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.

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

To make confirm that the Cat-SacB construct works, we streaked DH5alpha cells containing the cat-sacB on LA plates with chloramphenicol (12 µg/mL) or sucrose (1 mM) together with a negative control which does not contain the cassette. On the chloramphenicol plates the bacteria survives, and on sucrose plate they die.