Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Abstract

A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb ClaI/SphI chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum, SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.

Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains

“…For the preparation of clostridial competent cells, cells were grown on CG medium (Roos et al 1985), as described previously (Oultram et al 1988). When required, culture media were supplemented with ampicillin (100 μg/mL), chloramphenicol (30 μg/mL), erythromycin (50 μg/mL or 30 μg/mL for liquid cultures and plates, respectively).…”

“…Correct methylation was checked by restriction analysis with Fnu 4HI. Methylated pFC002, pFC005, pFC006, pFC007 and methylated pMTL500E plasmids (Table 1) were electroporated into C. acetobutylicum ATCC 824 as described by Oultram et al (Oultram et al 1988). Erythromycin-resistant colonies were cultivated in CGM liquid medium and total DNA was extracted as described above.…”

Spo0A directly controls the switch from acid to solvent production in solvent‐forming clostridia

“…Plasmids pRGus, pRG‐ADCwt, pRG‐ADCdown, pRG‐PTBwt, pRG‐PTBup and pRG‐PTBdown were transferred to C. beijerinckii NCIMB 8052 by electrotransformation, essentially as described previously (Oultram et al ., 1988). Plasmids were re‐extracted from transformants as described previously (Williams et al ., 1990) and verified by restriction enzyme digestion (ADC plasmids) or sequencing (PTB plasmids).…”

Recent advances in the genetics of the clostridia

“…This transformation method is currently proving widely applicable to many different bacterial genera [42,43]. Reports of transformation of intact cells of C. acetobutylicum NCIB 8052 [36] and C. perfringens [40,41] with various plasmids have already appeared (Table 3) and doubtless other species of Clostridium will be added to the list in the very near future.…”