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New enzymes by identification from metagenomic libraries and directed evolution using microfluidic microdroplets

Final Report Summary - METAPROMIS (New enzymes by identification from metagenomic libraries and directed evolution using microfluidic microdroplets)

This proposal aimed to miniaturize cell lysate screening assays to the single cell level in order to perform high throughput screening of metagenomic libraries in search for new biocatalysts and to perform directed enzyme evolution. As we described in the proposal the project had three milestones: (1) The set-up and optimization of the screening system. (2) Generation of libraries. (3) Screening the resulting libraries to isolate new enzymes and perform directed evolution. All three steps have been completed in the two years of the project, and we have published a peer-reviewed research article on the screening procedure, a review and a book chapter on the state-of-the-art of screening in microdroplets. Experiments for an additional research article, including the whole project up to the third milestone, is in preparation and will be published in the following year.
(1. milestone) We set up workflows that translate conventional screening procedure using E.coli cell lysate from microtitre plate to the microfluidic droplet scale (Figure 1A). The final procedure that was optimised in the first year of the fellowship is shown in Figure 1B. The key feature of this technology is a fluorescence-activated dielectrophoretic droplet sorter that enables the quantitative discrimination of enzyme variants in these artificial reaction compartments at a rate of 1000 s-1 .

Currently two spin-off companies (DropTech, Sphere Fluidics Limited) utilize the device at the University of Cambridge and we are building a partnership with a leading company in industrial enzymes, to exploit the potential in the technology. We have also developed an electroporation procedure which enable us to use the sample purified from the droplets directly to transform E. coli cells (step 5), and there is no need for PCR amplification and cloning. This feature makes the system much faster compared to previous protocols. Using an alternative droplet formulation, in which a Figure 1. The screening workflow starts with intracellular expresssion of an enzyme library in E. coli liquid culture (step 1). Single cells compartmentalised into monodisperse emulsion droplets along with the substrate and cell lysis agents (step 2). The sample is collected in a syringe (107 droplets) and incubated to let the reaction progress until the fluorescent product have accumulated (step 3). Subsequently the droplets are reinjected into the fluorescence activated droplet sorter (FADS) (step 4).
After releasing the aqueous phase of the sorted droplets, plasmid DNA is purified from the sample and electroporated into E. coli. second emulsification step is applied to form water-in-oil-in-water double emulsion droplets, the screening procedure has been adapted to sort the libraries with conventional cell sorters.