박테리아 형질변환: 열 충격 방법
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Q1: What is a plasmid and why is it used in bacterial transformation?
A plasmid is a small, circular, double-stranded DNA molecule that can reduce its size through supercoiling, allowing it to pass through bacterial cell membrane pores. Plasmids contain a multiple cloning site for inserting DNA fragments, an origin of replication for DNA copying, and an antibiotic resistance gene that lets transformed cells survive in antibiotic-containing media.
Q2: How does calcium chloride make bacterial cells competent?
Calcium chloride creates a calcium-rich environment where positive calcium ions neutralize negative charges on both the plasmid DNA and bacterial cell wall. This neutralization reduces electrostatic repulsion and weakens the cell wall, preparing cells to take up foreign DNA during the heat shock procedure.
Q3: What happens during the heat shock step of bacterial transformation?
A sudden temperature increase to 42°C for 30 seconds creates a pressure difference between the cell's interior and exterior, inducing pore formation in the bacterial membrane. Supercoiled plasmid DNA enters through these pores. After heat shock, cells are immediately cooled on ice, allowing the cell wall to self-heal and stabilize the plasmid inside.
Q4: What is transformation efficiency and how is it calculated?
Transformation efficiency measures the success rate of the transformation procedure. It is calculated by dividing the number of successful transformants (colonies containing the plasmid) by the total amount of DNA plated. This metric helps researchers evaluate how effectively the heat shock method introduced plasmids into bacterial cells.
Q5: How are chemically competent cells prepared before heat shock transformation?
Bacteria are grown to mid-log phase, then centrifuged and washed multiple times with cold calcium chloride solution to remove growth media. After the final wash, cells are resuspended in calcium chloride plus 15% glycerol, distributed into microfuge tubes, and stored at -80°C until needed for transformation.
Q6: Why is aseptic technique important during bacterial transformation?
Aseptic technique maintains sterility throughout the transformation procedure by preventing airborne contaminants from entering the workspace. A Bunsen burner sterilizes instruments and reagents while creating a convection current that keeps contaminants away. This ensures that only the desired transformed bacteria grow on agar plates.
Q7: What happens after transformed bacteria are plated and what is the next step?
Plates are incubated overnight at 37°C upside down to prevent condensation exposure. The next day, colonies containing the plasmid are visible and can be counted to calculate transformation efficiency. Selected colonies can then be grown in antibiotic-containing liquid medium for plasmid purification isolation and purification of plasmid DNA for further experimentation.