Lactobacillus transformation (Kim 2005): Difference between revisions
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==Procedure== | ==Procedure== | ||
===Prepare Electrocompetent cells | ===Prepare Electrocompetent cells=== | ||
#Use overnight culture (10<sup>6</sup> CFU/mL) to inoculate 100mL MRS containing 1% glycine. | #Use overnight culture (10<sup>6</sup> CFU/mL) to inoculate 100mL MRS containing 1% glycine. | ||
#Grow until OD<sub>660</sub> = 0.2-0.3. | #Grow until OD<sub>660</sub> = 0.2-0.3. | ||
| Line 20: | Line 20: | ||
##Resuspend in unsepcified amount of electroportion buffer. | ##Resuspend in unsepcified amount of electroportion buffer. | ||
#Keep on ice and transform cells within 30 minutes. | #Keep on ice and transform cells within 30 minutes. | ||
===Electroporation=== | ===Electroporation=== | ||
#Add 1μL (25 ng/μL) of plasmid DNA to 50 ul (10<sup>8</sup> CFU/ml) of ice-cold cell suspension. | #Add 1μL (25 ng/μL) of plasmid DNA to 50 ul (10<sup>8</sup> CFU/ml) of ice-cold cell suspension. | ||
Revision as of 19:11, 16 September 2011
| back to protocols | ||
Back to Protocols
Back to Electro-transformation of Lactobacillus spp.
Overview
Electrotransformation procedure for Lactobacillus acidophilus, Lactobacillus brevis, and Lactobacillus helveticus.
Procedure
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.
- Repeat.
- 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.
Electroporation
- Add 1μL (25 ng/μL) of plasmid DNA to 50 ul (108 CFU/ml) of ice-cold cell suspension.
- Electroporate at 12.5 kV/cm (pulse number = 10, puse interval = 500 ms).
- Dilute electroporated cells to 1ml in MRS broth and incubate at 37°CC for 3 hours.
- Plate bacteria onto MRS agar plates with appropriate antibiotic.
- 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.
