CELL-FREE SENSOR SYSTEMS
The present described inventions relate, inter alia, to methods and compositions that provide for improved detection of target molecules in, for example, bioengineering.
1 . A method of making an allosteric DNA-binding protein sensor and/or switch which binds to a target molecule, comprising:
(a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a DNA-binding protein sensor and/or switch for an ability to bind a target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield a candidate allosteric DNA-binding protein sensor and/or switch having an ability to bind a target molecule;
(b) providing a host cell with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and a nucleic acid encoding a reporter gene system and selecting for a cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system;
(c) isolating nucleic acids from the cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture, the IVT or IVTT mixture comprising a target molecule and a detection reagent; and
(d) interrogating the IVT or IVTT mixture for reporter response, the reporter response being indicative of target molecule binding to the candidate allosteric DNA-binding protein sensor and/or switch.
2 . The method of claim 1 , wherein the allosteric DNA-binding protein sensor and/or switch is an engineered prokaryotic transcriptional regulator family member optionally selected from a LysR, AraC/XylS, TetR, LuxR, Lacl, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.
3 . The method of claim 1 or 2 , wherein the target molecule is a small molecule that is not a native ligand of the wild type candidate allosteric DNA-binding protein sensor and/or switch.
4 . The method of any one of the above claims, wherein the target molecule is an antibiotic.
5 . The method of any one of the above claims, wherein step (a) comprises mutating an allosteric protein.
6 . The method of any one of the above claims, wherein the nucleic acid is provided to the host cell by one or more of electroporation, chemical transformation, ballistic transformation, pressure induced transformation, electrospray injection, mechanical shear forces induced, for example, in microfluids, and carbon nanotubes, nanotube puncture, induced natural competence mechanisms of an organism, merging of protoplasts, and conjugation with Agrobacterium.
7 . The method of any one of the above claims, wherein the host cell is selected from a eukaryotic or prokaryotic cell, selected from a bacterial, yeast, algal, plant, insect, mammalian cells, and immortalized cell.
8 . The method of any one of the above claims, wherein the reporter gene system comprises a protein having a unique spectral signature and/or assayable enzymatic activity.
9 . The method of any one of the above claims, wherein the IVT or IVTT mixture comprises a coupled or linked system.
10 . The method of any one of the above claims, wherein the reporterresponse is a direct amplification of the genotype of the allosteric protein.
11 . The method of any one of the above claims, wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the nucleic acid encoding the reporter gene system comprises a single nucleic acid vector.
12 . The method of any one of the above claims, wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the nucleic acid encoding the reporter gene system comprises two nucleic acid vectors.
13 . The method of any one of the above claims, further comprising: (e) isolating the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch.
14 . The method of claim 13 , wherein the isolating comprises the use of flasks, culture tubes, and plastic ware, microliter plates, patterned microwells, or microdroplets generated either in bulk or microfluidically.
15 . A method of making an allosteric DNA-binding protein sensor and/or switch which binds to a target molecule, comprising:
(a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a DNA-binding protein sensor and/or switch for an ability to bind a target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield a DNA-binding protein sensor and/or switch which has an ability to bind a target molecule;
(b) providing a host cell with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and a nucleic acid encoding a reporter gene system and selecting for a cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system;
(c) isolating nucleic acids from the cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture, the IVT or IVTT mixture comprising a target molecule and a detection reagent; and
(d) interrogating the IVT or IVTT mixture by nucleic acid sequencing before and after selection to determine those molecules that have become functionally enriched.
16 . The method of claim 15 , wherein the allosteric DNA-binding protein sensor and/or switch is an engineered prokaryotic transcriptional regulator family member optionally selected from a LysR, AraC/XylS, TetR, LuxR, Lacl, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.
17 . The method of claim 15 or 16 , wherein the target molecule is a small molecule that is not a native ligand of the wild type candidate allosteric DNA-binding protein sensor and/or switch.
18 . The method of any one of claims 15 - 17 , wherein the target molecule is an antibiotic.
19 . The method of any one of claims 15 - 18 , wherein step (a) comprises mutating an allosteric protein.
