# US 2020/0399632 A1 · Sensor systems

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- Number: US 2020/0399632 A1
- Document ID: US20200399632A1
- Status: Published application
- Kind: A1
- Application number: 16/970,132
- Filed: 2019-02-15
- Published: 2020-12-24
- Earliest claimed: 2018-02-15
- Assignees: enEvolv, Inc.
- Applicants: enEvolv, Inc.
- Inventors: Matthew R. DUNN; Kristin J. ADOLFSEN; Noah D. TAYLOR; James E. SPOONAMORE; Jay H. KONIECZKA; Caitlin D. ALLEN; Lindong WENG
- Web page: https://patentgrep.com/patent/US20200399632A1

## CPC

- C40B 40/02: Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
- B01F 23/41: Emulsifying
- C12N 1/02: Separating microorganisms from their culture media
- C12N 15/1034: {Isolating an individual clone by screening libraries}
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- C12N 15/1075: {by coupling phenotype to genotype, not provided for in other groups of this subclass}
- C12N 15/1086: {Preparation or screening of expression libraries, e.g. reporter assays}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12Q 1/6897: involving reporter genes operably linked to promoters
- G01N 21/64: Fluorescence; Phosphorescence
- G01N 33/582: {with fluorescent label}
- G01N 15/1459: {the analysis being performed on a sample stream}
- G01N 15/1492: within droplets
- G01N 2015/1006: {for cytology}
- G01N 2015/1481: {Optical analysis of particles within droplets (sorting particles within droplets G01N15/1492)}
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present technology relates to methods and compositions that provide for improved detection of target molecules in, for example, bioengineering.

## Claims

### Claim 1

A method for producing a population of engineered producer cells comprising:
encapsulating each producer cell from a pool of genetically varied producer cells in a droplet to form a plurality of droplets encapsulating engineered producer cells;
assaying the droplets for levels of a target molecule, wherein an engineered protein-based sensor provides a readout of the level of the desired target molecule produced by the producer cell through activation or repression of a reporter;
isolating droplets with producer cells that produce desired levels of the target molecule; and,
recovering the cells that produce desired levels of the target molecule to form the population of producer cells, wherein the population of producer cells is an enriched population that produce desired levels of the target molecule.

### Claim 2 (depends on 1)

The method of claim 1, wherein the recovery comprises:
(a) breaking the droplets,
(b) sorting the genetically varied producer cells, and
(c) growing the the producer cells on a growth medium.

### Claim 3 (depends on 2)

The method of claim 2, wherein the sorting is by fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS).

### Claim 4 (depends on 2, 3)

The method of claim 2 or 3, wherein the recovery comprises:
(a) sorting the droplets,
(b) sorting the genetically varied producer cells, and
(c) growing the the producer cells on a growth medium.

### Claim 5 (depends on 4)

The method of claim 4, wherein the sorting is by fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS).

### Claim 6 (depends on 2, 3, 4, 5)

The method of any one of claims 2-5, wherein breaking the droplets comprises breaking the droplets encapsulating isolated engineered producer cells that produce desired levels of the target molecule to form the population of engineered producer cells, wherein the population of engineered producer cells is an enriched population of engineered producer cells that produce desired levels of the target molecule.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The methods of any one of claims 1-6, wherein the DNA encoding the engineered protein-based sensor is encoded episomally.

### Claim 8 (depends on 7)

The method of claim 7, wherein the DNA encoding the engineered protein-based sensor is encoded on a plasmid.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6)

The methods of any one of claims 1-6, wherein the DNA encoding the engineered protein-based sensor is integrated in the genome of the producer cell.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The methods of any one of claims 1-9, wherein the engineered protein-based sensor is or has been transfected, transduced, transformed, or otherwise made available inside the producer cells.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The methods of any one of claims 1-10, wherein the reporter is a gene encoding a detectable marker that is activated in trans by the sensor-based protein.

### Claim 12 (depends on 11)

The method of claim 11, wherein the detectable marker is an enzyme or a selectable marker.

