Difference between revisions of "Lactobacillus transformation (Kim 2005)"

From OpenWetWare
Jump to: navigation, search
(Overview)
(Notes)
 
(7 intermediate revisions by the same user not shown)
Line 1: Line 1:
 
{{back to protocols}}
 
{{back to protocols}}
 +
Back to [[Protocols]]<br>
 +
Back to [[Electro-transformation of Lactobacillus spp.]]
 +
 
==Overview==
 
==Overview==
  
Line 5: Line 8:
  
 
==Procedure==
 
==Procedure==
#Prepare Electrocompetent cells
+
===Prepare Electrocompetent cells===
##Inoculate overnight culture at 10^6 CFU/ml in MRS containing 1% glycene
+
#Use overnight culture (10<sup>6</sup> CFU/mL)  to inoculate 100mL MRS containing 1% glycine.
##Harvest at early-log phase (OD660 0.2-0.3)
+
#Grow until OD<sub>660</sub> = 0.2-0.3.
##chill on ice for 10 minutes
+
#Place culture into two 50mL centrifuge tubes and chill on ice for 10 mins.
##wash twice in cold washing buffer (5 mmol 1^-1 sucrose, 3 mmol 1^-1 MgCl2, pH 7.4)
+
#Wash 2X with cold washing buffer (5 mM NaH<sub>2</sub>PO<sub>4</sub>, 1mM MgCl<sub>2</sub>, pH 7.4):
##Use cells within 30 minutes
+
##Centrifuge for 5min at 4000g.
#Electroporation
+
##Resuspend in unspecified amount of washing-buffer.
##add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
+
##Repeat.
##electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
+
#Concentrate in electroporation-buffer (1mM sucrose, 3mM MgCl<sub>2</sub>, pH 7.4):
##dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h
+
##Centrifuge for 5min at 4000g
##plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
+
##Resuspend in unsepcified amount of electroportion buffer.
##incubate under anaerobic conditions
+
#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==
 
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
 
  
'''*[[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
+
*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==
'''Relevant papers and books'''
+
 
 
<!-- 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 et al (Journal of App. Microbio. 2005, 99, 167-174)
+
*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 05:59, 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

  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.