ccdB cassette without ccdA-
BBa_P1016 encodes the positive selection marker ccdB. The CcdB protein, constitutively expressed by P1016, is lethal to most of the BioBrick cell strains. Only DB3.1 is resistant.
BioBrick standard vectors are designed be easy to use for their most common purpose: assembly of BioBrick standard biological parts. To meet this requirement, we included BBa_P1016 within the BioBrick cloning site of BioBrick standard vectors. BBa_P1016 encodes the positive selection marker ccdB. Positive selection markers prevent one of the most common problems during assembly of BioBrick parts: contamination of the ligation reaction with uncut plasmid DNA. Any cells transformed with the uncut plasmid DNA produce the lethal protein ccdB and die. Note, however, that the drawback of this solution is that inclusion of BBa_P1016 requires that users propagate both the base vector and any derived vectors in E. coli strains tolerant of ccdB expression such as DB3.1.
A new set of BioBrick standard vectors are available with the BBa_I52001 and BBa_I52002 inserts (both of which include BBa_P1016). See I52001 Physical DNA and I52002 Physical DNA for the current list of plasmids that are available in this form.
Usage and Biology
- For more information on how to use this BioBrick part, visit the Featured Parts:Cell Death page.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000INCOMPATIBLE WITH RFCIllegal BsaI site found at 323
- Bernard P, Gabant P, Bahassi EM, Couturier M, Université Libre de Bruxelles. Positive-selection vectors using the F plasmid ccdB killer gene." Gene 1994 Oct 11;148(1):71-4. pmid:7926841. Pubmed
- Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 5,910,438, 1999. Google Patents
- Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 6,180,407 B1, 2001. Google Patents
- Bernard P, Gabant P, Université Libre de Bruxelles. Cloning and/or sequencing vector US Patent Number 7,176,029 B2, 2007. Google Patents
- Bahassi, et al., Université Libre de Bruxelles. Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA., J. Biol. Chem. 1999 274: 10936-10944. pmid:10196173. Pubmed
- Shetty R, Endy D, Knight T. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering 2008 Apr 14;2:5 URL
- GenBank EU496090