Electrotransformation of Lactobacillus Spp.
Overview
General guidelines for the electro-transformation of Lactobacillus sake as described by Berthier et al. and as used by Alegre et al. with Lactobacillus plantarum.
Materials
50 ul of L.plantarum competent cells
In vitro modified plasmid DNA
MRS media + antibiotic
Stock solution of MgCl2
Stock Solution of Glucose
Procedure
Ensure that the in vitro modification of DNA protocol for Lactobacillus plantarum has been followed.
Thaw 50 ul of competent cells on ice from aliquots in -80°C freezer.
Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).
Electroporation done at 13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.
Add 500 ul of MRS media containing 80mM MgCl2 and 55mM Glucose
Incubate for 2 hours at 30°C.
Plate cells on MRS plates + antibiotic resistance.
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