Transferring pBE110 to S17-1

May 20, 2014
Created electrocompetent E. coli S17-1 cells using cold treatment. The goal is to transfer the pBE110 to this E. coli strain because it can more readily conjugated into Stenotrophomonas and other bacterial strains we are interested in studying. Right now, we only have the pBE110 in T1R E. coli cells (the chemically competent ones).


Making Electrocompetent Cells:

Note: Put sterile nuclease free water on ice to chill.

1. Streak culture onto appropriate media and incubate overnight2. Re-suspend one loop full of bacteria in 500 uL of ice-cold ddH₂O in 1.5 m centrifuge tubes (2-3 day old plates work best) 3. Spin to pellet (24,000 x g for 1 minute 4° C)4. Discard supernatant5. Re-suspend pellet in 500 uL of ice-cold ddH₂O6. Repeat steps 3 – 57. Repeat steps 3 - 48. Re-suspend pellet in 50 uL of ice-cold ddH₂O

Electroporation

Note: Put SOC media into 37C to warm it up before use in recovery after electroporation. Put cuvettes on ice to chill them. Ideally, pipets should be put in fridge to chill them as well.

      Controls : Added 1 uL of pUC19 DNA to 49 uL of competent S17-1 and put in a chilled 1 mm disposable electroproation cuvettes (Link). Added only 50 uL competent S17-1 cells to chilled cuvette.

     Experiment: Added 5 uL of pBE110 DNA to 45 uL of competent S17-1 cells. Transferred to chilled electroporation cuvette. Repeated this treatment group in triplicate.

1. Placed cuvettes into Bio-Rad MicroPulser Electroporator (Link) and ran per the manufacturer's instructions using the "Ec1" program at 1.8 kV for one pulse.

2. Immediately added 1 mL of warmed SOC media to each cuvette after electroporation

3. Incubated cuvettes at 37C static for 1 hour
       Note: put selective abx media in 37C to warm up media

4. Spread plated 200 uL of cuvette broth for each treatment onto appropriate antibiotic media. pUC19 will be plated onto LB Amp100. pBE110 experimental treatment will be plated onto LB Tet15 Tm200 (Tm = trimethoprim)
      Note: used 200 uL instead of the traditional 100 uL because I didn't have very high hopes of getting colony growth

5.  Incubated at 37C overnight.

May 21, 2014

Results:

Only had growth on the pUC19 growth plate (4780 CFU/mL). There was no colony growth on any of the pBE110 experiment treatment. The agar plates that were used for the experimental treatment were a month old, and there was some question about what antibiotics were actually on the agar. The markings on the plate contradicted the label on the bag.

Future:

Will make new agar for experimental treatment so I know that the antibiotics are correct. Then will repeat this experiment.