結合:アンピシリン耐性をドナーからレシピエント大腸菌に移す方法
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Q1: What is the sex pilus and what role does it play in bacterial conjugation?
The sex pilus is an extracellular filamentous structure generated by the donor cell to establish direct cell-to-cell contact with the recipient cell. This physical contact is essential for conjugation to proceed. The sex pilus is encoded by the F (fertility) factor and serves as the initial bridge between donor and recipient cells before DNA transfer begins.
Q2: How does the Type IV secretion system facilitate DNA transfer during conjugation?
After the sex pilus establishes contact, a conduit called the Type IV secretion system is built between the two cells. This multiprotein complex allows DNA to be actively transported from the donor to the recipient. The system works with coupling proteins to pump replicated DNA through the channel, enabling the transfer of genetic material encoding traits like ampicillin resistance.
Q3: Why is selective plating used to confirm successful conjugation in this experiment?
Selective plating uses antibiotics to isolate only successfully conjugated cells. The donor strain requires diaminopimelic acid (DAP) to survive, while the recipient lacks ampicillin resistance. Plating on media with both tetracycline and ampicillin without DAP eliminates donor cells and non-conjugated recipients, leaving only recipient cells that acquired ampicillin resistance through conjugation.
Q4: What is the origin of transfer (OriT) and why is it important for conjugation?
The origin of transfer (OriT) is a genetic element on the plasmid that signals where DNA replication and transfer should begin. A relaxase enzyme cleaves at the nic site within the OriT sequence and covalently attaches to the DNA strand, initiating the formation of the relaxosome complex. This complex is essential for directing which DNA gets replicated and transferred to the recipient cell.
Q5: How does PCR confirm the successful transfer of ampicillin resistance genes?
PCR amplifies a specific DNA sequence from the transferred plasmid using primers designed for the ampicillin resistance gene. Successfully conjugated cells contain this gene and produce a visible band of known size on an agarose gel. A housekeeping gene serves as a positive control, while recipient-only DNA should show no amplification, confirming the resistance gene was successfully transferred.
Q6: How does conjugation differ from other bacterial DNA transfer mechanisms?
Conjugation requires direct physical contact between donor and recipient cells through the sex pilus, unlike transduction or transformation. It is unidirectional, with DNA flowing from donor to recipient, and naturally occurs in bacteria. This process has contributed to the spread of antibiotic resistance and can be manipulated in laboratory settings using selective pressures for horizontal gene transfer.
Q7: What happens to transferred DNA once it enters the recipient cell?
Once transferred, the DNA can either form an extrachromosomal plasmid or integrate into the recipient cell's chromosome. Regardless of its final location, the genes encoded on the transferred DNA are then expressed in the recipient cell. This gene expression produces the new phenotype, such as ampicillin resistance, which can be detected through selective plating or PCR analysis.