### Claim 13 (depends on 12)

The method of claim 12, wherein the enzyme is selected from lacZ, luciferase, or alkaline phosphatase.

### Claim 14 (depends on 12)

The method of claim 12, wherein the selectable marker is an auxotroph, antibiotic, resistance marker, a toxin, or a spectrally detectable gene product.

### Claim 15 (depends on 12)

The method of claim 12, wherein the selectable marker is a fluorescent protein.

### Claim 16 (depends on 14)

The method of claim 14, wherein the spectrally detectable gene product is detected by spectroscopy or spectrometry.

### Claim 17 (depends on 11)

The method of claim 11, wherein the gene encoding the reporter is encoded episomally.

### Claim 18 (depends on 17)

The method of claim 17, wherein the gene encoding the reporter is encoded episomally on a plasmid.

### Claim 19 (depends on 18)

The method of claim 18, wherein the gene encoding the reporter is encoded on the same plasmid as the gene encoding the engineered protein-based sensor.

### Claim 20 (depends on 11, 12, 13, 14, 15, 16, 17, 18)

The method of any one of claims 11-18, wherein the gene encoding the reporter is integrated in the genome.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The method of any one of claims 1-20, further comprising producing an engineered producer strain library from which the pool of engineered producer cells is taken, wherein the engineered producer strain library is engineered to produce one or more target molecules.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)

The methods of any one of claims 1-21, wherein engineered producer strain library is generated through genomic diversifying technology selected from multiplex automated genome-engineering (MAGE), plasmid-based production variation, or by non-GMO methods, wherein non-GMO methods are selected from chemical mutagenesis, radiation, and transposons.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The methods of any one of claims 1-22, wherein the droplets encapsulating isolated engineered producer cells further comprise growth medium and any required inducing agents.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The methods of any one of claims 1-23, wherein the readout level provided by the engineered protein sensor is by a reporter.

### Claim 25 (depends on 24)

The method of claim 24, wherein the reporter is GFP.

### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)

The method of any one of claims 1-25, wherein the engineered protein sensor is a transcription factor.

### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

The method of any one of claims 1-26, wherein the transcription factor is an allosteric transcription factor (aTF).

### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

The method of any one of claims 1-27, wherein the engineered protein sensor is an engineered prokaryotic transcriptional regulator family member selected from a LysR, AraC/XylS, TetR, LuxR, LacI, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.

### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

The method of any one of claims 1-27, wherein the engineered protein sensor is an engineered aTF listed in Table 1 (aTF (“Chassis”).

### Claim 30 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)

The method of any one of claims 1-29, wherein the target molecule is selected from the target molecules listed in Table 1 (Target Molecule Property).

### Claim 31

A method for producing a population of engineered producer cells comprising:
transforming a pool of engineered producer cells with an engineered sensor plasmid, wherein the engineered sensor plasmid encodes an engineered protein sensor;
encapsulating each producer cell from the pool of genetically varied producer cells in a droplet to form a plurality of droplets encapsulating engineered producer cells;
assaying the droplets for levels of a target molecule, wherein an engineered protein-based sensor provides a readout of the level of the target molecule produced by the producer cell through activation or repression of a reporter;
isolating the droplets with producer cells that produce desired levels of the target molecule; and,
recovering the cells that produce desired levels of the target molecule to form the population of producer cells, wherein the population of producer cells is an enriched population that produce desired levels of the target molecule.

### Claim 32 (depends on 31)

The method of claim 31, wherein the recovery comprises:
(a) breaking the droplets,
(b) sorting the genetically varied producer cells, and
(c) growing the the producer cells on a growth medium.

### Claim 33 (depends on 32)

The method of claim 32, wherein the sorting is by fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS).

### Claim 34 (depends on 33)

The method of claim 33, wherein the recovery comprises:
(a) sorting the droplets,
(b) sorting the genetically varied producer cells, and
(c) growing the the producer cells on a growth medium.

### Claim 35 (depends on 34)

The method of claim 34, wherein the sorting is by fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS).

