Lactobacillus transformation (Berthier 1996)

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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
• Electroporation Buffer (0.5M Sucrose, 10% Glycerol)

Procedure

1. Grow cells overnight in MRS Broth at 30°C.
2. Use overnight culture to inoculate 100ml MRS Broth .
3. Incubate at 30°C until OD₆₀₀ = 0.4-0.6.
4. Wash 2x with wash-solution (10mM MgCl₂)
   1. Centrifuge for 5min at 3000g.
   2. Resuspend in 10ml wash-solution.
   3. Repeat.
5. Wash 1X in Electroporation Buffer (0.5M Sucrose, 10% Glycerol)
   1. Centrifuge for 5min at 3,000g
   2. Resuspend in 10ml E buffer
6. Concentrate in Electroporation Buffer
   1. Centrifuge for 5min at 3,000g
   2. Resuspend in 500μL E buffer
7. Make 50μL aliquots and store at -80°C until use
8. Thaw aliquots on ice for use
9. Add up to 5μL plasmid DNA
10. Electroporate at 9000kV/cm
11. Recover cells by adding 500μL of MRS with 80mM MgCl₂
12. Incubate for 2 hours at 30°C
13. Plate to select.

Notes

• Time constants for this protocol are between 11.3 and 13.8ms using a Bio-Rad gene pulser with a pulse controller set at 25μF and 600Ω.

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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