Electro-transformation of Lactobacillus spp.

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Contents

Overview

While there is a wide variety of methods used for the electrotransformation of Lactobacillus spp., they all share a typical format.

  • Dilution: This is the amount of overnight culture to add to new gorwth media
  • Growth: These are the conditions used to grow the cells on the day you plan to transform.
  • Washing: Cells are repeatedly centrifuged and resuspended in a solution of some type.
  • Buffer: This is what is used for the final resuspension and electroporation.

This page will list many of these protocols in a standard format so that you can compare them and choose the one that works for you. In some cases an apples to apples comparison may not be acceptable (e.g. the differences in electroporators can be dramatic) so be sure to check the actual paper.

Procedures

Josson (1989)

  • Species = L. plantarum, L. casei.
  • Dilution = 1/50
  • Growth = MRS + 20mM DL threonine, 37°C, OD600=0.5-1.0
  • Wash = 2x w/ water (RT°C)
  • Buffer = 30% PEG 1000
  • Voltage = 8,500 V/cm
  • Recovery = 30min ice / 2hrs MRS

Bringel (1990)

  • Species = L. plantarum
  • Dilution = to OD600=0.05
  • Growth = MRS + 1% Glycine, 0.75M Sorbitol, 30°C, Shake vigorously, OD600=0.3 (~4 hours)
  • Wash = 3x w/ water (RT°C)
  • Buffer = 30% PEG
  • Voltage = 12,500 V/cm
  • Recovery = 30min ice / 3hrs MRS

Posno (1991)

  • Species = L. casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis
  • Dilution = 1/50
  • Growth = MRS + 1% Glycine, 37°C, 3-4 hours
  • Wash = 5mM NaH2PO4 1mM MgCl2 (Ice Cold)
  • Buffer = 0.3M Sucrose 5mM NaH2PO4 1mM MgCl2 (Ice Cold)
  • Voltage = 7,000 V/cm
  • Recovery = 90min MRS

Aukrust (1992)

  • Species = L. plantarum, L. sake
  • Dilution = 1/50
  • Growth = 1% Glycine, 30°C, OD600.=0.6
  • Wash = 1mM MgCl2 (RT°C)
  • Buffer = 30% PEG 1500
  • Voltage = 7,500 V/cm
  • Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2

Wei (1995)

  • Species =
  • Dilution = 1/50
  • Growth = 1% Glycine, 37°C
  • Wash = 5mM NaH2PO4 1mM MgCl2 (0°C)
  • Buffer = 0.9M Sucrose 3mM MgCl2
  • Voltage = 12,500 V/cm
  • Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2

Berthier (1996)

  • Species = Lb. sake
  • Dilution = unspecified
  • Growth = MRS, 30°C
  • Wash = 10mM MgCl2 (0°C)
  • Buffer = 0.5M Sucrose, 10%Glycerol
  • Voltage = 7,000 V/cm
  • Recovery = 2hrs MRS, 80mM MgCl2

Thompson (1996)

  • Species = Lb. plantarum
  • Dilution = 1/20
  • Growth = MRS, 6% Glycine, 37°C
  • Wash = 2X water, 1X 50mM EDTA, 2X 0.3M Sucrose ALL ICE COLD
  • Buffer = 0.3M Sucrose
  • Voltage = 7,500 V/cm
  • Recovery = 2hrs MRS

Serror (2002)

  • Species = L. delbrueckii
  • Dilution = Unpecified
  • Growth = MRS, 42°C to OD600 = 1.7
  • Wash = 3X ice-cold EB buffer (1 mM MgCl2, 5mM KH2PO4)
  • Buffer = EB Buffer
  • Heat-Shock = 45°C for 20 mins, then ice for 10 mins
  • Voltage = 5,000V/cm
  • Recovery = 3hrs Milk Medium ((0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl2) 37°C

Alegre (2004)

  • Species = L. plantarum
  • Dilution = 1/10
  • Growth = LAB, 37°C, MRS, 30°C
  • Wash = 2X 10mL 10 mM chilled MgCl2, 1X 10mL chilled 0.5M Sucrose, 10% Glycerol
  • Buffer = TE Buffer
  • Voltage = 13,000 V/cm
  • Recovery = 4hrs ice

Mason (2005)

  • Species = Lb. casei, Lb. crispatus, Lb. delbreuckii, Lb. plantarum, Lb. salivarius
  • Dilution = 1/6
  • Growth = MRS, 8% Glycine, 90mins, 37°C
  • Wash = 2X water; 1X 50mM EDTA; 2X 0.3M Sucrose. ALL ICE COLD
  • Buffer = 0.3M Sucrose
  • Voltage = 7,500 V/cm
  • Recovery = 2hrs MRS

Notes

  1. Centrifugation at 4000 rpm for 2 minutes was sufficient to pellet the competent cells to give a clear supernatant.
  2. Most of these papers use the Bio-Rad Gene Pulser and have time constants in the range of 9-10 ms with the lower voltages (7-9kV/cm). If you're using a preset electroporator (like the Eppendorf 2510) you should try a higher voltage (~1200) as your time constand will be in thee range of 5-6 ms. If you're using PEG in your electroporation buffer expect your time constant to be significantly lower.
  3. If you get ZERO TRANSFORMANTS after trying multiple methods, chances are that your plasmid is incompatible with the strain you're transforming. This page exists because this happened to me; and after I had tried ALL the protocols listed here, I got a new plasmid and it worked beautifully -- Mike.

References

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
  • mas853@psu.edu


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