Electro-transformation of Lactobacillus spp.

Overview

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

Dilution: This determines the amount of overnight culture to add to new growth 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.
Concentration: This determines how much Electroporation buffer to add relative to the volume of the growth media.
Voltage: How hard to shock em.
Recovery: What to do with your cells after the shock.

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
Concentration = 10X cell pellet volume
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
Concentration = 1/125
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)
Concentration = 1/100
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
Concentration = 1/100
Voltage = 7,500 V/cm
Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl₂

Wei (1995)

Species = L. fermentum
Dilution = 1/50
Growth = 1% Glycine, 37°C
Wash = 5mM NaH₂PO₄ 1mM MgCl₂ (0°C)
Buffer = 0.9M Sucrose 3mM MgCl₂
Concentration = 1/100
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
Concentration = 1/200
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
Concentration = 1/50
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 = 0.4M Sucrose, 1mM MgCl₂, 5mM KH₂PO₄; PH: 6)
Concentration = 1/30
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 = 0.5M Sucrose, 10% Glycerol
Concentration = 1/30
Voltage = 13,000 V/cm
Recovery = 2hrs MRS, 80mM MgCl2

Kim (2005)

Species = L. acidophilus, L. helveticus, L. brevis
Dilution = 1/50
Growth = 1% Glycine, 37°C, OD₆₀₀ = 0.2-0.3
Wash = Ice 10mins, 2x w/ 5mM NaH₂PO₄ 1mM MgCl₂ (0°C)
Buffer = 1M Sucrose 3mM MgCl₂
Concentration = 1/100
Voltage = 12,500 V/cm
Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2

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
Concentration = 1/120
Voltage = 7,500 V/cm
Recovery = 2hrs MRS

Speer (2012)

Species = L. plantarum
Dilution = 1/50
Growth = 2% Glycine, MRS, 3 hrs, 37°C, shaking
Wash = 2X water; 1X 50mM EDTA; 2X E Buffer. ALL ICE COLD
Buffer = 0.5M Sucrose, 10%Glycerol
Concentration = 1/100
Voltage = 12,000 V/cm
Recovery = 2hrs MRS 30°C

Notes

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

Alegre, M.T, Rodrıguez, M.C., and J.M. Mesas. (2004) "Transformation of Lactobacillus plantarum by electroporation with in vitro modified plasmid DNA." FEMS Microbiology Letters 241: 73-77)

Aukrust, T. and H. Blom (1992). "Transformation of Lactobacillus strains used in meat and vegetable fermentaions." 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.

Bringel, F. and J.C. Hubert. (1990) "Optimized transformation by electroporation of Lactobacillus plantarum strains with plasmid vectors" Applied Microbiology and Biotechnology 33:664-670

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.

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

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 plasmids and segregational instability of the introduced vectors." Applied and Environmental Microbiology 57(6): 1822-1828.

Serror, P., Sasaki, T., Ehrlich, S.D. and Emmanuelle Maguin. (2002) "Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids." Applied and Environmental Microbiology, p.46–52

Speer, M.A. and T.L. Richard (2012) Will Soon Exist.

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

Tom Richard (tlr20@psu.edu)
Mike Speer (mas853@psu.edu)
Matthew Orton (morto077@uottawa.ca)

or instead, discuss this protocol. -->