Lactobacillus transformation (Kim 2005)

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Contents

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 10ml washing-buffer.
      3. Repeat.
    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.

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