Part:BBa_K1945001:Experience

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Applications of BBa_K1945001

We were able to use this part successfully to integrate into our cyanobacteria. We used a strain that already had http://partsregistry.org/Part:BBa_K1945002 integrated into its slr0168 neutral site. (This sequence is kanamycin resistance and nickel-inducible MazF, protein synthesis inhibitor. After transforming this plasmid, we followed the protocols detailed on our wiki to exert selective pressure by growing the cells on nickel plates. In the image below, the red arrows indicate the colonies that have finished segregating across all chromosome copies to incorporate this piece as growing on nickel is no longer lethal and it has lost kanamycin resistance.

The colony PCR results in the gel below further indicate that the double recombination process was successful. We used primers to amplify the slr0168 neutral site, and the band that would indicate the presence of MazF and kanamycin in the genome is not present.

For proof of concept of the bio brick assembly process in BBa_K1945001 we digested and ligated BBa_I712019 into BBa_K1945001 with standard iGEM protocol

Which was sequenced confirmed with iGEM sequence verification primers.

User Reviews

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