Team:Uppsala University/Catsac

In our project we have extensively used recombineering onto the bacterial chromosome. To aid this work, we have built a improved Cat-SacB selection-counterselection casette, which provides easy and reliable recombineering, and the possibility of scarless gene deletion.

Using λ-red recombineering you can make sure that your construct have inserted in the right place by first insert the cat-sacB cassette in the place you want your wanted insert and plate on chloramphenicol which selects for all bacteria containing the cassette. Then do another λ-red with your wanted insert and then slect on sucrose plate, only the bacteria which has lost the cassette have got the right construct on the right place.

To make sure that the cat-sacB construct work we plated DH5alpha cells containing the cat-sacB on chloramphenicol plates and on sucrose plates with a negative control who does not contain the cassette. On the chloramphenicol plates the bacteria survives and on sucrose plate they die.