Electro-transformation of Lactobacillus spp.: Difference between revisions

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Line 27: Line 27:
*Dilution = 1/50
*Dilution = 1/50
*Growth = 1% Glycine or Threonine, 37°C
*Growth = 1% Glycine or Threonine, 37°C
*Wash = 5mM NaH<sub>2</sub>PO<sub>4</sub> 1mM MgCl<sub>2</sub> (0°C)
*Wash = 5mM NaH<sub>2</sub>PO<sub>4</sub> 1mM MgCl<sub>2</sub> (Ice Cold)
*Buffer = 0.3M Sucrose 5mM NaH2PO4 1mM MgCl2 (0°C)
*Buffer = 0.3M Sucrose 5mM NaH<sub>2</sub>PO<sub>4</sub> 1mM MgCl<sub>2</sub> (Ice Cold)
*Voltage = 7,000 V/cm
*Voltage = 7,000 V/cm
*Recovery = 90min MRS
*Recovery = 90min MRS
Line 36: Line 36:
*Dilution = 1/50
*Dilution = 1/50
*Growth = 1% Glycine, Shake
*Growth = 1% Glycine, Shake
*Wash = 1mM MgCl2 (RT°C)
*Wash = 1mM MgCl<sub>2</sub> (RT°C)
*Buffer = 30% PEG 1500
*Buffer = 30% PEG 1500
*Voltage = 7,500 V/cm
*Voltage = 7,500 V/cm
*Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2
*Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl<sub>2</sub>


===Wei (1995)===
===Wei (1995)===
Line 45: Line 45:
*Dilution = 1/50
*Dilution = 1/50
*Growth = 1% Glycine, 37°C
*Growth = 1% Glycine, 37°C
*Wash = 5mM NaH2PO4 1mM MgCl2 (0°C)
*Wash = 5mM NaH<sub>2</sub>PO<sub>4</sub> 1mM MgCl<sub>2</sub> (0°C)
*Buffer = 0.9M Sucrose 3mM MgCl2
*Buffer = 0.9M Sucrose 3mM MgCl<sub>2</sub>
*Voltage = 12,500 V/cm
*Voltage = 12,500 V/cm
*Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2
*Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2
Line 54: Line 54:
*Dilution = unspecified
*Dilution = unspecified
*Growth = MRS, 30°C
*Growth = MRS, 30°C
*Wash = 10mM MgCl2 (0°C)
*Wash = 10mM MgCl<sub>2</sub> (0°C)
*Buffer = 0.5M Sucrose, 10%Glycerol
*Buffer = 0.5M Sucrose, 10%Glycerol
*Voltage = 7,000 V/cm
*Voltage = 7,000 V/cm
*Recovery = 2hrs MRS, 80mM MgCl2
*Recovery = 2hrs MRS, 80mM MgCl<sub>2</sub>


===Thompson (1996)===
===Thompson (1996)===

Revision as of 08:15, 29 March 2011

Overview

While there is a wide variety of methods used for the electrotransformation of Lactobacillus spp., there are a few steps all of them share. 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 =
  • Dilution = 1/50
  • Growth = 1% Glycine, 37°C
  • Wash = 2x w/ water (RT°C)
  • Buffer = 30% PEG 1000
  • Voltage = 8,500 V/cm
  • Recovery = 30min ice / 2hrs MRS

Bringel (1990)

  • Species =
  • Dilution = unspecified
  • Growth = 1% Glycine, 0.75M Sorbitol, 30°C, Shake
  • Wash = 2x w/ water (RT°C)
  • Buffer = 30% PEG
  • Voltage = 12,500 V/cm
  • Recovery = 30min ice / 3hrs MRS

Posno (1991)

  • Species =
  • Dilution = 1/50
  • Growth = 1% Glycine or Threonine, 37°C
  • 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 =
  • Dilution = 1/50
  • Growth = 1% Glycine, Shake
  • 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 =
  • 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 =
  • 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 =
  • Species =
  • Dilution =
  • Growth =
  • Wash =
  • Buffer =
  • Voltage =
  • Recovery =

Alegre (2004)

  • Species =
  • Species =
  • Dilution =
  • Growth =
  • Wash =
  • Buffer =
  • Voltage =
  • Recovery =

Mason (2005)

  • Species =
  • 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

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.
  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 (7.5-9kV/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.

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


or instead, discuss this protocol. -->

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