Lactobacillus transformation-PDF

Overview

Electrotransformation procedure for Lactobacillus acidophilus, Lactobacillus brevis, and Lactobacillus helveticus.

Procedure

Prepare Electrocompetent cells

1. Use overnight culture (10⁶ CFU/mL) to inoculate 100 mL MRS containing 1% glycine.
2. Grow until OD₆₆₀ = 0.2-0.3.
3. Place culture into two 50mL centrifuge tubes and chill on ice for 10 mins.
4. Wash 2X with cold washing buffer (5 mM NaH₂PO₄, 1mM MgCl₂, pH 7.4):
   1. Centrifuge for 5min at 4000g.
   2. Resuspend in an unspecified amount of washing buffer.
   3. Repeat.
5. Concentrate in electroporation-buffer (1mM sucrose, 3mM MgCl₂, pH 7.4):
   1. Centrifuge for 5min at 4000g
   2. Resuspend in an unspecified amount of electroporation buffer.
6. Keep on ice and transform cells within 30 minutes.

Electroporation

1. Add 1μL (25 ng/μL) of plasmid DNA to 50 ul (10⁸ CFU/ml) of ice-cold cell suspension.
2. Electroporate at 12.5 kV/cm (pulse number = 10, puse interval = 500 ms).
3. Dilute electroporated cells to 1ml in MRS broth and incubate at 37°CC for 3 hours.
4. Plate bacteria onto MRS agar plates with the appropriate antibiotic.
5. Incubate under anaerobic conditions.

References

• Kim, Y.H., Han, K.S., Oh, s., You, S. and S.H. Kim (2005) “Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation.” Journal of Applied Microbiology 99: 167–174