Electro-transformation of Lactobacillus spp.

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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 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.
 * Concentration: This determines how much 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.

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 (2011)

 * 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

Contact

 * morto077@uottawa.ca


 * mas853@psu.edu

or instead, discuss this protocol.

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