Monitoring of in vitro protein synthesis
Provided herein are methods, and compositions for the real-time detection of protein synthesis. The methods are applicable to monitoring on a microfluidic device.
1 . A method for the monitoring of cell free protein synthesis in a droplet on a digital microfluidic device comprising:
a. cell free transcription and translation of a protein of interest fused to a peptide tag where the peptide tag is a component of a fluorescent protein; and
b. monitoring, in the digital microfluidic device, the presence of the peptide tag using a further polypeptide, said further polypeptide being a complementary portion of the fluorescent protein, which in the presence of the peptide tag produces a detectable signal; wherein the droplets are in an oil layer and the oil layer contains surfactant.
2 . The method as claimed in claim 1 , wherein the transcription and translation occurs in human lysate system.
3 . The method as claimed in claim 1 , wherein the transcription and translation occurs in a rabbit reticulocyte lysate (RRL) system or a Chinese Hamster Ovary lysate system.
4 . The method as claimed in claim 1 , wherein the cell free transcription and translation occurs in a wheat germ cell-free system.
5 . The method as claimed in claim 1 , wherein herein the cell free transcription and translation occurs a E. coli whole cell lysate system.
6 . The method as claimed in claim 1 , wherein the cell free transcription and translation occurs in a system of purified recombinant elements (PURE).
7 . The method as claimed in claim 1 , wherein the cell free transcription and translation are coupled.
8 . The method as claimed in claim 1 , wherein the cell free transcription and translation are uncoupled.
9 . The method as claimed in claim 1 , wherein the peptide tag is one component of a green fluorescent protein (GFP) or one component of sfCherry.
10 . The method as claimed in claim 9 wherein the peptide tag is GFP 11 and the further polypeptide is GFP 1-10 or wherein the peptide tag is sfCherry11 and the further polypeptide is sfCherry 1-10 .
11 . The method as claimed in claim 1 , wherein the protein of interest is fused to multiple peptide tags.
12 . The method as claimed in claim 11 wherein the protein of interest is fused to multiple GFP 11 peptide tags or multiple sfCherry 11 peptide tags.
13 . The method as claimed in claim 11 , wherein the protein of interest is fused to one or more sfCherry 11 peptide tags and one or more GFP 11 peptide tags.
14 . The method as claimed in claim 11 , wherein the multiple peptide tags are fused to the peptide of interest in a tandem fashion.
15 . The method as claimed in claim 1 , wherein the protein of interest is a single.
16 . The method as claimed in claim 15 , wherein the single protein of interest is a TdT, IFN beta 1-alpha, VEGF or is a protein part of a protein-protein complex or larger assembly.
17 . The method of claim 1 , wherein the surfactant in the oil layer is a non-ionic surfactant.
18 . The method according to claim 1 , wherein the surfactant is a sorbitan ester.
19 . The method according to claim 18 , wherein the surfactant is sorbitan trioleate.
20 . The method as claimed in claim 16 , wherein the protein of interest is a terminal deoxynucleotidyl transferase (TdT) enzyme or a truncated version thereof or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species or the homologous amino acid sequence of Polμ, Polβ, Polλ, and Polθ of any species or the homologous amino acid sequence of X family polymerases of any species.