Methods for generating and screening compartmentalised peptide libraries

A library of at least three (3) million gel beads, each gel bead comprising:

• a) a plurality of polynucleotides that have a sequence that comprises, from 5′ to 3′:
• a sequence encoding an N-terminal intein fragment,
• a sequence encoding the polypeptide that is to be cyclized, and
• a sequence encoding a C-terminal intein fragment;
• and
• b) the corresponding cyclic polypeptide.

A library at least three (3) million gel beads made according to a method comprising

• a) forming a plurality of multicompartment systems comprising a plurality of water-in-oil-in-water (w/o/w) emulsion droplets on the hydrophilic microfluidic device, wherein each of the plurality of multi-compartment systems comprises:
• (i) a first compartment comprising an aqueous droplet comprising an in vitro transcription and translation (IVTT) system, a detectable fluorescent reporter, and a gel matrix formed from a gel forming agent comprising an ultra low gelling temperature agarose,
• wherein the gel matrix comprises a polynucleotide sequence that encodes a cyclic polypeptide, and the polynucleotide sequence comprises, from 5′ to 3′, a sequence encoding an N-terminal intein fragment, a sequence encoding the polypeptide that is to be cyclised, and a sequence encoding a C-terminal intein fragment,
• wherein detectable fluorescent reporters are selected from the group consisting of fluorescein, fluorescent proteins and fluorophores,
• wherein the IVTT system comprises a tRNA charged with a natural or non-natural amino acid;
• (ii) a second compartment comprising a fluorinated oil surrounding the first compartment, such that the first compartment and the second compartment form a water-in-oil emulsion droplet;
• and
• (iii) a third compartment comprising a discontinuous external aqueous phase comprising a surfactant, wherein the discontinuous external aqueous phase surrounds the water-in-oil emulsion droplet to form a water-in-oil-in-water emulsion droplet;
• wherein prior to formation of compartment (iii), the method comprises,
• b) amplifying the polynucleotide sequence that encodes a cyclic polypeptide within each first compartments to produce a plurality of amplicons, each comprising a polynucleotide sequence encoding the polypeptide that is to be cyclized within each of the gel matrix, such that during amplification an external source of heat is constantly and evenly applied to a reaction vessel and/or applied to a reaction container in which amplification is being carried out, wherein the application of heat to the reaction vessel and/or the container maintains the gel matrix in a liquid phase for amplification of the polynucleotide sequence prior to in vitro polypeptide expression, and such that more than three (3) million droplets are generated;
• c) following amplification, solidifying the liquid phase to form a gel matrix that traps the plurality of amplicons in the gel matrix emulsion droplet;
• and
• d) in vitro expressing a plurality of polypeptides from the plurality of amplicons within the first compartment formed in (a) to produce a plurality of expressed polypeptides, such that the plurality of amplicons encoding the plurality of polypeptides and the expressed polypeptides are contained in the first compartment;
• wherein the plurality of expressed polypeptides in the first compartment are cyclised.