Platform for enzymatic cloning

LCAB project
LCAB group members

  Candidate enzymes for screening are most often selected by sequence homology (even those with weak homology, to augment diversity) with reference proteins (known proteins with a specific activity).

The candidate genes are amplified from DNA from a collection of strains from Genoscope and the “Cloaca maxima” metagenome, then cloned in 96-well microtiter plates by a cloning method which employs restriction and ligation enzymes.
 

The cloned genes are then over-expressed in E. coli BL21 in 96-well microtiter plates followed by the preparation of cellular lysates. Enzymatic screening of these plates is carried out in 96 or 384-well format.

This platform, which has been functioning for more than a year, has a current output of about 5000 clonings per year.