Conjugation with E. coli WM3064

Strain WM3064

Genotype: thrB1004 pro thi rpsL hsdS lacZΔM15 RP4-1360 Δ(araBAD)567 ΔdapA1341::[erm pir]

Purpose Is auxotrophic for DAP (diaminopimelic acid - a lysine precursor). The auxotrophy enables removal of this strain from a bi-parental mating setup after conjugation. This strain can be used for conjugation experiments and replication of plasmids that require Pir protein.

Strain was derived from E. coli B2155 by William Metcalf at UIUC.

Protocol

Preparation:

• E. coli WM3064 carrying plasmid of interest e.g. pUFR80, pST-Blue, or pKMW3 derivatives
• Media:
  • LB + 300 uM DAP + antibiotic
  • CPG + 300 uM DAP
  • CPG+DAP-Glc Modified CPG plates: (no glucose or TZC, + 300 uM DAP) 4 plates per transformation
  • CPG +TZC + antibiotic plates
• Cut 80 mm nitrocellulose filters (0.45 um pore size) into 6ths and autoclave. Overlay filters on CPG+DAP-Glc plates. If doing multiple replicates of the same donor/recipient, you can add up to 4 filters/plate. Keep different strains on separate plates to avoid cross-contamination–sometimes liquid droplets bounce/spray.

Methods

1. Day 0: Streak Ralstonia strain on CPG plate. Incubate at 28C for 2 days
2. Day 1: Optional Streak out E. coli WM3064+plasmid onto LB+DAP+antibiotic plate. Incubate at 37C overnight.
3. Day 2: Inoculate a single colony of Ralstonia into 5 ml CPG broth. Incubate overnight 28C with 180 rpm shaking.
   • Optional - Start overnight culture of E. coli WM3064+plasmid in 3 ml LB+DAP+antibiotic broth.
4. Day 3: Grow cells to mid-log before initiating transformation.
   1. E. coli: Either subculture 500 ul of the overnight culture into 4.5 ml LB+DAP+antibiotic in XXX mm test tube or thaw 1 ml -80C aliquot on ice before subculturing it into 25 ml pre-warmed LB+DAP+antibiotic in 125 ml flask. Grow at 37C with shaking until mid-log (OD 0.5-0.8).
   2. Ralstonia: Dilute 2 ml of overnight culture into 25 ml pre-warmed CPG broth in 125 ml flask. Incubate at 28C with shaking until E. coli is ready. Record OD of Ralstonia isolate in notebook.
5. Pellet cultures (8k xg for 5 min). Resuspend in 1 ml CPG+DAP. Transfer to 1.5 ml tube and pellet (>13k xg for 1 min). Repeat wash with additional 1 ml CPG+DAP.
   Resupend in 1 ml CPG+DAP.
6. Measure OD of 1:10 or 1:100 dilution of cell suspensions. Use bacterial_density_workbook to adjust Ralstonia density to OD = 3 and E. coli density to OD = 1.
7. Mix even volumes of Ralstonia and E. coli (creating a 3:1 ratio of recipient:donor strain).
8. Plate 100 ul of the suspension on the center of a nitrocellulose filter on CPG+DAP-Glc plate. Allow spot to dry on filter (incubate without lid in Biosafety Cabinet or laminar flow hood to accelerate drying)
9. Incubate at 28 C for 2 - 4 h.
10. Select transconjugants.
11. Transfer filter with conjugation spot to 2.0 ml tube with 1 ml CPG.
12. Vortex 10-30 s to dislodge bacteria.
13. Plate 100 ul of a dilution series: 10⁰, 10^-1, 10^-2, 10^-3, and 10¹(the remaining cells resuspended in 100 ul).
14. Incubate plates at 28 C for 2 days (up to 4 days). As soon as colonies appear, restreak for purity. Follow up with colony pcr to genotype.

Note about transformation efficiency: Transformation with pKMW3 derivative in 2018-11 yielded efficiencies (# transformants = # colonies on antibiotci plate / total # cells = # colonies on CPG plate) of 3.9x10^-5 to 8.6x10^-3.