CcdB

Overview

ccdB is a lethal gene that targets DNA gyrase. A strain of E. coli with a specific mutation in the gyrase (such as DB3.1, see E. coli genotypes) can be used to propagate ccdB. The lethal gene is useful for ensuring that the plasmid containing it cannot be propagated in standard E. coli strains (for cloning purposes). The ccdB positive-selection marker acts by killing the background of cells with no cloned DNA, only cells containing a recombinant DNA giving rise to viable clones (insertional inactivation of ccdB).

Notes

• Austin 16:35, 7 Oct 2005 (EDT): I observed a strange result where when DB3.1 and non-DB3.1 strains were transformed with non-ccdB plasmids (three different plasmids), the transformation efficiency of DB3.1 was markedly lower. When the same strains were transformed with ccdB plasmids, as expected, the DB3.1 strain had many colonies and non-DB3.1 had zero or close to zero. I'm not sure why the DB3.1 transformation appears to need ccdB as the strain is propagated fine without any plasmid.

References

1. Bernard P and Couturier M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J Mol Biol. 1992 Aug 5;226(3):735-45. DOI:10.1016/0022-2836(92)90629-x |
2. Miki T, Park JA, Nagao K, Murayama N, and Horiuchi T. Control of segregation of chromosomal DNA by sex factor F in Escherichia coli. Mutants of DNA gyrase subunit A suppress letD (ccdB) product growth inhibition. J Mol Biol. 1992 May 5;225(1):39-52. DOI:10.1016/0022-2836(92)91024-j |
3. Bernard P, Kézdy KE, Van Melderen L, Steyaert J, Wyns L, Pato ML, Higgins PN, and Couturier M. The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase. J Mol Biol. 1993 Dec 5;234(3):534-41. DOI:10.1006/jmbi.1993.1609 |
4. Bernard P, Gabant P, Bahassi EM, and Couturier M. Positive-selection vectors using the F plasmid ccdB killer gene. Gene. 1994 Oct 11;148(1):71-4. DOI:10.1016/0378-1119(94)90235-6 |
5. Bernard P. New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers. Gene. 1995 Aug 30;162(1):159-60. DOI:10.1016/0378-1119(95)00314-v |
6. Bernard P. Positive selection of recombinant DNA by CcdB. Biotechniques. 1996 Aug;21(2):320-3. DOI:10.2144/96212pf01 |
7. Maki S, Takiguchi S, Horiuchi T, Sekimizu K, and Miki T. Partner switching mechanisms in inactivation and rejuvenation of Escherichia coli DNA gyrase by F plasmid proteins LetD (CcdB) and LetA (CcdA). J Mol Biol. 1996 Mar 1;256(3):473-82. DOI:10.1006/jmbi.1996.0102 |
8. Van Reeth T, Drèze PL, Szpirer J, Szpirer C, and Gabant P. Positive selection vectors to generate fused genes for the expression of his-tagged proteins. Biotechniques. 1998 Nov;25(5):898-904. DOI:10.2144/98255pf01 |