Lactobacillus transformation (Berthier 1996)

Overview

General guidelines for the electro-transformation of Lactobacillus sake as described by Berthier et al. and as used by Alegre et al. with Lactobacillus plantarum.

Materials

• 50 ul of L.plantarum competent cells
• In vitro modified plasmid DNA
• MRS media + antibiotic
• Stock solution of MgCl₂
• Stock Solution of Glucose

Procedure

1. Ensure that the in vitro modification of DNA protocol for Lactobacillus plantarum has been followed.
2. Thaw 50 ul of competent cells on ice from aliquots in -80°C freezer.
3. Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).
4. Electroporation done at 13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.
5. Add 500 ul of MRS media containing 80mM MgCl₂ and 55mM Glucose
6. Incubate for 2 hours at 30°C.
7. Plate cells on MRS plates + antibiotic resistance.

Notes

All questions, input and feedback are welcome!

References

Berthier, F., Zagorec, M., Champomier-Verge`s, M., Ehrlich, S.D. and Morel-Deville, F. (1996) Efficient transformation of Lactobacillus sake by electroporation. Microbiology 142, 1273–1279.

Alegre et al. (FEMS Microbiology Letters 241 (2004) 73–77)

Contact

• morto077@uottawa.ca

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