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

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 ΔCotA) 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. Fluorescence-activated droplet sorting was then used to sort the droplets containing the desired activity and the corresponding cells were then recultivated and identified using colorimetric assays. We demonstrated that E. coli CotA cells were enriched 191-fold from a 1 : 9 initial ratio of E. coli CotA to E. coli ΔCotA cells (or 437-fold from a 1 : 99 initial ratio) using a sorting rate of 400 droplets per s. This system allows screening of 10(6) cells in only 4 h, compared to 11 days for screening using microtitre plate-based systems. Besides this low error rate sorting mode, the system can also be used at higher throughputs in "enrichment" screening mode to make an initial purification of a library before further steps of selection. Analysis mode, without sorting, was used to rapidly quantify the activity of a CotA library constructed using error-prone PCR. This mode allows analysis of 10(6) cells in only 1.5 h.

[1]  J. Walker,et al.  Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. , 1996, Journal of molecular biology.

[2]  G. Whitesides,et al.  Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). , 1998, Analytical chemistry.

[3]  Dan S. Tawfik,et al.  Man-made cell-like compartments for molecular evolution , 1998, Nature Biotechnology.

[4]  A. Danchin,et al.  CotA of Bacillus subtilis Is a Copper-Dependent Laccase , 2001, Journal of bacteriology.

[5]  M. A. Carrondo,et al.  Crystal Structure of a Bacterial Endospore Coat Component , 2003, Journal of Biological Chemistry.

[6]  Andrew D Griffiths,et al.  Miniaturising the laboratory in emulsion droplets. , 2006, Trends in biotechnology.

[7]  Charles L. Wilkins,et al.  Problems with the “omics” , 2006 .

[8]  David A. Weitz,et al.  Electrocoalescence of drops synchronized by size-dependent flow in microfluidic channels , 2006 .

[9]  Dan S. Tawfik,et al.  Protein engineers turned evolutionists , 2007, Nature Methods.

[10]  George M. Whitesides,et al.  Microsolidics: Fabrication of Three‐Dimensional Metallic Microstructures in Poly(dimethylsiloxane) , 2007 .

[11]  Lorenz M Mayr,et al.  The Future of High-Throughput Screening , 2008, Journal of biomolecular screening.

[12]  J. S. Johnson,et al.  Biocompatible surfactants for water-in-fluorocarbon emulsions. , 2008, Lab on a chip.

[13]  Andrew D Griffiths,et al.  Droplet-based microreactors for the synthesis of magnetic iron oxide nanoparticles. , 2008, Angewandte Chemie.

[14]  Dan S. Tawfik,et al.  Advances in laboratory evolution of enzymes. , 2008, Current opinion in chemical biology.

[15]  J. Keasling.  Synthetic biology for synthetic chemistry. , 2008, ACS chemical biology.

[16]  Mehmet Toner,et al.  Controlled encapsulation of single-cells into monodisperse picolitre drops. , 2008, Lab on a chip.

[17]  Manuela M. Pereira,et al.  Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants. , 2008, The Biochemical journal.

[18]  Andrew D Griffiths,et al.  A fast and efficient microfluidic system for highly selective one-to-one droplet fusion. , 2009, Lab on a chip.

[19]  D. Weitz,et al.  Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. , 2009, Lab on a chip.

[20]  A. Griffiths,et al.  Reliable microfluidic on-chip incubation of droplets in delay-lines. , 2009, Lab on a chip.

[21]  Jean-Christophe Baret,et al.  Kinetic aspects of emulsion stabilization by surfactants: a microfluidic analysis. , 2009, Langmuir : the ACS journal of surfaces and colloids.

[22]  N. Mano,et al.  Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells. , 2010, Biochemical and biophysical research communications.

[23]  A. Abate,et al.  High-throughput injection with microfluidics using picoinjectors , 2010, Proceedings of the National Academy of Sciences.

[24]  A. Abate,et al.  Ultrahigh-throughput screening in drop-based microfluidics for directed evolution , 2010, Proceedings of the National Academy of Sciences.

[25]  L. Mazutis,et al.  Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. , 2011, Lab on a chip.

[26]  Philippe Cinquin,et al.  Mediatorless high-power glucose biofuel cells based on compressed carbon nanotube-enzyme electrodes , 2011, Nature communications.

[27]  Andrew D. Griffiths,et al.  Immobilization of CotA, an extremophilic laccase from Bacillus subtilis, on glassy carbon electrodes for biofuel cell applications , 2011 .

[28]  D. Weitz,et al.  Droplet microfluidics for high-throughput biological assays. , 2012, Lab on a chip.

[29]  Sindy K. Y. Tang,et al.  Characterization of sensitivity and specificity in leaky droplet-based assays. , 2012, Lab on a chip.

[30]  Andrew D Griffiths,et al.  A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution. , 2012, Lab on a chip.

[31]  S. Herminghaus,et al.  Droplet based microfluidics , 2012, Reports on progress in physics. Physical Society.

[32]  Fabienne Courtois,et al.  Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution. , 2012, Chemistry & biology.

[33]  A. Griffiths,et al.  Membraneless glucose/O2 microfluidic biofuel cells using covalently bound enzymes. , 2013, Chemical communications.

[34]  M. Konrad,et al.  Ultra-high throughput detection of single cell β-galactosidase activity in droplets using micro-optical lens array. , 2013, Applied physics letters.

[35]  M. A. Sanromán,et al.  Recent developments and applications of immobilized laccase. , 2013, Biotechnology advances.

[36]  Haakan N Joensson,et al.  Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors. , 2013, Lab on a chip.

[37]  Andrew D. Griffiths,et al.  New glycosidase substrates for droplet-based microfluidic screening. , 2013, Analytical Chemistry.

[38]  J. Nielsen,et al.  High-throughput screening for industrial enzyme production hosts by droplet microfluidics. , 2014, Lab on a chip.