Part:BBa_K1413041:Design

Mutation of OriVR6Kgamma origin of replication (ori)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part follows rules of RFC92, compatible with RFC10. This oriVR6K gamma is controlled by the pi protein, which is encoded by the pir gene. Indeed, the pi protein allows the replication of the plasmid by binding a particular site in the Ori sequence. Hence, the oriVR6Kgamma can only be replicated in a bacterial strain producing the pi protein, in our case DH5alpha pir. This ori was already used in the iGEM competition in 2009 by a french team. The gamma origin is adjacent to the pi protein binding site and other sites bounded by the host cell proteins involved in its own reproduction.

Mutation of oRiVR6Kgamma The first issue we encountered was the presence of XbaI restriction sites in the OriVR6Kgamma sequence.We had to induce mutations in that site of the plasmid using PCR mutagenesis. pNK2 plasmid was amplified with primers allowing us to modify the XbaI site : 5' TCTAGA 3' to 5' ACTAGA 3'. After amplification of the plasmid, we purified the PCR products obtained and transformed it into DH5alpha pir. Colonies were inoculated in liquid cultures supplemented with kanamycin (50µg/mL) and the pNK2 plasmid was finally extracted. Those plasmids were digested by XbaI in order to verify the mutation of the XbaI site.

1,2,3 is plasmid pNK2 with mutation and 4 is plasmid original pNK2

Source

This part was given by a member of the institute of systems and synthetic biology (Evry, France)

References

Matsumoto-Mashimo C1, Guerout AM, Mazel D. (2004) A new family of conditional replicating plasmids and their cognate Escherichia coli host strains, Research in Microbiology