20 . The method of any one of claims 15 - 19 , wherein the nucleic acid is provided to the host cell by one or more of electroporation, chemical transformation, ballistic transformation, pressure induced transformation, electrospray injection, mechanical shear forces induced, for example, in microfluids, and carbon nanotubes, nanotube puncture, induced natural competence mechanisms of an organism, merging of protoplasts, and conjugation with Agrobacterium.
21 . The method of any one of claims 15 - 20 , wherein the host cell is selected from a eukaryotic or prokaryotic cell, selected from a bacterial, yeast, algal, plant, insect, mammalian cells, and immortalized cell.
22 . The method of any one of claims 15 - 21 , wherein the reporter gene system comprises a protein having a unique spectral signature and/or assayable enzymatic activity.
23 . The method of any one of claims 15 - 22 , wherein the IVT or IVTT mixture comprises a coupled or linked system.
24 . The method of any one of claims 15 - 23 , wherein the reporter response is a direct amplification of the genotype of the allosteric protein.
25 . The method of any one of claims 15 - 24 , wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the nucleic acid encoding the reporter gene system comprises a single nucleic acid vector.
26 . The method of any one of claims 15 - 25 , wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the nucleic acid encoding the reporter gene system comprises two nucleic acid vectors.
27 . The method of any one of claims 15 - 26 , further comprising: (e) isolating the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch.
28 . The method of claim 27 , wherein the isolating comprises the use of flasks, culture tubes, and plastic ware, microliter plates, patterned microwells, or microdroplets generated either in bulk or microfluidically.
29 . A method of making an allosteric DNA-binding protein sensor and/or switch which binds to a target molecule, comprising:
(a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a DNA-binding protein sensor and/or switch for an ability to bind a target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield the candidate allosteric DNA-binding protein sensor and/or switch having an ability to bind a target molecule;
(b) contacting a solid support with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and selecting for a solid support comprising the candidate allosteric DNA-binding protein sensor and/or switch;
(c) isolating nucleic acids from the solid support comprising the candidate allosteric DNA-binding protein sensor and/or switch and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture;
(d) introducing a reporter gene system, detection reagent, and target molecule, and interrogating the mixture for a reporter response, the reporter response being indicative of the target molecule binding to the candidate allosteric DNA-binding protein sensor and/or switch.
30 . The method of claim 29 , wherein the solid support is a nanoparticle and a microparticle.
31 . The method of claim 29 , wherein the solid support is a bead, selected from a nanobead and a microbead.
32 . The method of claim 29 , wherein the solid support is an array.
33 . The method of any one of claims 29 - 32 , wherein the candidate allosteric DNA-binding protein sensor and/or switch is an engineered prokaryotic transcriptional regulator family member optionally selected from a LysR, AraC/XylS, TetR, LuxR, Lacl, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.
34 . The method of any one of claims 29 - 33 , wherein the target molecule is a small molecule that is not a native ligand of the wild type candidate allosteric DNA-binding protein sensor and/or switch.
35 . The method of any one of claims 29 - 33 , wherein the target molecule is an antibiotic.
36 . The method of any one of claims 29 - 35 , wherein step (a) comprises mutating an allosteric protein.
37 . The method of any one of claims 29 - 36 , wherein the reporter gene system comprises a protein having a unique spectral signature and/or assayable enzymatic activity.
38 . The method of any one of claims 29 - 37 , wherein the IVT or IVTT mixture comprises a coupled or linked system.
39 . The method of any one of claims 29 - 38 , wherein the reporter response is a direct amplification of the genotype of the allosteric protein.
40 . The method of any one of claims 29 - 39 , wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the nucleic acid encoding the reporter gene system comprises a single nucleic acid vector.
41 . The method of any one of claims 29 - 39 , wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the nucleic acid encoding the reporter gene system comprises two nucleic acid vectors.
42 . The method of any one of claims 29 - 41 , wherein the nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch comprises a synthetic DNA, amplified DNA, or amplified RNA.
43 . The method of any one of claims 29 - 42 , further comprising: (e) isolating the nucleic acid encoding the allosteric DNA-binding protein sensor and/or switch.
