Difference between revisions of "Part:BBa K2491030"

Revision as of 03:55, 28 October 2017

RK2 Broad Host Range Vector with Kanamycin Resistance

The goal of this improvement was to replace p15a origin of replication in pSB3K3 (https://parts.igem.org/Part:pSB3K3) with the RK2 ori to generate a shuttle vector as a broad host range vector (i.e. can replicate in E.coli and other microorganisms).

The RK2 origin of replication is a broad-host-range plasmid belonging to the incP incompatibility group that can be maintained in a large number of bacteria. The minimal region for replication and maintenance consists of an origin of replication, oriV, and the plasmid replication initiator protein TrfA protein, that activates oriV. The copy number of RK2 is about 4-7 per cell in E. coli, 3 in Pseudomonas aeruginosa, and 4-7 in Agrobacterium

Proof Of Concept

Clone verification. The following primers annealing in the TRFA region of the RK2 backbone vector and the kanamycin region were used to verify the construct by amplification and sequencing of the backbone: TRFA_F: 5’-CCGACGGATGTTCGACTATTT-3’, TRFA_R: 5’-AGTAAAGCGCTGGCTGAA-3’, KM_F: 5’-AAAGCCGTTTCTGTAATGAAGG-3’, and KM_R: 5’-CCTGTTGAACAAGTCTGGAAAG-3’. The data showed that the 2 regions were present. In addition, cultures containing the RK2 backbone harboring the RFP gene [biobrick part BBa_K2491030 containing biobrick part BBa_J04450] showed a red pellet (right vial) compared to a control (left vial) without the RFP gene.

Sequence and Features