Droplet-based selection by injection

A method for the identification of a polynucleotide encoding an enzyme of interest in a microfluidic device, said method comprising the steps of:

• a) providing an emulsion of microfluidic droplets comprising a library of polynucleotides encoding one or more enzyme, wherein each droplet comprises at most a single member of the library comprised within a single prokaryotic host cell, and expressing the library of polynucleotides to produce the one or more enzyme;
• b) introducing an aliquot comprising a substrate for the one or more enzyme into each droplet, wherein the presence of one or more active enzyme of interest will convert the substrate into a screenable product;
• c) determining positive droplets that contain the screenable product above a predetermined threshold level which, in turn, also determines the negative droplets;
• d) introducing an aliquot of concentrated PCR solution comprising nucleotides, a suitable DNA polymerase and a set of PCR primers which allows PCR amplification of the polynucleotides encoding the one or more enzyme of interest into each positive droplets determined in step (c);
• and/of introducing an aliquot of a solution which results in DNA denaturation as well as enzyme denaturation and/or cell lysis into the negative droplets determined in step (c);
• e) cloning or PCR amplifying the polynucleotide encoding the one or more enzyme of interest from the positive droplets;
• and
• f) identifying a polynucleotide encoding an enzyme of interest from the cloned or PCR amplified polynucleotides in step (e).