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

1. Set-Up

1. 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).
2. 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.
3. Ensure that some glycerol and a box of pre-sterilized plastic pipette tips are close at hand.
4. 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.
5. Once finished, sterilize all surfaces and gloves with 70% ethanol and wash hands.

2. Donor and Recipient Strain Preparation

1. 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.
2. Spin down 1 mL of both cultures (~3000 rpm for 5 minutes) and wash the cells with PBS.
3. Resuspend the cells in 500 µL of phosphate buffered saline (PBS).

3. Conjugation

1. Combine 50 µL of recipient cells with 50 µL of donor cells in a microcentrifuge tube and mix by pipetting.
2. Incubate the cell mixture for 1 hour at 37 °C. This will enhance the efficiency of conjugation before and during the next plating step.
3. 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.
4. 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.
5. Incubate both plates overnight at 37 °C.
6. 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.
7. Vortex the cells and gently spin them down (~3000 rpm for 5 minutes).
8. Resuspend the cells in 1 mL of PBS.
9. 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.
10. 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.
11. Incubate the plates overnight at 37 °C.
12. 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

1. Isolate DNA from the previously prepared cultures using 4.5 mL of the total culture volume by DNA miniprep.
2. To do this, elute the DNA using 35 µL of nuclease-free water.
3. Pure DNA will generate an absorbance ratio (A_260/280) of approximately 1.8.
4. 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.
5. Store the freezer stocks at -80 °C.

5. Confirmation of Plasmid Transfer Conjugation by PCR

1. 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
2. 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
3. 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
4. Load all six PCR reactions onto a 1% agarose gel and run at ~150V for about 20 minutes.
5. Visualize the PC product using a UV illuminator.