Table 1.

Changes in the optimized transformation protocol for C. ljungdahlii

Step or parameter	Original procedure (3, 48)	Optimized procedure
Preparation of competent cells
Growth phase for harvesting (OD600)	0.3–0.7	0.2–0.3
pH of wash buffer	7.4	6
Resuspension buffer	SMPa (pH 7.4)	SMPa (pH 6) with 10% DMSOb
Cell density of competent cells	∼80× concentrated from the original cultures	1010–1011 cells/ml (∼1,000× concentrated)
Freeze-thaw	No	Yes
Thawing of cells	No	1 min on ice
Preparation of plasmids
In vivo methylation	Yes (20)	No
E. coli strain for plasmid preparation	Kc strain (ER2275)	Bc strain (NEB Expressd)
Electroporation procedures
Cell vol (μl)	600	25
Preincubation with plasmid	5 min on ice	No
Amount of plasmid DNA (μg)	0.1–1.5	1–5
Electroporation cuvette gap (cm)	0.4	0.1
Electric pulse	2.5 kV, 600 Ω, 25 μF	0.625 kV, 600 Ω, 25 μF
Recovery	5 ml PETCe; 37°C, until clear growth occurs	10 ml PETC; 37°C, 9–12 h
Plating	Liquid cultures on a solid agar plate	Liquid cultures mixed with molten agar
Antibiotic concn (μg/ml)	Clarithromycin (5), thiamphenicol (20)	Clarithromycin (4), thiamphenicol (5)

^aSMP, 270 mM sucrose, 1 mM MgCl₂, and 7 mM phosphate buffer.

^bDMSO, dimethyl sulfoxide.

^cK strain, Dcm⁺ Dam⁻; B strain, Dcm⁻ Dam⁺.

^dNew England Biolabs, Ipswich, MA.

^ePETC, American Type Culture Collection (ATCC) PETC 1754 medium.