Eubacterium maltosivorans SA11 transformation protocol (BSI)

Based on protocol for E. limosum described in Sanford & Woolston (2022) [1], with some adjustments made based on results from initial electroporation tests which gave a transformation efficiency of approx. 6,000 CFU/µg in a best attempt.

Electrocompetent cell preparation

1. Grow six 10 ml SA11 cultures (or upscale as required) to early log phase (OD ~0.5)
2. Cool cultures on ice for 20 min.
3. Bring cultures into chamber on ice, pour 30 ml culture into two O-ring sealed Oak Ridge centrifuge tubes, ensure lids are sealed tightly and remove from anaerobic chamber, keeping on ice.
4. Centrifuge at 15,000 × g  at 4 °C for 10 min.
5. Bring tubes back into chamber and pour off culture supernatant and add 30 ml ice cold 270 mM sucrose buffer, gently resuspending with a ≥5 ml pipette tip.
6. Repeat buffer wash and centrifugation steps under the same conditions, but also including 20% (v/v) glycerol in the wash buffer.
7. Bring washed pellets back into chamber, decant supernatant and remove as much excess buffer as possible without disturbing pellet.
8. Add 300 µl 270 mM sucrose/20% glycerol buffer and gently resuspend as minimally as needed.
9. Dispense preferred volumes into chilled Kimax tubes ensuring lids are securely fastened before removing from chamber.  
10. Store aliquots at -80 °C for later use.

Electroporation

1. Thaw electrocompetent cell aliquots on ice, bring into anaerobic chamber with pre-chilled 2 mm electroporation cuvettes, CO₂ -filled Kimax tubes for recoveries, anaerobic M2GSC recovery medium, and pJIR750 plasmid DNA.
2. Add 50 µl electrocompetent cells and 100-200 ng of pJIR750 plasmid to an electroporation cuvette, flick to mix and chill on ice for 3 min.
3. Electroporate using the following settings: exponential decay, 2.5 kV, 400 Ω, 25 μF.
4. Pipette 950 µl of M2GSC medium into the cuvette, mix gently and pipette into Kimax tube.
5. Recover at 39 °C for six hours before plating 100 µl on M2GSC agar plates with 5 µg/ml thiamphenicol and incubate at 39 °C. Colonies usually form on the second day of incubation.

External Links

Sanford PA, Woolston BM. 2022. Expanding the genetic engineering toolbox for the metabolically flexible acetogen Eubacterium limosum. J Ind Microbiol Biotechnol 49: kuac019.