# US 2021/0239694 A1 · Phage constructs for detecting bacteria in a fluid, microfluidic devices for use with constructs, and related methods

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- Number: US 2021/0239694 A1
- Document ID: US20210239694A1
- Status: Published application
- Kind: A1
- Application number: 17/049,280
- Filed: 2019-04-19
- Published: 2021-08-05
- Earliest claimed: 2018-04-20
- Applicants: Cornell University; Tokitae LLC
- Inventors: Luis F. ALONZO; Spencer GARING; Troy HINKLEY; Anne-Laure M. LE NY; Damian MADAN; Kevin Paul Flood NICHOLS; Sam Rasmussen NUGEN
- Web page: https://patentgrep.com/patent/US20210239694A1

## CPC

- C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)}
- G01N 33/56911: {Bacteria}
- C12N 7/025: {Packaging cell lines, e.g. transcomplementing cell lines, for production of virus}
- C12N 15/73: Expression systems using phage (lambda) regulatory sequences
- C12N 15/86: Viral vectors
- C12Q 1/10: Enterobacteria
- C12Q 1/66: involving luciferase
- G01N 33/54366: {Apparatus specially adapted for solid-phase testing}
- C12N 2795/10231: Uses of virus other than therapeutic or vaccine, e.g. disinfectant
- C12N 2795/10243: viral genome or elements thereof as genetic vector
- G01N 2333/245: Escherichia (G)
- G01N 2333/30: from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Generally, this disclosure relates to expression constructs that encode a reporter enzyme-affinity binding tag fusion protein that is produced after the construct is inserted into bacteriophage and the bacteriophage infects bacteria. In some embodiments, the fusion protein is captured and produces a detectable signal. Signal intensity may correlate with the number of bacterial cells in a fluid sample. Methods of detecting bacteria using the expression constructs, and microfluidic devices for detecting bacteria using the expression constructs are also disclosed.

## Claims

### Claim 1

A device for concentrating bacteria and bacterial products, the device comprising:
a first inlet fluidly coupled to a first immobilization region, the first immobilization region constructed of a material configured to concentrate the bacteria;
a second inlet fluidly coupled to the first immobilization region;
a third inlet fluidly coupled to the first immobilization region;
a fourth inlet fluidly coupled to a second immobilization region, the second immobilization region constructed of a material configured to concentrate bacterial products and different from the material of the first immobilization region.

### Claim 2 (depends on 1)

The device of claim 1, wherein the material of the first immobilization region includes a non-cellulosic material.

### Claim 3 (depends on 2)

The device of claim 2, wherein the non-cellulosic material includes polyvinylidene difluoride.

### Claim 4 (depends on 1)

The device of claim 1, wherein a surface area of the first immobilization region is greater than a surface area of the second immobilization region.

### Claim 5 (depends on 1)

The device of claim 1, wherein the bacterial products include a protein affinity tag.

### Claim 6 (depends on 5)

The device of claim 5, wherein the material of the first immobilization region is unable to bind the protein affinity tag.

### Claim 7 (depends on 5)

The device of claim 5, wherein the protein affinity tag includes a cellulose binding motif.

### Claim 8 (depends on 7)

The device of claim 7, wherein the material of the second immobilization region includes a cellulose-based material.

### Claim 9 (depends on 1)

The device of claim 1, wherein the first inlet is configured to accept a sample including bacteria.

### Claim 10 (depends on 1)

The device of claim 1, wherein the second inlet is configured to accept media for culturing the bacteria.

### Claim 11 (depends on 1)

The device of claim 1, wherein the third inlet is configured to accept bacteriophage capable of infecting the bacteria.

### Claim 12 (depends on 1)

The device of claim 1, wherein the bacterial products include a reporter enzyme and the fourth inlet is configured to accept a substrate compound for the reporter enzyme.

### Claim 13 (depends on 1)

The device of claim 1, wherein at least one of the first inlet fluidly coupled to the first immobilization region, the second inlet fluidly coupled to the first immobilization region, the third inlet fluidly coupled to the first immobilization region, and the fourth inlet fluidly coupled to the second immobilization region is fluidly coupled without a valve.

### Claim 14 (depends on 1)

The device of claim 1, wherein at least one of the first inlet fluidly coupled to the first immobilization region, the second inlet fluidly coupled to the first immobilization region, the third inlet fluidly coupled to the first immobilization region, and the fourth inlet fluidly coupled to the second immobilization region is fluidly coupled with a valve.

### Claim 15 (depends on 1)

The device of claim 1, further comprising at least one of a first outlet fluidly coupled to the first immobilization region, a second outlet fluidly coupled to the first immobilization region, and a third outlet fluidly coupled to the second immobilization region.

### Claim 16 (depends on 15)

The device of claim 15, wherein at least one of the first outlet fluidly coupled to the first immobilization region, the second outlet fluidly coupled to the first immobilization region, and the third outlet fluidly coupled to the second immobilization region is fluidly coupled without a valve.

