Part:BBa_K1744002:Experience

Applications of BBa_K1744002

BBa_K1744002 was not tested yet, but it is designed to achieve clean deletion in a genome or in a plasmid. The first step is to amplify this part with appropriate primers. These have to contain an additional tail (>=80bp) corresponding to the upstream homology to the region to delete for the forward primer and to the downstream homology for the reverse primer.

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Then this cassette with the right homologies is used to recombinate (through recombineering) and delete the targeted region of the plasmid/genome. After what the recombinants will be selected with kanamycin, since the gene aph is present in the cassette. In addition to the resistance to kanamycin, the recombinants should appear blue because of the amilCP gene, but results obtained with BBa_K1744002 indicated that the coloration was really slow to become visible. Note that during all the culture steps implicated, the cells has to be in a medium with glucose (we use 5%w/v) to keep the expression of the toxin vcrx028, that is present in the cassette, repressed. The next step is to amplify both region on each side of the targeted sequence. Then a fusion PCR of those 2 regions has to be done.

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With the cassette obtained, another recombination is done that will delete the part BBa_K1744000 and only the cassette used to delete it will remain. To get only the recombinants of this step, the cells must be put on a medium containing arabinose (we used 1%w/v of final concentration) to trigger the killswitch in cells that wouldn't have lost the part. After all these steps, you will get a clean deletion in your plasmid/genome.

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