Lactobacillus transformation (Kim 2005)
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Electrotransformation procedure for Lactobacillus acidophilus, Lactobacillus brevis, and Lactobacillus helveticus.
- Prepare Electrocompetent cells:
- Use overnight culture (106 CFU/mL) to inoculate 100mL MRS containing 1% glycine.
- Grow until OD660 = 0.2-0.3.
- Place culture into two 50mL centrifuge tubes and chill on ice for 10 mins.
- Wash 2X with cold washing buffer (5 mM NaH2PO4, 1mM MgCl2, pH 7.4):
- Centrifuge for 5min at 4000g.
- Resuspend in unspecified amount of washing-buffer.
- Concentrate in electroporation-buffer (1mM sucrose, 3mM MgCl2, pH 7.4):
- Centrifuge for 5min at 4000g
- Resuspend in unsepcified amount of electroportion buffer.
- Keep on ice and transform 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.