key requirements for performing phage transduction in the laboratory?

Accepted Answer

Phage transduction requires a donor strain containing the gene of interest, a recipient strain lacking it, a phage capable of infecting both strains, and a selective growth media that supports transduced bacteria while inhibiting non-transduced ones. The selective media is essential for screening and identifying which bacterial cells successfully received the transferred genetic material.

Question 3

Why is chloroform added to the phage lysate during preparation?

Accepted Answer

Chloroform is added to the phage lysate to inhibit bacterial growth and preserve the phage particles. After centrifugation removes debris, the supernatant containing phage is treated with chloroform and stored at four degrees Celsius for no more than one day, ensuring the phage remains viable for the transduction procedure.

Question 4

What role do magnesium sulfate and calcium chloride play in transduction?

Accepted Answer

Magnesium sulfate and calcium chloride are added to the recipient strain suspension to enhance phage attachment and DNA transfer efficiency. These divalent cations stabilize the phage-bacterial cell interaction, increasing the likelihood of successful transduction by promoting optimal conditions for DNA uptake by recipient cells.

Question 5

How does qPCR confirm successful transduction of the ampicillin resistance gene?

Accepted Answer

qPCR measures cycle quantification (Cq) values for the ampicillin resistance gene and a housekeeping gene. Low Cq values below 29 cycles indicate high target sequence amounts in transduced samples. Delta Cq values normalize results against the housekeeping gene, and normalized transduction efficiency is calculated to confirm the gene transfer occurred in recipient cells.

Question 6

What is the purpose of the negative control in phage transduction experiments?

Accepted Answer

The negative control combines recipient strain with LB medium containing magnesium sulfate and calcium chloride, but without phage lysate. This control undergoes identical steps as the transduction reaction, allowing researchers to verify that any ampicillin resistance detected in transduced samples resulted from phage-mediated gene transfer, not contamination or spontaneous resistance.

Question 7

Why is sodium citrate used during the transduction procedure?

Accepted Answer

Sodium citrate is added after the one-hour incubation period to chelate divalent cations and halt phage-mediated DNA transfer. This stops the transduction reaction at a defined time point, preventing continued gene transfer and allowing researchers to accurately assess the efficiency of the transduction event that occurred during the incubation window.

Transdução fágica: um método para transferir a resistência à ampicilina da E. coli doadora para a receptora - Questionário de avaliação