| CPC G01N 33/5008 (2013.01) [C07K 1/047 (2013.01); C07K 7/64 (2013.01); C12N 15/1075 (2013.01); C40B 40/04 (2013.01); C40B 40/10 (2013.01)] | 11 Claims |
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1. A method for co-compartmentalizing cyclic polypeptides in a hydrophilic microfluidic device, the method comprising in order:
(a) forming a plurality of multicompartment systems comprising a plurality of monodisperse water-in-oil-in-water (w/o/w) emulsion droplets on the hydrophilic microfluidic device, wherein each of the plurality of multicompartment systems comprises:
(i) a first compartment comprising an aqueous droplet, comprising an in vitro transcription and translation (IVTT) system, detectable fluorescent reporters, and a gel forming agent comprising an ultra-low gelling temperature agarose, wherein the an ultra-low gelling temperature agarose is fluid at 37° C. and has a transition gelling point between 8-17° C., wherein the gel forming agent comprises a polynucleotide sequence that encodes a cyclic polypeptide, and wherein 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 cyclized, 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 transfer RNA (tRNA) charged with natural or non-natural amino acids, an RNA polymerase, a ribosome, nucleotide phosphates, and translation factors;
(ii) a second compartment comprising a fluorinated oil surrounding the first compartment, such that the first compartment and the second compartment form a plurality of monodisperse water-in-oil (w/o) emulsion droplets, and;
(iii) a third compartment comprising a discontinuous external aqueous phase comprising a surfactant, wherein the discontinuous external aqueous phase surrounds each of the plurality of monodisperse water-in-oil emulsion droplets to form a plurality of monodisperse water-in-oil-in-water emulsion droplets, wherein prior to formation of the third compartment, the method comprises,
(b) amplifying the polynucleotide sequence that encodes a cyclic polypeptide within each first compartment to produce a plurality of amplicons, each first compartments comprising a plurality polynucleotide sequences encoding the polypeptides that are to be cyclized 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 constant and even application of heat to the reaction vessel and/or to the reaction container maintains the gel forming agent in a liquid phase for amplification of the polynucleotide sequence prior to in vitro polypeptide expression, such that more than three (3) million monodisperse w/o emulsion droplets are generated;
(c) following amplification, solidifying the gel forming agent by lowering the temperature of the reaction vessel and/or the reaction container to form a gel matrix that traps the plurality of amplicons in the gel matrix of the monodisperse w/o emulsion droplet, and
(d) in vitro expressing a plurality of polypeptides from the plurality of amplicons within the gel matrix formed in (c) 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 gel matrix of the first compartment; wherein the plurality of expressed polypeptides in each of the plurality of first compartments self-cyclize to form a plurality of cyclic polypeptides within each of the plurality of monodisperse w/o emulsion droplets.
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