Dandekar & Chandler:Pseudomonas Mating

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Pseudomonas Mating Protocol

Master compilation of protocols from Sudha, Thao and Brook - Assembled by Moe T.

Before starting, make sure you have all the appropriate plates prepared:

  • LB
  • LB + antibiotic required for deletion construct (needed for multiple days)
  • Pseudomonas isolation agar (PIA) + antibiotic (needed for multiple days)
  • No Salt LB + 10% sucrose (needed for multiple days)

Pseudomonas requires higher concentrations of antibiotic than E. coli to maintain a plasmid; for gentamicin use 100 μg/mL

Day 1

Transform E. coli S17-1 with deletion construct(s) needed to make the knockout. **S17-1 has tra genes required for conjugation integrated on the chromosome; if your construct was made in DH5 alpha remember to move it into S17-1!**

Day 2

Streak S17-1 with construct to LB+ab for singles and leave at 37°C O/N

Day 3

Grow PAO1 in LB and S17-1 with construct in LB+ab tubes overnight at 37°C

Day 4

Dilute the S17-1 culture 1:100 in a tube with 5mL LB+ab and grow at 37°C to mid-log phase (OD600 ~0.3-0.6) with shaking

Dilute PAO1 into 50 mL flask 1:10 with 10mL LB and place at 42°C* with NO shaking to increase the frequency of recombination of foreign DNA *This is the small incubator by heat blocks in EPG lab

When S17-1 reaches OD ~0.3-0.6, gently mix cultures at 1:1 and 3:1 (donor: recipient) in 15mL conical tubes; spin at 2000 rpm at RT for 10 minutes

Remove supernatant and resuspend each pellet in 40 μL LB

Spot each culture mix onto LB agar plates with no antibiotic; incubate the plate O/N at 30°C* *This is the bottom incubator in EPG lab

Day 5

Scrape the cells from the spot and resuspend in 1 mL 1x PBS

Plate onto PIA+ab:

  • 100 μL of suspension directly
  • 100 μL of 1:10 dilution
  • 100 μL of 1:100 dilution
  • The remaining pellet of cells (This always makes a lawn for me, so I omit – Nicole)

Incubate O/N at 30°C

Day 6/7

Colonies may be small – either go another 24 hrs at 30°C or transfer to 37°C in the morning and colonies should be large enough by end of day

Patch colonies that appear on the PIA+ab plates again to PIA+ab to confirm single cross integration of plasmid onto PAO1 chromosome Incubate O/N at 30°C

Day 8

Patch colonies again to LB+ab to confirm single cross integration of plasmid backbone onto the chromosome

Day 9

Scrape up patches (at least 3-5) for each mating with an inoculating loop and resuspend each patch separately in 1ml 1x PBS

Plate onto No Salt LB+10% sucrose:

  • 100 μL of suspension directly
  • 100 μL of 1:10 dilution
  • 100 μL of 1:100 dilution
  • The remaining pellet of cells (NES omits)

Incubate O/N at 30°C

Hopefully plating onto sucrose induces the double crossover event:

Deletion constructs generally have antibiotic + sacB which makes growth on sucrose toxic
Plating on sucrose plates with no antibiotic will counterselect for those strains that have lost the plasmid backbone carrying the antibiotic resistance marker and the sacB gene
Expression of the sacB gene from Bacillus subtilis is toxic for gram-negative bacteria when grown in the presence of 5% sucrose, providing a direct selection for loss of the plasmid; the resulting sucR colonies are screened for the simultaneous loss of abR to ensure
that that the sucrose-resistant phenotype is actually due to loss of the integrated plasmid and not a mutation
If there is a lawn of colonies on your No Salt LB+ab you have likely picked up a mutation in the sacB gene and should start over :(

Day 10

Patch each colony again to No Salt LB+10% sucrose, LB+ab and LB with no ab to confirm loss of construct

The presence of colonies that are sucR and abS indicate that there was a double crossover event, meaning that the plasmid backbone, antibiotic resistance cassette and sacB gene are all lost. Tentative congratulations! You may have won the game!

Day 11 and beyond

PCR check the deletion mutant and include gDNA as (+) control

Use outside primers designed to amplify far upstream and downstream of the deleted gene; use inside primers as well to get clean sequences

Start O/N to freeze down glycerols of (+) strains

Send to sequence any construct that contains bands of the appropriate size