Laboratory for enzymatic cloning and screening

Group members

The mission of the high throughput cloning and enzymatic screening platform includes two types of projects:

Large-scale functional genomics project

Several years ago Genoscope started to take advantage of its savoir-faire in “high-throughput” technology to develop tools for large-scale studies of “functional genomics” for the purpose of fundamental research (enrichment of our knowledge of the function of enzymes) or the discovery of new biocatalysts which could be utilized in industry. This project fits in with the desire of Genoscope to contribute actively and massively to the inventory of chemical reactions catalyzed by microbial enzymes (UMR8030 “Metabolic Genomics”), whose sequences are accumulating in public databases.
 
  Furthermore, enzymes whose functions have already been characterized have usually been studied in a specific metabolic context. However, it has been shown repeatedly that enzymes are less substrate-specific than previously thought, and it is not rare for an enzyme to be bi-functional or for a substrate to be the target of several metabolic enzymes with a large spectrum of activity (example: dehydrogenases). Furthermore, the screening of enzymes, even those with “known” function, on unnatural substrates should increase the catalog of possible reactions of these enzymes, even when the activity detected is marginal. In fact, the efficacy of these enzymes can then be increased by various methods of evolution (mutagenesis, evolution of strains, etc.).

Platform for enzymatic cloning and screening

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 (LIC).
 
 
 
  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.