CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics

Thomas Beneyton (1, 2) , Faith Coldren (1) , Jean-Christophe Baret (3, 4) , Andrew D. Griffiths (1, 2) , Valérie Taly (1, 5)

1 ISIS - Institut de Science et d'ingénierie supramoléculaires
2 ESPCI Paris - Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris
3 CRPP - Centre de recherches Paul Pascal
4 MPIDS - Max Planck Institute for Dynamics and Self-Organization
5 Centre universitaire des Saints-Pères

We present a high-throughput droplet-based microfluidic analysis/screening platform for directed evolution of CotA laccase: droplet-based microfluidic modules were combined to develop an efficient system that allows cell detection and sorting based on the enzymatic activity. This platform was run on two different operating modes: the “analysis” mode allowing the analysis of the enzymatic activity in droplets at very high rates (>1000 Hz) and the “screening” mode allowing sorting of active droplets at 400 Hz. The screening mode was validated for the directed evolution of the cytoplasmic CotA laccase from B. subtilis, a potential interesting thermophilic cathodic catalyst for biofuel cells. Single E. coli cells expressing either the active CotA laccase (E. coli CotA) or an inactive frameshifted variant (E. coli DCotA) were compartmentalized in aqueous droplets containing expression medium. After cell growth and protein expression within the droplets, a fluorogenic substrate was “picoinjected” in each droplet.....

Mots clés: Microfluidic

Domaines: Matière Condensée [cond-mat]

Thomas Beneyton, Faith Coldren, Jean-Christophe Baret, Andrew D. Griffiths, Valérie Taly. CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics. Analyst, 2014, 139 (13), pp. 3314-3323. ⟨10.1039/c4an00228h⟩. ⟨hal-01128349⟩