### Claim 36 (depends on 32, 33, 34, 35)

The method of any one of claims 32-35, wherein breaking the droplets comprises breaking the droplets encapsulating isolated engineered producer cells that produce desired levels of the target molecule to form the population of engineered producer cells, wherein the population of engineered producer cells is an enriched population of engineered producer cells that produce desired levels of the target molecule.

### Claim 37 (depends on 31, 32, 33, 34, 35, 36)

The methods of any one of claims 31-36, wherein the DNA encoding the engineered protein-based sensor is encoded episomally.

### Claim 38 (depends on 37)

The method of claim 37, wherein the DNA encoding the engineered protein-based sensor is encoded on a plasmid.

### Claim 39 (depends on 31, 32, 33, 34, 35, 36)

The methods of any one of claims 31-36, wherein the DNA encoding the engineered protein-based sensor is integrated in the genome of the producer cell.

### Claim 40 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39)

The methods of any one of claims 31-39, wherein the engineered protein-based sensor is or has been transfected, transduced, transformed, or otherwise made available inside the producer cells.

### Claim 41 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)

The methods of any one of claims 31-40, wherein the reporter is a gene encoding a detectable marker that is activated in trans by the sensor-based protein.

### Claim 42 (depends on 41)

The method of claim 41, wherein the detectable marker is an enzyme or a selectable marker.

### Claim 43 (depends on 42)

The method of claim 42, wherein the enzyme is selected from lacZ, luciferase, or alkaline phosphatase.

### Claim 44 (depends on 42)

The method of claim 42, wherein the selectable marker is an auxotroph, antibiotic, resistance marker, a toxin, or a spectrally detectable gene product.

### Claim 45 (depends on 42)

The method of claim 42, wherein the selectable marker is a fluorescent protein.

### Claim 46 (depends on 44)

The method of claim 44, wherein the spectrally detectable gene product is detected by spectroscopy or spectrometry.

### Claim 47 (depends on 41)

The method of claim 41, wherein the gene encoding the reporter is encoded episomally.

### Claim 48 (depends on 47)

The method of claim 47, wherein the gene encoding the reporter is encoded episomally on a plasmid.

### Claim 49 (depends on 48)

The method of claim 48, wherein the gene encoding the reporter is encoded on the same plasmid as the gene encoding the engineered protein-based sensor.

### Claim 50 (depends on 41, 42, 43, 44, 45, 46, 47, 48)

The method of any one of claims 41-48, wherein the gene encoding the reporter is integrated in the genome.

### Claim 51 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)

The method of any one of claims 31-50, further comprising producing an engineered producer strain library from which the pool of engineered producer cells is taken, wherein the engineered producer strain library is engineered to produce one or more target molecules.

### Claim 52 (depends on 51)

The method of claim 51, wherein the engineered producer strain library is produced before transforming the pool of engineered producer cells with an engineered sensor plasmid.

### Claim 53 (depends on 51)

The method of claim 51, wherein the engineered producer strain library is produced after transforming the pool of engineered producer cells with an engineered sensor plasmid.

### Claim 54 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53)

The method of any one of claims 31-53, wherein engineered producer strain library is generated through genomic diversifying technology selected from multiplex automated genome-engineering (MAGE), plasmid-based production variation, or by non-GMO methods, wherein non-GMO methods are selected from chemical mutagenesis, radiation, and transposons.

### Claim 55 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)

The methods of any one of claims 31-54, wherein the droplets encapsulating isolated engineered producer cells further comprise growth medium and any required inducing agents.

### Claim 56 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55)

The methods of any one of claims 31-55, wherein the readout level provided by the engineered protein sensor is by a reporter.

### Claim 57 (depends on 56)

The method of claim 56, wherein the reporter is GFP.

### Claim 58 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57)

The method of any one of claims 31-57, wherein the engineered protein sensor is a transcription factor.

### Claim 59 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The method of any one of claims 31-58, wherein the transcription factor is an allosteric transcription factor (aTF).

### Claim 60 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)

The method of any one of claims 31-59, wherein the engineered protein sensor is an engineered prokaryotic transcriptional regulator family member selected from a LysR, AraC/XylS, TetR, LuxR, LacI, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.

