Lactobacillus transformation (Kim 2005)

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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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###Centrifuge for 5min at 4000g.
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###Resuspend in 10ml washing-buffer.
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###Repeat.
##Use cells within 30 minutes
##Use cells within 30 minutes
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#Electroporation
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#'''Electroporation:'''
##add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
##add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
##electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
##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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##plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
##plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
##incubate under anaerobic conditions
##incubate under anaerobic conditions
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==Notes==
==Notes==

Revision as of 21:25, 16 September 2011

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Contents

Overview

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

Procedure

  1. 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 10ml washing-buffer.
      3. Repeat.
    5. Use cells within 30 minutes
  2. Electroporation:
    1. add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
    2. electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
    3. dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h
    4. plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
    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

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.

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