Lactobacillus transformation (Kim 2005)

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Back to [[Protocols]]<br>
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Back to [[Electro-transformation of Lactobacillus spp.]]
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==Overview==
==Overview==
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Electrotransformation procedure for ''Lactobacillus acidophilus''
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Electrotransformation procedure for ''Lactobacillus acidophilus'', ''Lactobacillus brevis'', and ''Lactobacillus helveticus''.
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==Procedure==
==Procedure==
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#Prepare Electrocompetent cells
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===Prepare Electrocompetent cells===
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##Inoculate overnight culture at 10^6 CFU/ml in MRS containing 1% glycene
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#Use overnight culture (10<sup>6</sup> CFU/mL)  to inoculate 100mL MRS containing 1% glycine.
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##Harvest at early-log phase (OD660 0.2-0.3)
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#Grow until OD<sub>660</sub> = 0.2-0.3.
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##chill on ice for 10 minutes
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#Place culture into two 50mL centrifuge tubes and chill on ice for 10 mins.
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##wash twice in cold washing buffer (5 mmol 1^-1 sucrose, 3 mmol 1^-1 MgCl2, pH 7.4)
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#Wash 2X with cold washing buffer (5 mM NaH<sub>2</sub>PO<sub>4</sub>, 1mM MgCl<sub>2</sub>, pH 7.4):
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##Use cells within 30 minutes
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##Centrifuge for 5min at 4000g.
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#Electroporation
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##Resuspend in unspecified amount of washing-buffer.
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##add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
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##Repeat.
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##electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
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#Concentrate in electroporation-buffer (1mM sucrose, 3mM MgCl<sub>2</sub>, pH 7.4):
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##dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h
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##Centrifuge for 5min at 4000g
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##plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
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##Resuspend in unsepcified amount of electroportion buffer.
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##incubate under anaerobic conditions
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#Keep on ice and transform cells within 30 minutes.
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===Electroporation===
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#Add 1μL (25 ng/μL) of plasmid DNA to 50 ul (10<sup>8</sup> CFU/ml) of ice-cold cell suspension.
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#Electroporate at 12.5 kV/cm (pulse number = 10, puse interval = 500 ms).
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#Dilute electroporated cells to 1ml in MRS broth and incubate at 37°CC for 3 hours.
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#Plate bacteria onto MRS agar plates with appropriate antibiotic.
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#Incubate under anaerobic conditions.
==Notes==
==Notes==
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Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
 
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'''*[[User:Derek Ju|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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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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*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)''':
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*Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
==References==
==References==
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'''Relevant papers and books'''
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<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
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Kim et al (Journal of App. Microbio. 2005, 99, 167-174)
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*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==

Current revision

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Back to Protocols
Back to Electro-transformation of Lactobacillus spp.

Contents

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

  • 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.

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