44 . The method of claim 43 , wherein the isolating comprises the use of flasks, culture tubes, and plastic ware, microliter plates, patterned microwells, or microdroplets generated either in bulk or microfluidically.
45 . A method for making a target molecule in a biological cell, comprising:
(a) engineering the biological cell to produce the target molecule;
(b) introducing an allosteric DNA-binding protein sensor and/or switch which binds to the target molecule in the biological cell; and
(c) screening for target molecule production.
46 . The method of claim 45 , wherein the biological cell is engineered to produce the target molecule by a multiplex genome engineering technique and/or a method involving a double-strand break (DSB) or single-strand break or nick.
47 . The method of claim 45 or 46 , wherein the allosteric DNA-binding protein sensor and/or switch which binds to the target molecule is produced by a method comprising:
(a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a candidate allosteric DNA-binding protein sensor and/or switch for an ability to bind the target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield the candidate allosteric DNA-binding protein sensor and/or switch having an ability to bind the target molecule;
(b) providing a host cell with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and a nucleic acid encoding the reporter gene system and selecting for a cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system;
(c) isolating nucleic acids from the cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture, the IVT or IVTT mixture comprising a target molecule and a detection reagent; and
(d) interrogating the IVT or IVTT mixture for reporter response, the reporter response being indicative of target molecule binding to the allosteric DNA-binding protein sensor and/or switch.
48 . The method of claim 45 or 46 , wherein the allosteric DNA-binding protein sensor and/or switch which binds to the target molecule is produced by a method comprising:
(a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a candidate allosteric DNA-binding protein sensor and/or switch for an ability to bind the target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield the candidate allosteric DNA-binding protein sensor and/or switch which has an ability to bind the target molecule;
(b) providing a host cell with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system and selecting for a cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system;
(c) isolating nucleic acids from the cell comprising the candidate allosteric DNA-binding protein sensor and/or switch and the reporter gene system and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture, the IVT or IVTT mixture comprising a target molecule and a detection reagent; and
(d) interrogating the IVT or IVTT mixture by nucleic acid sequencing before and after selection to determine those molecules that have become functionally enriched.
49 . The method of claim 45 or 46 , wherein the allosteric DNA-binding protein sensor and/or switch which binds to a target molecule is produced by a method comprising:
(a) constructing a candidate allosteric DNA-binding protein sensor and/or switch, the constructing comprising (i) designing a candidate allosteric DNA-binding protein sensor and/or switch for an ability to bind the target molecule, the designing optionally being in silico or (ii) undertaking directed or random mutagenesis to yield the candidate allosteric DNA-binding protein sensor and/or switch having an ability to bind the target molecule;
(b) contacting a solid support with a nucleic acid encoding the candidate allosteric DNA-binding protein sensor and/or switch and selecting for a solid support comprising the candidate allosteric DNA-binding protein sensor and/or switch;
(c) isolating nucleic acids from the solid support comprising the candidate allosteric DNA-binding protein sensor and/or switch and contacting the isolated nucleic acids with an in vitro transcription (IVT) or an in vitro transcription and translation (IVTT) mixture;
(d) introducing a reporter gene system, detection reagent, and target molecule, and interrogating the mixture for a reporter response, the reporter response being indicative of target molecule binding to the candidate allosteric DNA-binding protein sensor and/or switch.
50 . The method of claim 49 , wherein the solid support is a nanoparticle and a microparticle.
51 . The method of claim 49 , wherein the solid support is a bead, selected from a nanobead and a microbead.
52 . The method of claim 49 , wherein the solid support is an array.
53 . The method of any one of claims 45 - 52 , wherein the allosteric DNA-binding protein sensor and/or switch is an engineered prokaryotic transcriptional regulator family member optionally selected from a LysR, AraC/XylS, TetR, LuxR, Lacl, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.
55 . The method of any one of claims 45 - 53 , wherein the screening for target molecule comprises a positive or negative screen.
56 . The method of any one of claims 45 - 55 , wherein the allosteric DNA-binding protein sensor and/or switch is one or more of those of Table 1 and has about 1, or 2, or 3, or 4, or 5, or 10 mutations.