Difference between revisions of "Electro-transformation of Lactobacillus spp."

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*MRS media
 
*MRS media
 
*Culture of ''L. plantarum'' cells
 
*Culture of ''L. plantarum'' cells
*MgCL<sub>2</sub> (10mM)
+
*Glycine
 
*sucrose  
 
*sucrose  
 
*glycerol
 
*glycerol
*Centrifuge capable of holding four 50mL centrifuge tubes.  
+
*Centrifuge capable of holding two 50mL centrifuge tubes.
  
 
=='''Procedure'''==
 
=='''Procedure'''==

Revision as of 19:38, 23 September 2010

Overview

Instructions on how to prepare Lactobacillus plantarum competent cells before electrotransformation. This protocol prepares 10 100μL aliquots of L. plantarum competent cells. These cells need to be used the day of preparation.

Materials

  • MRS media
  • Culture of L. plantarum cells
  • Glycine
  • sucrose
  • glycerol
  • Centrifuge capable of holding two 50mL centrifuge tubes.

Procedure

Day 1

  1. Prepare the following:
  • 25mL MRS media
  • 25mL Treatment Media (MRS media with 4g glycine(16%) and 15g (1.8M) sucrose added).
  • 50mL Water
  • 50ml 50mM EDTA
  • 25mL Electroporation Buffer (0.9M Sucrose, 10%(v/v) glycerol)
  1. Cap the flasks with foil and autoclave.
  2. Once the MRS has cooled, inoculate the flask without glycine with L.plantarum culture and grow overnight at 30°C.

Day 2

  1. Put the buffers on ice and pre-chill the centrifuge.
  2. Add the 25mL Treatment Media to the 25ml overnight culture.
  3. Incubate cells for 1-1.5 hr at 30°C.
  4. Divide culture into two 50mL centrifuge tubes.
  5. Centrifuge for 2 minutes at 5000g.
  6. Pour off supernatant and resuspend pellet in 10mL ice-cold water.
  7. Centrifuge for 2 minutes at 5000g.
  8. Pour off supernatant and resuspend pellet in 10mL ice-cold water.
  9. Centrifuge for 2 minutes at 5000g.
  10. Pour off supernatant and resuspend pellet in 25mL EDTA solution.
  11. Let cell suspension sit on ice for 30 mins.
  12. Centrifuge for 2 minutes at 5000g.
  13. Pour off supernatant and resuspend pellet in 10mL ice-cold Electroporation Buffer.
  14. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
  15. Pour off supernatant and resuspend cells in 500μL ice-cold Electroporation Buffer.
  16. Dispense into 100μL aliquots.
  17. Store at -20°C for use that day.

Notes

All questions, input and feedback are welcome!

  1. Centrifugation at 4000 rpm for 2 minutes was sufficient to pellet the competent cells to give a clear supernatant.

References

Relevant Papers and Books

This protocol is based mostly on the protocols described by Thompson and Collins (1996) and Mason et al. (2004) but other literature are also listed.

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

Aukrust, T. and H. Blom (1992). "TRANSFORMATION OF LACTOBACILLUS STRAINS USED IN MEAT AND VEGETABLE FERMENTATIONS." Food Research International 25(4): 253-261.

Berthier, F., M. Zagorec, et al. (1996). "Efficient transformation of Lactobacillus sake by electroporation." Microbiology-Uk 142: 1273-1279.

Josson, K., T. Scheirlinck, et al. (1989). "CHARACTERIZATION OF A GRAM-POSITIVE BROAD-HOST-RANGE PLASMID ISOLATED FROM LACTOBACILLUS-HILGARDII." Plasmid 21(1): 9-20.

Mason, C. K., M. A. Collins, et al. (2005). "Modified electroporation protocol for Lactobacilli isolated frorn the chicken crop facilitates transformation and the use of a genetic tool." Journal of Microbiological Methods 60(3): 353-363.

Posno, M., R. J. Leer, et al. (1991). "INCOMPATIBILITY OF LACTOBACILLUS VECTORS WITH REPLICONS DERIVED FROM SMALL CRYPTIC LACTOBACILLUS PLASMIDS AND SEGREGATIONAL INSTABILITY OF THE INTRODUCED VECTORS." Applied and Environmental Microbiology 57(6): 1822-1828.

Thompson, K. and M. A. Collins (1996). "Improvement in electroporation efficiency for Lactobacillus plantarum by the inclusion of high concentrations of glycine in the growth medium." Journal of Microbiological Methods 26(1-2): 73-79.

Wei, M. Q., C. M. Rush, et al. (1995). "AN IMPROVED METHOD FOR THE TRANSFORMATION OF LACTOBACILLUS STRAINS USING ELECTROPORATION." Journal of Microbiological Methods 21(1): 97-109.

Contact

  • morto077@uottawa.ca

or instead, discuss this protocol. -->