Table 2.

Electroporation protocol for M. plutonius developed in this study

Step no.	Protocol
1	Grow M. plutonius strains on KSBHI agar at 35°C for 3 days under anaerobic conditions
2	Harvest cells, suspend them in sterilized deionized distilled water to an OD600e of 0.8, and inoculate 2 ml of the bacterial suspension into 500 mla of KSBHI broth containing 40 mM dl-threonine
3	Grow M. plutonius cells at 35°C under anaerobic conditions
4	Stop culturing M. plutonius at early logarithmic phase (OD600 = 0.05 to 0.1)
5	Chill the culture on ice for 15 to 30 min and collect cells by centrifugation at 12,000 × g for 15 min at 4°Cb
6	Wash the cells once with 1/50 to 1/100 culture vol of ice-cold CTB (55 mM MnCl2, 15 mM CaCl2, 250 mM KCl, and 10 mM PIPES, pH 6.7), resuspend the cells in 1/50 to 1/100 culture vol of ice-cold CTB, and incubate the suspension on ice for 30 min
7	Collect the cells by centrifugation at 12,000 × g for 5 to 15 min at 4°C
8	Wash the cells three times with 1/50 to 1/100 culture vol of ice-cold EB (2 mM potassium phosphate containing 10% sucrose, pH 8.4) and once with 1/Xc culture vol of ice-cold EB containing 15% glycerol and resuspend the final cell pellet in 1/Xc culture vol of ice-cold EB containing 15% glycerold
9	Add plasmid DNA (1 μg) to an 80-μl aliquot of ice-cold cells and mix gently
10	Pulse the cell-DNA mixture (20.0 kV/cm, 200 Ω, and 25 μF) in a cold 0.1-cm-gap electroporation cuvette, add 920 μl of room-temp KSBHI broth containing 10% glucose, and disperse the cells with gentle pipetting
11	Plate on KSBHI agar containing 4 μg/ml chloramphenicol immediately and incubate at 35°C for 4 to 5 days under anaerobic conditions

^aScale up or scale down, depending on the application.

^bIt is important to keep the cells at 4°C or on ice for the remainder of the procedure. The cells, and any bottles or solutions that they come in contact with, must be prechilled on ice.

^cX = 50/OD₆₀₀ of M. plutonius culture at step 4.

^dAlthough cells are suspended in the buffer containing glycerol, freeze-thaw procedures drastically reduce the transformation efficiency.

^eOD₆₀₀, optical density at 600 nm.