Part:BBa_K1744001:Experience

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Applications of BBa_K1744001

This part can be used for recombineering experiment. In this context, amilCP serves us as a plasmidic marker to avoid plasmid background and KanR serves us as a positive recombineering marker to help us select the good recombinants. If the colonies are blue, it is plasmidic background, if they are white, then they are recombinants (since the expression of amilCP in single copy is not visible). Both KanR gene and amilCP gene can serve as reporters in several experiments such as plasmid construction, transformation control, recombineering and many more!

In order to assay the expression of amilCP in single copy, it was inserted through recombineering in lacZ gene in E. coli K-12 substr. BW25113. The result shown above demonstrate that the insertion had a 100% rate of success. In fact, we screened with two primer pairs. The first one (at left) produce a 875 bp amplicon if insertion of the part in the genome is successful but no bands if it failed. The second primer pair (at right) produces a 1.7 kb band if kanR-amilCP was successfully inserted in the genome and a 1.2 kb band if it did not. Using both results, we can conclude that 100% of the clones are positive. The cells were selected on LB Kanamycin 50 µg/mL and ampicilin 100 µg/ml (for the selection of the pSIM6 plasmid important for recombineering). The cells were photographied to show the difference between cells with the plasmidic form of amilCP gene and the inserted version of the gene:

In this previous image, you can see the appearance of colonies on LB agar when they carry plasmidic or genomic amilCP-KanR cassette.

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