Lactobacillus transformation (Kim 2005): Difference between revisions
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Back to [[Protocols]]<br> | |||
Back to [[Electro-transformation of Lactobacillus spp.]] | |||
==Overview== | ==Overview== | ||
Electrotransformation procedure for ''Lactobacillus acidophilus'' | Electrotransformation procedure for ''Lactobacillus acidophilus'', ''Lactobacillus brevis'', and ''Lactobacillus helveticus''. | ||
==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== | |||
*Used plasmid pNZ123, works with L. acidophilus strains 43121, 4356, NCFM, 30SC, A4, 107A, GP4A; L. helveticus KU107; L. brevis 3102 '''*[[User:Derek Ju|Derek Ju]] 04:44, 29 October 2008 (EDT)''': | |||
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | *Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | ||
==References== | ==References== | ||
<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. --> | <!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. --> | ||
Kim | *Kim, Y.H., Han, K.S., Oh, s., You, S. and S.H. Kim (2005) "Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation." Journal of Applied Microbiology 99: 167–174 | ||
==Contact== | ==Contact== |
Latest revision as of 06:59, 6 October 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
- Used plasmid pNZ123, works with L. acidophilus strains 43121, 4356, NCFM, 30SC, A4, 107A, GP4A; L. helveticus KU107; L. brevis 3102 *Derek Ju 04:44, 29 October 2008 (EDT):
- Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
- Kim, Y.H., Han, K.S., Oh, s., You, S. and S.H. Kim (2005) "Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation." Journal of Applied Microbiology 99: 167–174
Contact
- Derek Ju (derekju [at] mit.edu)
or instead, discuss this protocol.