Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: HO Yuan Heng Trevor
Date created: 2012-08-18 11:00:00
Date modified: 2015-06-15 12:38:08
Modified ccdB for plasmid assembly on standard vectors
| Types | DnaRegion |
| Roles | engineered_region Generator |
| Sequences | BBa_K524201_sequence (Version 1) |
Description
This part was modified from the existing BioBrick BBa_P1010 (ccdB cell death gene), flanked by restriction sites AvrII (followed after RFC10 prefix) and NheI (preceding RFC10 suffix).One of the many ways to construct plasmids from BioBricks would be to perform standard assembly on a standard vector, excise the insert out using XbaI and SpeI, and then allow the linearized plasmid to self ligate. Such strategy usually involves the purification of desired fragment by electrophoresis followed by gel purification. If the insert is larger than the standard vector, the excised gel fragment might be contaminated by the vector. Contaminant standard vectors would self-ligate and possess higher transformation efficiency than the desired constructed plasmid. In cases where such 'co-transformation' are not desired, the modified ccdB cell death gene can be added at either end of insert by standard assembly (in such situation a ccdB tolerant strain should be used to harbor the construct), and the intended fragment can be excised using either a combination of XbaI + AvrII or SpeI + NheI. As transformation proceeds, any undesired fragments carrying the ccdB part would kill the host cells and the remaining population would only hold the plasmid of interest.
Notes
The AvrII site and NheI site were encouraged to exclude from standard biobricks. The addition of two sites but not one is the result considering that either site might be unavoidable in some parts. The arrangement that the AvrII site precedes the NheI site is plainly arbitrary.Source
Modified P1010 was constructed from BBa_P1010 (ccdB cell death gene) on pSB1C3 by PCR amplification.| Sequence Annotation | Location | Component / Role(s) |
| AvrII site NheI site ccdB ccdA BBa_P1010 -10 -35 | 1,6 682,687 35,340 342,562 7,681 610,615 624,629 | feature/dna sequence_feature sequence_feature feature/dna feature/cds CDS feature/cds CDS engineered_region feature/BioBrick feature/promoter promoter feature/promoter promoter |