Final Report Summary - PHAGE-BEADS (Hydrogel-Phage Composite Materials and Droplet Microfluidics)
Bead-emulsion technology for directed evolution of enzymes
Directed evolution, a technology to improve proteins towards novel desirable properties, is based on introducing mutations on the genetic level, subsequent screening for improved variants, and iteration. With enzymes, assays usually require reaction vessels which limits the number of clones that can be screened with conventional methods. Using microfluidic, picoliter-sized emulsion droplets for assays enables directed evolution experiments to have a much higher throughput and thus improved chances of success. We developed two novel formats for emulsion-based directed evolution campaigns, the first based on transforming monodisperse water-in-oil (w/o) emulsion droplets into water-in-oil-in-water (w/o/w) double emulsions, the second transforming emulsions into gel-shell beads. Both approaches allow to analyze and sort the resulting microcompartments with fluorescence-activated cell sorting (FACS). The resulting workflows, due to their exceptional high throughput, provide novel possibilities for research while being simple enough to be adapted by laboratories not specialized in microfluidic technology.
From enzyme assays in emulsion droplets to library screens
Monodisperse emulsion droplets are produced in microfluidic chips with high throughput (up to 10 kHz) so that single cells (containing enzyme alongside encoding plasmid) are compartmentalized together with substrate. For a lysate assay, cells are destroyed directly after droplet formation, starting the enzymatic reaction. Monodispersity of the sample allows precise and quantitative readout reflecting the amount of substrate turned over to fluorescent product. Typically the level of stringency in our selections is adjusted with heat inactivation: the assays are stopped where the “parent”- enzyme shows little or no measurable turnover yet, so that hits can be confidently distinguished. Currently, we can screen libraries of about one million clones per experiment.
Sorting of droplets with Flow Cytometry
In contrast to using sophisticated microfluidic chips for sorting (Agresti J et al., PNAS, 2010, 4004) we transform emulsions into double emulsions or gel-shell beads without losing the required genotypephenotype linkage. FACS can then be used to screen for improved variants. After sorting the encoding plasmids of selected clones are extracted, amplified and recloned for being applied to another round of screening, mutation or in-depth monoclonal analysis using 96-well plates. In this way, the microfluidic tools necessary for directed evolution experiments are reduced to operating a microfluidic droplet generator; all equipment is inexpensive and commercially available. Flow cytometers are widely used for sorting cells and sorting with exceptional high throughput (about 108 events/h) is routine with this mature technology.
Double emulsion compartments
The initial emulsion containing enzymatic assays of single library members is transformed into a double emulsion by processing with a hydrophilic flowfocusing chip. This second droplet generator transforms the w/o emulsion into w/o/w double emulsion without decoupling the genotype-phenotype linkage. Size and monodispersity of the first emulsion is preserved. These samples can then be sorted with FACS.
Directed evolution, a technology to improve proteins towards novel desirable properties, is based on introducing mutations on the genetic level, subsequent screening for improved variants, and iteration. With enzymes, assays usually require reaction vessels which limits the number of clones that can be screened with conventional methods. Using microfluidic, picoliter-sized emulsion droplets for assays enables directed evolution experiments to have a much higher throughput and thus improved chances of success. We developed two novel formats for emulsion-based directed evolution campaigns, the first based on transforming monodisperse water-in-oil (w/o) emulsion droplets into water-in-oil-in-water (w/o/w) double emulsions, the second transforming emulsions into gel-shell beads. Both approaches allow to analyze and sort the resulting microcompartments with fluorescence-activated cell sorting (FACS). The resulting workflows, due to their exceptional high throughput, provide novel possibilities for research while being simple enough to be adapted by laboratories not specialized in microfluidic technology.
From enzyme assays in emulsion droplets to library screens
Monodisperse emulsion droplets are produced in microfluidic chips with high throughput (up to 10 kHz) so that single cells (containing enzyme alongside encoding plasmid) are compartmentalized together with substrate. For a lysate assay, cells are destroyed directly after droplet formation, starting the enzymatic reaction. Monodispersity of the sample allows precise and quantitative readout reflecting the amount of substrate turned over to fluorescent product. Typically the level of stringency in our selections is adjusted with heat inactivation: the assays are stopped where the “parent”- enzyme shows little or no measurable turnover yet, so that hits can be confidently distinguished. Currently, we can screen libraries of about one million clones per experiment.
Sorting of droplets with Flow Cytometry
In contrast to using sophisticated microfluidic chips for sorting (Agresti J et al., PNAS, 2010, 4004) we transform emulsions into double emulsions or gel-shell beads without losing the required genotypephenotype linkage. FACS can then be used to screen for improved variants. After sorting the encoding plasmids of selected clones are extracted, amplified and recloned for being applied to another round of screening, mutation or in-depth monoclonal analysis using 96-well plates. In this way, the microfluidic tools necessary for directed evolution experiments are reduced to operating a microfluidic droplet generator; all equipment is inexpensive and commercially available. Flow cytometers are widely used for sorting cells and sorting with exceptional high throughput (about 108 events/h) is routine with this mature technology.
Double emulsion compartments
The initial emulsion containing enzymatic assays of single library members is transformed into a double emulsion by processing with a hydrophilic flowfocusing chip. This second droplet generator transforms the w/o emulsion into w/o/w double emulsion without decoupling the genotype-phenotype linkage. Size and monodispersity of the first emulsion is preserved. These samples can then be sorted with FACS.