### Claim 61 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The method of any one of claims 31-58, wherein the engineered protein sensor is an engineered aTF listed in Table 1 (aTF (“Chassis”).

### Claim 62 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

The method of any one of claims 31-61, wherein the target molecule is selected from the target molecules listed in Table 1 (Target Molecule Property).

### Claim 63

A method for producing a population of engineered producer cells comprising:
encapsulating each producer cell from the pool of genetically varied producer cells in a droplet to form a plurality of droplets encapsulating engineered producer cells;
merging each droplet containing the producer cell with a droplet encapsulating an engineered-protein based sensor cell, wherein the engineered sensor cell produces an engineered protein sensor;
assaying the droplets for levels of a target molecule, wherein an engineered protein-based sensor provides a readout of the level of the desired target molecule produced by the producer cell through activation or repression of a reporter;
sorting the merged droplets to isolate droplets containing producer cells that produce desired levels of the target molecule; and,
recovering the cells that produce desired levels of the target molecule to form the population of producer cells, wherein the population of producer cells is an enriched population that produce desired levels of the target molecule.

### Claim 64 (depends on 63)

The method of claim 63, wherein the recovery comprises:
(a) breaking the droplets,
(b) sorting the genetically varied producer cells, and
(c) growing the the producer cells on a growth medium.

### Claim 65 (depends on 64)

The method of claim 64, wherein the sorting is by fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS).

### Claim 66 (depends on 65)

The method of claim 65, wherein the recovery comprises:
(a) sorting the droplets,
(b) sorting the genetically varied producer cells, and
(c) growing the the producer cells on a growth medium.

### Claim 67 (depends on 66)

The method of claim 66, wherein the sorting is by fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS).

### Claim 68 (depends on 64, 65, 66, 67)

The method of any one of claims 64-67, wherein breaking the droplets comprises breaking the droplets encapsulating isolated engineered producer cells that produce desired levels of the target molecule to form the population of engineered producer cells, wherein the population of engineered producer cells is an enriched population of engineered producer cells that produce desired levels of the target molecule.

### Claim 69 (depends on 63, 64, 65, 66, 67, 68)

The methods of any one of claims 63-68, wherein the DNA encoding the engineered protein-based sensor is encoded episomally.

### Claim 70 (depends on 69)

The method of claim 69, wherein the DNA encoding the engineered protein-based sensor is encoded on a plasmid.

### Claim 71 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78)

The methods of any one of claims 63-78, wherein the DNA encoding the engineered protein-based sensor is integrated in the genome of the producer cell.

### Claim 72 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71)

The methods of any one of claims 1-71, wherein the engineered protein-based sensor is or has been transfected, transduced, transformed, or otherwise made available inside the producer cells.

### Claim 73 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72)

The methods of any one of claims 1-72, wherein the reporter is a gene encoding a detectable marker that is activated in trans by the sensor-based protein.

### Claim 74 (depends on 73)

The method of claim 73, wherein the detectable marker is an enzyme or a selectable marker.

### Claim 75 (depends on 74)

The method of claim 74, wherein the enzyme is selected from lacZ, luciferase, or alkaline phosphatase.

### Claim 76 (depends on 74)

The method of claim 74, wherein the selectable marker is an auxotroph, antibiotic, resistance marker, a toxin, or a spectrally detectable gene product.

### Claim 77 (depends on 74)

The method of claim 74, the selectable marker is a fluorescent protein.

### Claim 78 (depends on 76)

The method of claim 76, wherein the spectrally detectable gene product is detected by spectroscopy or spectrometry.

### Claim 79 (depends on 73)

The method of claim 73, wherein the gene encoding the reporter is encoded episomally.

### Claim 80 (depends on 79)

The method of claim 79, wherein the gene encoding the reporter is encoded episomally on a plasmid.

### Claim 81 (depends on 80)

The method of claim 80, wherein the gene encoding the reporter is encoded on the same plasmid as the gene encoding the engineered protein-based sensor.

