Lactobacillus transformation (Kim 2005)
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==Procedure== | ==Procedure== | ||
| - | + | ===Prepare Electrocompetent cells:=== | |
| - | + | #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. | |
| - | + | #Place culture into two 50mL centrifuge tubes and chill on ice for 10 mins. | |
| - | + | #Wash 2X with cold washing buffer (5 mM NaH<sub>2</sub>PO<sub>4</sub>, 1mM MgCl<sub>2</sub>, pH 7.4): | |
| - | + | ##Centrifuge for 5min at 4000g. | |
| - | + | ##Resuspend in unspecified amount of washing-buffer. | |
| - | + | ##Repeat. | |
| - | + | #Concentrate in electroporation-buffer (1mM sucrose, 3mM MgCl<sub>2</sub>, 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 (10<sup>8</sup> 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== | ==Notes== | ||
Revision as of 22:10, 16 September 2011
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Back to Electro-transformation of Lactobacillus spp.
Contents |
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.
