Lactobacillus transformation (Kim 2005)

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

Procedure

 * 1) Prepare Electrocompetent cells
 * 2) Inoculate overnight culture at 10^6 CFU/ml in MRS containing 1% glycene
 * 3) Harvest at early-log phase (OD660 0.2-0.3)
 * 4) chill on ice for 10 minutes
 * 5) wash twice in cold washing buffer (5 mmol 1^-1 sucrose, 3 mmol 1^-1 MgCl2, pH 7.4)
 * 6) Use cells within 30 minutes
 * 7) Electroporation
 * 8) add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
 * 9) electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
 * 10) dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h
 * 11) plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
 * 12) incubate under anaerobic conditions

Contact

 * Derek Ju (derekju [at] mit.edu)

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