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.

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.

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.

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.

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.

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.

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.

Conjugation: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

0 views12:52 min

1. Set-Up

Autoclave approximately 1L of Luria-Bertani medium (LB). This sterile LB will be used to make approximately 5 mL of LB containing 0.3 mM diaminopimelic acid (DAP).
Collect the following plates: LB agar plates with 1X Tet and 0.3 mM DAP, LB agar plates with 1X Tet only, and LB agar plates with 1X Amp/Tet only.
Ensure that some glycerol and a box of pre-sterilized plastic pipette tips are close at hand.
Prior to starting any work involving microbes, sterilize the workspace with 70% ethanol. Always wear the necessary personal protective equipment, including a lab coat and gloves.
Once finished, sterilize all surfaces and gloves with 70% ethanol and wash hands.

2. Donor and Recipient Strain Preparation

Prepare 5 mL bacterial cultures of the donor and recipient strains and grow them overnight at 37 °C with aeration and shaking at 220 rpm. The donor strain should be grown in LB with 0.3 mM DAP.
Spin down 1 mL of both cultures (~3000 rpm for 5 minutes) and wash the cells with PBS.
Resuspend the cells in 500 µL of phosphate buffered saline (PBS).

3. Conjugation

Combine 50 µL of recipient cells with 50 µL of donor cells in a microcentrifuge tube and mix by pipetting.
Incubate the cell mixture for 1 hour at 37 °C. This will enhance the efficiency of conjugation before and during the next plating step.
Pipet 100 µL of the cell mixture onto an agar plate with 1X Tet and 0.3 mM DAP. Do not spread on the plate.
Pipet 100 µL of recipient cell culture onto an agar plate with 1X Tet and 0.3 mM DAP. Do not spread on the plate.
Incubate both plates overnight at 37 °C.
Scrape up the conjugation cell mixture and the recipient cell culture with a sterile cell scraper. Transfer the cells to sterile microcentrifuge tube and resuspend the cells in 1 mL of PBS.
Vortex the cells and gently spin them down (~3000 rpm for 5 minutes).
Resuspend the cells in 1 mL of PBS.
Plate the conjugation reaction cell mixture on an LB agar plate with 1X Amp/Tet only. On this plate, only the recipient bacteria that successfully gained the Amp resistance gene via conjugation are expected to grow.
Plate the recipient cells onto an LB agar plate with 1X Tet only. On this plate, only unconjugated recipient bacteria, which do not have the Amp resistance gene, are expected to grow.
Incubate the plates overnight at 37 °C.
Pick single colonies from both plates and use them to grow overnight cultures in 5 mL of media (37 °C with aeration at 220 rpm).

4. DNA Isolation

Isolate DNA from the previously prepared cultures using 4.5 mL of the total culture volume by DNA miniprep.
To do this, elute the DNA using 35 µL of nuclease-free water.
Pure DNA will generate an absorbance ratio (A_260/280) of approximately 1.8.
Use the remaining 0.5 mL of each culture to prepare glycerol stocks by making a 1:1 mixture of bacterial culture and 100% glycerol.
Store the freezer stocks at -80 °C.

5. Confirmation of Plasmid Transfer Conjugation by PCR

Prepare two PCR master mixes, each with a different set of forward and reverse primers, one targeting a 500 base-pair segment within the ampicillin resistance gene and the other targeting a segment within a housekeeping gene.
1. Housekeeping gene primers were designed to amplify a segment of DNA within the bacterial gene encoding for DNA gyrase B (14).
2. The following reagent volumes were used to prepare 90 μL of each master mix:
  7.5 μL of 10 μM forward primer
  7.5 μL of 10 μM reverse primer
  75 μL of 2X PCR Master Mix
Prepare the following six PCR reactions using 15 μL master mix, 10 ng of template DNA, and nuclease-free water up to a final volume of 25 μL.
Conjugation reaction DNA and ampicillin primers
Conjugation reaction DNA and housekeeping primers
Negative control DNA and ampicillin primers
Negative control DNA and housekeeping primers
No DNA and ampicillin primers
No DNA and housekeeping primers
Transfer these reactions to a PCR machine with the block preheated to 98 C and begin thermocycling under the following conditions:
98 °C for 30 seconds
25-35 cycles of 98 °C for 5-10 seconds, 45-72 °C for 10 to 30 seconds, and 72 °C for 15-30 seconds per kb
72 °C for 5-10 minutes
Hold at 4 °C
Load all six PCR reactions onto a 1% agarose gel and run at ~150V for about 20 minutes.
Visualize the PC product using a UV illuminator.

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Source: Alexander S. Gold¹, Tonya M. Colpitts¹
¹ Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseas…