Lactobacillus transformation (Berthier 1996): Difference between revisions

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(New page: ==Overview== General guidelines for the electrotransformation of ''Lactobacillus plantarum'' as described by Alegre et al. ==Materials== *50 ul of L.plantarum competent cells *Plasmid DNA...)
 
 
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==Overview==
==Overview==
General guidelines for the electrotransformation of ''Lactobacillus plantarum'' as described by Alegre et al.
General guidelines for the electro-transformation of ''Lactobacillus sake'' as described by Berthier et al. and as used by Alegre et al. with ''Lactobacillus plantarum''.


==Materials==
==Materials==
*50 ul of L.plantarum competent cells
*50 ul of ''L.plantarum'' competent cells
*Plasmid DNA
*In vitro modified plasmid DNA
*MRS media + antibiotic
*MRS media + antibiotic
*Stock solution of MgCl2
*Stock solution of MgCl<sub>2</sub>
*Stock Solution of Glucose
*Electroporation Buffer (0.5M Sucrose, 10% Glycerol)


==Procedure==
==Procedure==
#Ensure that the in vitro modification of DNA protocol for ''Lactobacillus plantarum'' has been followed.
#Grow cells overnight in MRS Broth at 30°C.
#Thaw 50 ul of competent cells on ice from aliquots in -80°C freezer.
#Use overnight culture to inoculate 100ml MRS Broth .
#Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).
#Incubate at 30°C until OD<sub>600</sub> = 0.4-0.6.
#Electroporation done at 13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.
#Wash 2x with wash-solution (10mM MgCl<sub>2</sub>)
#Add 500 ul of MRS media containing 80 mM MgCl2
##Centrifuge for 5min at 3000g.
#Incubate for 2 hours at 30°C.
##Resuspend in 10ml wash-solution.
#Plate cells on MRS plates + antibiotic resistance.
##Repeat.
#Wash 1X in Electroporation Buffer (0.5M Sucrose, 10% Glycerol)
##Centrifuge for 5min at 3,000g
##Resuspend in 10ml E buffer
#Concentrate in Electroporation Buffer
##Centrifuge for 5min at 3,000g
##Resuspend in 500μL E buffer
#Make 50μL aliquots and store at -80°C until use
#Thaw aliquots on ice for use
#Add up to 5μL plasmid DNA
#Electroporate at 9000kV/cm
#Recover cells by adding 500μL of MRS with 80mM MgCl<sub>2</sub>
#Incubate for 2 hours at 30°C
#Plate to select.


==Notes==
==Notes==
All questions, input and feedback are welcome!
*Time constants for this protocol are between 11.3 and 13.8ms using a Bio-Rad gene pulser with a pulse controller set at 25μF and 600Ω.


==References==
==References==
Berthier, F., Zagorec, M., Champomier-Verge`s, M., Ehrlich, S.D. and Morel-Deville, F. (1996) Efficient transformation of ''Lactobacillus sake'' by electroporation. ''Microbiology'' 142, 1273–1279.
Alegre et al. (FEMS Microbiology Letters 241 (2004) 73–77)
Alegre et al. (FEMS Microbiology Letters 241 (2004) 73–77)


Latest revision as of 18:13, 16 September 2011

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

Overview

General guidelines for the electro-transformation of Lactobacillus sake as described by Berthier et al. and as used by Alegre et al. with Lactobacillus plantarum.

Materials

  • 50 ul of L.plantarum competent cells
  • In vitro modified plasmid DNA
  • MRS media + antibiotic
  • Stock solution of MgCl2
  • Stock Solution of Glucose
  • Electroporation Buffer (0.5M Sucrose, 10% Glycerol)

Procedure

  1. Grow cells overnight in MRS Broth at 30°C.
  2. Use overnight culture to inoculate 100ml MRS Broth .
  3. Incubate at 30°C until OD600 = 0.4-0.6.
  4. Wash 2x with wash-solution (10mM MgCl2)
    1. Centrifuge for 5min at 3000g.
    2. Resuspend in 10ml wash-solution.
    3. Repeat.
  5. Wash 1X in Electroporation Buffer (0.5M Sucrose, 10% Glycerol)
    1. Centrifuge for 5min at 3,000g
    2. Resuspend in 10ml E buffer
  6. Concentrate in Electroporation Buffer
    1. Centrifuge for 5min at 3,000g
    2. Resuspend in 500μL E buffer
  7. Make 50μL aliquots and store at -80°C until use
  8. Thaw aliquots on ice for use
  9. Add up to 5μL plasmid DNA
  10. Electroporate at 9000kV/cm
  11. Recover cells by adding 500μL of MRS with 80mM MgCl2
  12. Incubate for 2 hours at 30°C
  13. Plate to select.

Notes

  • Time constants for this protocol are between 11.3 and 13.8ms using a Bio-Rad gene pulser with a pulse controller set at 25μF and 600Ω.

References

Berthier, F., Zagorec, M., Champomier-Verge`s, M., Ehrlich, S.D. and Morel-Deville, F. (1996) Efficient transformation of Lactobacillus sake by electroporation. Microbiology 142, 1273–1279.

Alegre et al. (FEMS Microbiology Letters 241 (2004) 73–77)

Contact

  • morto077@uottawa.ca

or instead, discuss this protocol. -->

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