### Claim 82 (depends on 73, 74, 75, 76, 77, 78, 79, 80)

The method of any one of claims 73-80, wherein the gene encoding the reporter is integrated in the genome.

### Claim 83 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)

The method of any one of claims 63-82, further comprising producing an engineered producer strain library from which the pool of engineered producer cells is taken, wherein the engineered producer strain library is engineered to produce one or more target molecules.

### Claim 84 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83)

The methods of any one of claims 63-83, wherein engineered producer strain library is generated through genomic diversifying technology selected from multiplex automated genome-engineering (MAGE), plasmid-based production variation, or by non-GMO methods, wherein non-GMO methods are selected from chemical mutagenesis, radiation, and transposons.

### Claim 85 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84)

The methods of any one of claims 63-84, wherein the droplets encapsulating isolated engineered producer cells further comprise growth medium and any required inducing agents.

### Claim 86 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85)

The methods of any one of claims 63-85, wherein the readout level provided by the engineered protein sensor is by a reporter.

### Claim 87 (depends on 86)

The method of claim 86, wherein the reporter is GFP.

### Claim 88 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87)

The method of any one of claims 63-87, wherein the engineered protein sensor is a transcription factor.

### Claim 89 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88)

The method of any one of claims 63-88, wherein the transcription factor is an allosteric transcription factor (aTF).

### Claim 90 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89)

The method of any one of claims 63-89, wherein the engineered protein sensor is an engineered prokaryotic transcriptional regulator family member selected from a LysR, AraC/XylS, TetR, LuxR, LacI, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.

### Claim 91 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89)

The method of any one of claims 63-89, wherein the engineered protein sensor is an engineered aTF listed in Table 1 (aTF (“Chassis”).

### Claim 92 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91)

The method of any one of claims 63-91, wherein the target molecule is selected from the target molecules listed in Table 1 (Target Molecule Property).

### Claim 93

A method for producing a population of engineered producer cells comprising:
transforming a pool of engineered producer cells with an engineered sensor plasmid, wherein the engineered sensor plasmid encodes an engineered protein sensor;
encapsulating each producer cell from a pool of genetically varied producer cells in a droplet to form a plurality of droplets encapsulating engineered producer cells;
wherein each droplet is surrounded by an immiscible continuous phase that comprises a fluorinated-based oil or emulsion;
assaying the droplets for levels of a target molecule, wherein an engineered protein-based sensor provides a readout of the level of the desired target molecule produced by the producer cell through activation or repression of a reporter;
isolating droplets with producer cells that produce desired levels of the target molecule;
recovering the cells that produce desired levels of the target molecule to form the population of producer cells, wherein the population of producer cells is an enriched population that produce desired levels of the target molecule.

### Claim 94

A method for producing a population of engineered producer cells comprising:
encapsulating each producer cell from the pool of genetically varied producer cells in a droplet to form a plurality of droplets encapsulating engineered producer cells; wherein each droplet is:
(a) surrounded by an immiscible continuous phase that comprises a fluorinated-based oil or emulsion, and
(b) comprises an engineered sensor cell, wherein the engineered sensor cell produces an engineered protein sensor;
assaying the droplets for levels of a target molecule, wherein an engineered protein-based sensor provides a readout of the level of the target molecule produced by the producer cell through activation or repression of a reporter;
isolating the droplets with producer cells that produce desired levels of the target molecule;
recovering the cells that produce desired levels of the target molecule to form the population of producer cells, wherein the population of producer cells is an enriched population that produce desired levels of the target molecule.

### Claim 95

A method for producing a population of engineered producer cells comprising:
encapsulating each producer cell from the pool of genetically varied producer cells in a droplet to form a plurality of droplets encapsulating engineered producer cells;
wherein each droplet is surrounded by an immiscible continuous phase that comprises a fluorinated-based oil or emulsion;
merging each droplet containing the producer cell with a droplet encapsulating an engineered-protein based sensor cell, wherein the engineered sensor cell produces an engineered protein sensor;
assaying the droplets for levels of a target molecule, wherein an engineered protein-based sensor provides a readout of the level of the desired target molecule produced by the producer cell through activation or repression of a reporter;
sorting the merged droplets to isolate droplets containing producer cells that produce desired levels of the target molecule; and,
recovering the cells that produce desired levels of the target molecule to form the population of producer cells, wherein the population of producer cells is an enriched population that produce desired levels of the target molecule.

