Lactobacillus transformation (Kim 2005)

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Overview

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

Procedure

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.

Electroporation

1. Add 1μL (25 ng/μL) of plasmid DNA to 50 ul (10⁸ CFU/ml) of ice-cold cell suspension.
2. Electroporate at 12.5 kV/cm (pulse number = 10, puse interval = 500 ms).
3. Dilute electroporated cells to 1ml in MRS broth and incubate at 37°CC for 3 hours.
4. Plate bacteria onto MRS agar plates with appropriate antibiotic.
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.