박테리아 형질변환: 전기 천공법
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Q1: What is a plasmid and why is it used in bacterial transformation?
A plasmid is a small, circular, extrachromosomal piece of DNA that acts as a vector to carry your specific DNA sequence into bacterial cells. It contains a multiple cloning site where restriction enzymes insert your target gene, an origin of replication for DNA copying, and an antibiotic resistance gene that allows transformed bacteria to survive on antibiotic-containing media.
Q2: How does electroporation create pores in bacterial cell membranes?
Electroporation applies a high-voltage electric field of 1,000 to 10,000 volts per centimeter for milliseconds, causing the voltage across the membrane to reach 0.5-1 volts. This rearranges the phospholipid bilayer, creating temporary pores through which plasmid DNA passes. Once pulsing stops, the bilayer repairs itself, trapping the DNA inside the cell.
Q3: What are electrocompetent cells and how are they prepared?
Electrocompetent cells are bacteria prepared to readily take up DNA during electroporation. E. coli cells are grown to mid-log phase, then washed multiple times in deionized water and 10% glycerol solution to remove salt. Salt removal is critical because high salt content causes arcing, an electrical discharge that kills cells and indicates transformation failure.
Q4: Why is the time constant important in predicting electroporation success?
The time constant measures how quickly voltage decays after the electric pulse is applied. Good time constants for bacteria range from 5-10 milliseconds. Rapid decay indicates high salt concentration, causing arcing and cell death. Monitoring the time constant helps predict whether your transformation will succeed before observing colony growth.
Q5: How does electroporation compare to heat shock transformation?
Heat shock transformation uses calcium chloride and heat exposure instead of electrical pulses, making it gentler on bacteria and cheaper than electroporation. However, heat shock produces lower transformation efficiencies, takes longer, and works only on bacteria, yeast, and plant protoplasts. Electroporation achieves higher efficiency and applies to mammalian cells through a process called transfection.
Q6: What steps follow electroporation to identify successfully transformed bacteria?
After pulsing, add recovery media to cells and incubate at 37°C for one hour with shaking. Plate cells on antibiotic-containing agar and incubate overnight at 37°C. Only bacteria that successfully took up the plasmid survive the antibiotic. Count resulting colonies and calculate transformation efficiency by dividing successful transformants by total DNA plated.
Q7: What are the applications of electroporation beyond bacterial transformation?
Electroporation applies to mammalian cells through transfection, using lower field strengths and higher time constants than bacterial cells. Scientists also use electroporation in whole organisms like developing chicken embryos by injecting plasmid DNA into brain tissue and applying an electric field. This allows observation of fluorescent protein expression and structural changes in developing neural tissue.