### Claim 96 (depends on 93, 94, 95)

The method of any one claims 93-95, wherein the fluorinated-based oil or emulsion is an organic oil, a fluorinated oil, a fluorinated polymer, a water-in fluorocarbon emulsion, a water-in perfluorocarbon emulsion, or combinations thereof.

### Claim 97 (depends on 93, 94, 95, 96)

The method of any one claims 93-96, wherein the fluorinated-based oil or emulsion is stabilized by a particle.

### Claim 98 (depends on 97)

The method of claim 97, wherein the particle is a partially fluorinated nanoparticle or a partially hydrophobic nanoparticle.

### Claim 99 (depends on 98)

The method of claim 98, wherein the partially fluorinated nanoparticle or partially hydrophobic nanoparticle is a silica-based nanoparticle.

### Claim 100 (depends on 93, 94, 95, 96, 97, 98, 99)

The method of any one claims 93-99, wherein the droplet is under microfluidic control.

### Claim 101 (depends on 93, 94, 95, 96, 97, 98, 99, 100)

The methods of any one of claims 93-100, further comprising producing an engineered producer strain library from which the pool of engineered producer cells is taken, wherein the engineered producer strain library is engineered to produce one or more target molecules.

### Claim 102 (depends on 101)

The method of claim 101, wherein engineered producer strain library is generated through genomic diversifying technology selected from multiplex automated genome-engineering (MAGE), plasmid-based production variation, or by non-GMO methods, wherein non-GMO methods are selected from chemical mutagenesis, radiation, and transposons.

### Claim 103 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102)

The methods of any one of claims 93-102, wherein the droplets encapsulating isolated engineered producer cells further comprise growth medium and any required inducing agents.

### Claim 104 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103)

The methods of any one of claims 93-103, wherein the readout level provided by the engineered protein sensor is by a reporter.

### Claim 105 (depends on 104)

The method of claim 104, wherein the reporter is GFP.

### Claim 106 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105)

The method of any one of claims 93-105, wherein the engineered protein sensor is a transcription factor.

### Claim 107 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106)

The method of any one of claims 93-106, wherein the transcription factor is an allosteric transcription factor (aTF).

### Claim 108 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107)

The method of any one of claims 93-107, wherein the engineered protein sensor is an engineered prokaryotic transcriptional regulator family member selected from a LysR, AraC/XylS, TetR, LuxR, LacI, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.

### Claim 109 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108)

The method of any one of claims 93-108, wherein the engineered protein sensor is an engineered aTF listed in Table 1.

### Claim 110 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109)

The method of any one of claims 93-109, wherein the target molecule is selected from the target molecules listed in Table 1 (Target Molecule Property).

### Claim 111 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110)

The method of any one of claims 93-110, wherein the recovery comprises:
(a) breaking the droplets,
(b) sorting the genetically varied producer cells, and
(c) growing the the producer cells on a growth medium.

### Claim 112 (depends on 111)

The method of claim 111, wherein the sorting is by fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS).

### Claim 113 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112)

The method of any one of claims 93-112, wherein the recovery comprises:
(a) sorting the droplets,
(b) sorting the genetically varied producer cells, and
(c) growing the the producer cells on a growth medium.

### Claim 114 (depends on 113)

The method of claim 113, wherein the sorting is by fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS).

### Claim 115 (depends on 111, 112, 113, 114)

The method of any one of claims 111-114, wherein breaking the droplets comprises breaking the droplets encapsulating isolated engineered producer cells that produce desired levels of the target molecule to form the population of engineered producer cells, wherein the population of engineered producer cells is an enriched population of engineered producer cells that produce desired levels of the target molecule.

### Claim 116 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115)

The methods of any one of claims 93-115, wherein the DNA encoding the engineered protein-based sensor is encoded episomally.

