Lactobacillus transformation

互联网2013-09-06

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Overview

This page details a electrotransformation protocol for Lactobacillus bacteria, specifically Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus delbruckii subsp. lactis . The protocol is based on Serror et al. (Applied and Environmental Microbiology 2002)

Materials

List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers'' page on that material are also appropriate.

MRS media

Electroporation Buffer:(0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4; PH 6)

Milk Medium: (0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM MgCl2)

Erythromycin, Chloramphenicol, other antibiotics may be needed for selection

Gene Pulser and Pulse Controller Electrophoration apparatus

Gaspak or method to generate anaerobic conditions

Strain of compatible Lactobacillus - For list of compatible strains/plasmids, please click [here]

Procedure

Estimated time for procedure: 3-4 days

CELL CULTURE
1. Inoculate serial dilutions of fresh bacterial culture into 100 ml of MRS containing 0.1% glycine and incubate at 42°C overnight.
2. Harvest 10 ml of culture cells at beginning of stationary phase (optical density at 600 nm, 1.7) by centrifugation (need ~2.3mL of culture per electroporation)

WASH BUFFER
1. `Wash bacteria once with 100 ml of cold electroporation buffer (EB) (0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4; PH 6)
2. Wash bacteria twice with 30 ml of cold EB

THERMAL SHOCK
1. Resuspend cells in EB to an optical density at 600 nm of about 50
2. Incubate cell suspension at 45°C for 20 min then keep on ice for 10 min

ELECTRICAL PULSE
1. Mix 80 µl of cell suspension with 0.3 to 2 µg of plasmid DNA
2. Subject sample to a 1-kV, 800-Ω, 25-µF electric pulse in a 0.2-cm cuvette by using a Gene Pulser and a Pulse Controller apparatus.

EXPRESSION
1. Immediately add 2 milliliters of milk medium (0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM MgCl2)

Derek Ju 04:28, 29 October 2008 (EDT) : I have found that adding MRS works just as well (and saves the step of making the milk medium)

PLATING, SELECTION
1. Incubate cells for 3h at 37°C before plating on MRS agar supplemented with antibiotics. Add antibiotic erythromycin at concentration 7.5 µg/ml and chloramphenicol at concentration 7.5 µg/ml depending on plasmid resistance gene
2. Incubate plates at 37°C for 2 to 3 days under anaerobic conditions in jars containing GasPak

:An easy and cheap alternative to gaspak is using an airtight box and putting in lit candles until they go out