Table 1. Comparison of published procedures and new transformation protocol for C. ljungdahlii.

	Köpke et al., 2010	Leang et al., 2012	Reeves, 2014	New protocol
protocol No.	1	2	3	4
preparation of cells and plasmids
OD600 (harvesting)	0.3–0.7	0.2–0.3	0.3–0.7	0.2–0.7
wash-buffer	SMPa	SMP	SMP	glycerol (10%)
pH wash-buffer	7.4	6	6	6
centrifugation steps	inside the chamber	outside of chamber	outside of chamber	inside the chamber
resuspension-buffer	SMP	SMP with 10% DMSO	SMP with 15% DMSO	glycerol (10%)
pH of res.-buffer	7.4	7.4	6.1	6
cell density at transformation	80-foldb	1000-fold	100-fold	30-fold
freeze/thaw	no	yes	yes	no
plasmid-methylation	yes32	no	yes (Clostridium spec. Type I; Reeves31)	yes32
strain plasmid-prep	K strain (ER2275)	B strain (NEB express)	severalc	K strain (DH5αMCR)
electroporation process
preincubation with plasmid on ice	5 min.	no	no	1–2 min.
volume of cells [μL]	600	25	50	200
plasmid-amount [μg]	0.1–1.5	1–5	1	2–3
electric pulse	2.5 kV, 600 Ω, 25 μF	0.625 kV, 600 Ω, 25 μF	1.5–2.5 kV, 600 Ω, 25 μF	2.5 kV, 600 Ω, 25 μF
electroporation cuvettes gap [cm]	0.4	0.1	0.2	0.2
cultivation after transformation	5 ml PETC, 37 °C	10 ml PETC, 37 °C	4 ml fermentation medium, 37 °C	5 ml RCM, 37 °C
outgrowth-cultivation time after transformation	until growth occurs	9–12 h	the next day	24–48 h
plating	liquid culture on solid agar	liquid culture mix with molten agar	liquid culture on solid agar	liquid culture mix with molten agar
antibiotics [μg/mL]	thiamphenicol: 20	thiamphenicol: 5	NId	thiamphanicol: 5
	clarithromycin: 5	clarithromycin: 4	NI	clarithromycin: 4
organisms transformed with procedure	C. ljungdahlii	C. ljungdahlii	C. autoethanogenum	C. ljungdahlii
				C. acetobutylicum
				C. perfringens
				C. pasteurianum

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

^bCalculated from the cell density in the beginning.

^cDH10B; BL21; GM2163; DH5α; ER2275.

^dNI = not indicated.