Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum

Table 3 Bacterial strains and plasmids used in this study

Strain or plasmid	Relevant characteristics^a	Source or reference^b
Bacterial strains
E. coli
TOP10		Invitrogen
ER2275	RecA⁻ McrBC⁻	NEB
C. acetobutylicum
ATCC824	Wild type	ATCC
MGCΔcac1502	ΔCA_C1502	This study
MGCΔcac1502Δupp	ΔCA_C 1502Δ CA_C 2879	This study
MGCΔcac1502ΔuppΔcac3535	ΔCA_C 1502ΔCA_C 2879ΔCA_C 3535	This study
MGCΔcac1502ΔuppΔcac3535ΔctfAB	ΔCA_C 1502ΔCA_C 2879ΔCA_C 3535ΔCA_P0162-3	This study
MGCΔcac1502ΔuppΔcac3535ΔldhA	ΔCA_C 1502ΔCA_C 2879ΔCA_C 3535ΔCA_C 0267	This study
Plasmids
pAN1	Cm^r, φ3TI, p15A origin	[27]
pKD4	Ap^r Km^r	[18]
pETSPO	Cm^r MLS^r	[4]
pUC18	Ap^r	Fermentas
pUC18-FRT-MLS2	Ap^r MLS^r	This study
pCons2-1	Cm^r	This study
pCR-BluntII-TOPO	Zeo^r Km^r	Invitrogen
pCIP2-1	Cm^r	This study
pREPcac15	Cm^r MLS^r ΔCA_C1502	This study
pCIPcac15	Cm^r MLS^r ΔCA_C1502	This study
pREPupp	Cm^r MLS^r Δupp	This study
pCP20	Ap^r Cm^r FLP	[29]
pSOS95	Ap^r MLS^r, acetone operon, repL gene, ColE1 origin	[32]
pCLF1	Cm^r FLP	This study
pCR4-TOPO-Blunt	Ap^r Km^r	Invitrogen
pCons::upp	Cm^r MLS^r upp	This study
pREPCAC3535::upp	Cm^r MLS^r upp ΔCA_C3535	This study
pREPctfAB::upp	Cm^r MLS^r upp ΔctfAB	This study
pREPldhA::upp	Cm^r MLS^r upp ΔldhA	This study

^a RecA ⁻ homologous recombination abolished, McrBC ⁻ lacking methylcytosine-specific restriction system, Cm ^r chloramphenicol resistance, Ap ^r ampicillin resistance, MLS ^r macrolide lincosamide and streptogramin B resistance, Zeo ^r zeomycin resistance, φ3TI φ3TI methyltransferase, repL Gram-positive origin of replication from pIM13
^b NEB New England BioLabs, ATCC American Type Culture Collection (Rockville, MD)

ISSN: 2731-3654