Lactobacillus transformation (Kim 2005)

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Overview

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

Procedure

1. Prepare Electrocompetent cells:
   1. Use overnight culture (10⁶ CFU/mL) to inoculate 100mL 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 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 unsepcified amount of electroportion buffer.
   6. Keep on ice and transform 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

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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.