Lactobacillus transformation (Kim 2005)

Overview

Electrotransformation procedure for Lactobacillus acidophilus

Procedure

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

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! *Derek Ju 04:44, 29 October 2008 (EDT):used plasmid pNZ123, works with L. acidophilus strains 43121, 4356, NCFM, 30SC, A4, 107A, GP4A; L. helveticus KU107; L. brevis 3102

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books Kim et al (Journal of App. Microbio. 2005, 99, 167-174)

Contact

• Derek Ju (derekju [at] mit.edu)

or instead, discuss this protocol.