Hello.
I am currently working on transformation of Enterococcus faecalis to do some knockout experiments. Specifically, I want to remove a gene encoding an enzyme that is useful for probiotic application in order to confirm its function.
Is there any established protocol for heat-shock transformation of Enterococcus faecalis with high transformation efficiency? Currently, electroporation is not an option so I am limited to heat-shock transformation only.
I hope you can help. Thank you!
Electroporation following a cell wall weakening procedure is the only practical way to transform E. faecalis I am aware of.
Unfortunately chemical transformation (heat shock transformation) rarely works outside of E. coli and a few related organisms.
Heat-shock transformation is less common for Enterococcus faecalis compared to electroporation due to typically lower efficiency. However, you can still try optimizing a protocol based on general principles and related protocols for Gram-positive bacteria. Here are some steps you could consider:
General Protocol for Heat-Shock Transformation of Enterococcus faecalis
Tips for Optimization
- Calcium Concentration: Higher calcium concentrations can sometimes improve transformation efficiency.
- Incubation Times: Vary the duration of heat-shock and ice incubation to find optimal conditions.
- Competent Cell Preparation: The quality of competent cells significantly affects transformation efficiency. Ensure cells are in mid-log phase and handle them gently to avoid stress
Mo-Hyeon Kang
After some digging I found two papers which transformed Enterococcus faecalis without electroporation. I have no direct experience with either so I cannot speak to their actual efficacy, but they might work.
The older paper refers to E. faecalis as Streptococcus faecalis because all the Enterococcus species were formerly placed in the Streptococcus genus, but it is the same species.
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