Lactobacillus transformation (Kim 2005)
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Revision as of 12:11, 23 February 2009
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Electrotransformation procedure for Lactobacillus acidophilus
- Prepare Electrocompetent cells
- Inoculate overnight culture at 10^6 CFU/ml in MRS containing 1% glycene
- Harvest at early-log phase (OD660 0.2-0.3)
- chill on ice for 10 minutes
- wash twice in cold washing buffer (5 mmol 1^-1 sucrose, 3 mmol 1^-1 MgCl2, pH 7.4)
- Use cells within 30 minutes
- add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
- electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
- dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h
- plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
- incubate under anaerobic conditions
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
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Relevant papers and books
Kim et al (Journal of App. Microbio. 2005, 99, 167-174)
- Derek Ju (derekju [at] mit.edu)
or instead, discuss this protocol.