Part:BBa_K1413041:Experience

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Applications of BBa_K1413041

We used OriVR6Kgamma in the pNK2 (BBa_K141344). This plasmid contains a particular origin of replication OriVR6Kgamma. 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 oriVR6K gamma can only be replicated in a bacterial strain producing the pi protein. Also we used the cells DH5alpha pir, which replicated this plasmid. With other bacteria like BL21, or DH5alpha there was not replication observed.

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

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