Part:BBa_K1744000:Experience

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Applications of BBa_K1744000

BBa_K1744000 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 the priming sites sequence indicated on each side of the part with an additional tail (~38-80bp) corresponding to the upstream homology to the region to delete for the forward primer and to the downstream homology for the reverse primer.

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 ampicillin, since the gene bla is present in the cassette. 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 above gel shows a representative success rate of BBa_K1744000. The result is detailed in the figure. Note that not all clones are positive even if selected with ampicilin. This could be an effect of two things, either ampicilin is degraded fast in the medium by real positive adjacent colonnies leading to fast grown satelite colony, or the cassette is able to insert itself in a non-specific genomic target. The first hypothesis could be investigated using more efficient antibiotic marker such as kanamycin. The second hypothesis leads us to some technical tips and trick. Through-ut the project, we observed that casette syze, homology syze and phosphothiolate protected 5' bonds were very important for method specificity. In fact, the bigger the cassette, the lower yeild of colonies you will get. The longuer the homologies, the more specific your recombineering will be. Finally, phosphothiolate bonds have a great protective role and could prevent non-specific DNA degradation. Adding those to your homology bearing primers should boost method efficiency for large fragment like that. But, as you can see, even without all that, the efficiency is high enough to get most of the time at least one good clone out of 3 and screening three clones is not mutch effort.

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. 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.

The above picture shows the screening effort used to obtain good recombinant. From our experience, it seems all recombinants after clean deletion of BBa_K1744000 are left. This means that vcrx028 can be successfully used as a highly efficient killswitch to counter select cells that did not recombine. The success rate of the method is, so far, of 100%, which is better than ampicilin since it has the tendancy to be degraded overtime.

After all these steps, you will get a clean deletion in your plasmid/genome.

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