### Claim 117 (depends on 116)

The method of claim 116, wherein the DNA encoding the engineered protein-based sensor is encoded on a plasmid.

### Claim 118 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115)

The methods of any one of claims 93-115, wherein the DNA encoding the engineered protein-based sensor is integrated in the genome of the producer cell.

### Claim 119 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118)

The methods of any one of claims 93-118, wherein the engineered protein-based sensor is or has been transfected, transduced, transformed, or otherwise made available inside the producer cells.

### Claim 120 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119)

The methods of any one of claims 93-119, wherein the reporter is a gene encoding a detectable marker that is activated in trans by the sensor-based protein.

### Claim 121 (depends on 120)

The method of claim 120, wherein the detectable marker is an enzyme or a selectable marker.

### Claim 122 (depends on 121)

The method of claim 121, wherein the enzyme is selected from lacZ, luciferase, or alkaline phosphatase.

### Claim 123 (depends on 122)

The method of claim 122, wherein the selectable marker is an auxotroph, antibiotic, resistance marker, a toxin, or a spectrally detectable gene product.

### Claim 124 (depends on 120)

The method of claim 120, wherein the selectable marker is a fluorescent protein.

### Claim 125 (depends on 124)

The method of claim 124, wherein the spectrally detectable gene product is detected by spectroscopy or spectrometry.

### Claim 126 (depends on 120)

The method of claim 120, wherein the gene encoding the reporter is encoded episomally.

### Claim 127 (depends on 126)

The method of claim 126, wherein the gene encoding the reporter is encoded episomally on a plasmid.

### Claim 128 (depends on 127)

The method of claim 127, wherein the gene encoding the reporter is encoded on the same plasmid as the gene encoding the engineered protein-based sensor.

### Claim 129 (depends on 120, 121, 122, 123, 124, 125, 126)

The method of any one of claims 120-126, wherein the gene encoding the reporter is integrated in the genome.

### Claim 130 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)

The method of any one of claims 1-30, wherein the engineered protein-based sensor and reporter are encoded within the producer cell.

### Claim 131 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)

The method of any one of claims 1-30, wherein the engineered protein-based sensor and reporter are encoded within a co-encapsulated sensor cell.

### Claim 132 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)

The method of any one of claims 1-30, wherein the engineered protein-based sensor and reporter are encoded within a sensor cell which is encapsulated in a separate droplet, which is then merged with the droplet containing an engineered producer cell.

### Claim 133 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)

The method of any one of claims 31-62, wherein the engineered protein-based sensor and reporter are encoded within the producer cell.

### Claim 134 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)

The method of any one of claims 31-62, wherein the engineered protein-based sensor and reporter are encoded within a co-encapsulated sensor cell.

### Claim 135 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)

The method of any one of claims 31-62, wherein the engineered protein-based sensor and reporter are encoded within a sensor cell which is encapsulated in a separate droplet, which is then merged with the droplet containing an engineered producer cell.

### Claim 136 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92)

The method of any one of claims 63-92, wherein the engineered protein-based sensor and reporter are encoded within the producer cell.

### Claim 137 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92)

The method of any one of claims 63-92, wherein the engineered protein-based sensor and reporter are encoded within a co-encapsulated sensor cell.

### Claim 138 (depends on 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92)

The method of any one of claims 63-92, wherein the engineered protein-based sensor and reporter are encoded within a sensor cell which is encapsulated in a separate droplet, which is then merged with the droplet containing an engineered producer cell.

### Claim 139 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129)

The method of any one of claims 93-129, wherein the engineered protein-based sensor and reporter are encoded within the producer cell.

### Claim 140 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129)

The method of any one of claims 93-129, wherein the engineered protein-based sensor and reporter are encoded within a co-encapsulated sensor cell.

### Claim 141 (depends on 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129)

The method of any one of claims 93-129, wherein the engineered protein-based sensor and reporter are encoded within a sensor cell which is encapsulated in a separate droplet, which is then merged with the droplet containing an engineered producer cell.