Team:Uppsala University/Catsac

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

When doing λ-red recombineering, you can make sure that your construct have inserted in the right place by plating on chloramphenicol which selects for all bacteria containing the cassette. Then, do another λ-red with your insert of interest that will replace the cat-sacB cassette and select on a sucrose plate, which will ensure that only the bacteria which has lost the cat-SacB cassette will survive.

To confirm that the cat-SacB construct works, we streaked MG1655 cells containing the cat-sacB integrated on the chromosome on LA plates with chloramphenicol (12 µg/mL) or sucrose (1 mM) together with a negative control (wild type MG1655) which does not contain the cassette. On the chloramphenicol plates the bacteria carrying the cat-sacB survives, while on the sucrose plate they die.