### Claim 17 (depends on 15)

The device of claim 15, wherein at least one of the first outlet fluidly coupled to the first immobilization region, the second outlet fluidly coupled to the first immobilization region, and the third outlet fluidly coupled to the second immobilization region is fluidly coupled with a valve.

### Claim 18

A method of detecting bacteria, the method comprising:
providing a sample including bacteria;
isolating the bacteria;
incubating the bacteria for a first incubation period;
adding a bacteriophage to the bacteria, the bacteriophage including an expression construct that encodes a fusion protein;
incubating the bacteria for a second incubation period sufficient for the bacteriophage to infect the bacteria and the bacteria to express the fusion protein;
capturing the fusion protein; and
detecting the bacteria by detecting the fusion protein.

### Claim 19 (depends on 18)

The method of claim 18, wherein the method is performed on a device, the device including:
a first inlet for receiving the sample including bacteria;
a first immobilization region for isolating the bacteria, incubating the bacteria for the first incubation period, and incubating the bacteria for the second incubation period sufficient for the bacteriophage to infect the bacteria and the bacteria to express the fusion protein;
a third inlet for receiving bacteriophage to be added to the bacteria; and
a second immobilization region for capturing the fusion protein.

### Claim 20 (depends on 18)

The method of claim 18, wherein the fusion protein includes a reporter enzyme linked to a protein affinity tag.

### Claim 21 (depends on 20)

The method of claim 20, wherein capturing the fusion protein includes capturing the fusion protein by an interaction between the protein affinity tag and a complementary substrate.

### Claim 22 (depends on 18)

The method of claim 18, wherein during the first incubation period, at least one of a metabolic activity or a number of cells of the bacteria increases.

### Claim 23 (depends on 18)

The method of claim 18, wherein the first incubation period is for less than about 3 hours.

### Claim 24 (depends on 23)

The method of claim 23, wherein the first incubation period is for about 2 hours to about 3 hours.

### Claim 25 (depends on 18)

The method of claim 18, wherein the second incubation period is for less than about 60 minutes.

### Claim 26 (depends on 25)

The method of claim 25, wherein the second incubation period is for about 30 minutes to about 45 minutes.

### Claim 27 (depends on 18)

The method of claim 18, wherein the detecting the bacteria by detecting the fusion protein includes detecting the fusion protein by visualization with the naked eye, a luminometer, a fluorometer, or a phosphorimeter.

### Claim 28 (depends on 18)

The method of claim 18, wherein the detecting the bacteria by detecting the fusion protein has a lower detection limit of 1 CFU/100 mL to 10 CFU/100 mL.

### Claim 29 (depends on 18)

The method of claim 18, wherein the bacteriophage is specific to at least one bacterial species or strain of interest.

### Claim 30 (depends on 29)

The method of claim 29, wherein the bacteriophage includes a plurality of bacteriophage specific to a plurality of bacterial species or strains of interest.

### Claim 31

A method of immobilizing bacterial products for detection, the method comprising:
exposing a bacteriophage including an expression construct to bacteria, wherein the expression construct includes a reporter enzyme and a protein affinity tag;
incubating the bacteriophage and the bacteria for an incubation time sufficient for the bacteriophage to infect the bacteria and the bacteria to produce the reporter enzyme and the protein affinity tag as a fusion protein;
immobilizing the fusion protein by binding the protein affinity tag to a complementary substrate; and
detecting the bacteria by detecting the reporter enzyme of the fusion protein.

### Claim 32 (depends on 31)

The method of claim 31, wherein the reporter enzyme includes alkaline phosphatase, β-galactosidase, β-glucuronidase, green fluorescent protein, luciferase, neuraminidase, or a derivative of any of the foregoing.

### Claim 33 (depends on 31)

The method of claim 31, wherein the protein affinity tag includes a carbohydrate binding module, a chitin binding protein, glutathione-S-transferase, a His tag, a maltose binding protein, or a Strep-tag.

### Claim 34 (depends on 31)

The method of claim 31, wherein the protein affinity tag includes a carbohydrate binding module and the complementary substrate is cellulose.

### Claim 35 (depends on 31)

The method of claim 31, wherein the incubation time is less than about 60 minutes.

### Claim 36 (depends on 35)

The method of claim 35, wherein the incubation time is about 30 minutes to about 45 minutes.

### Claim 37 (depends on 31)

The method of claim 31, wherein the detecting the bacteria by detecting the reporter enzyme of the fusion protein includes detecting the reporter enzyme by visualization with the naked eye, a luminometer, a fluorometer, or a phosphorimeter.

### Claim 38 (depends on 31)

The method of claim 31, wherein the detecting has a lower detection limit of 1 CFU/100 mL to 10 CFU/100 mL.

### Claim 39

An expression construct, comprising:
a phage-specific promoter;
a reporter enzyme gene operably linked to the promoter, the reporter enzyme gene selected from alkaline phosphatase, β-galactosidase, β-glucuronidase, green fluorescent protein, luciferase, neuraminidase, and a derivative of any of the foregoing; and
at least a portion of a protein affinity tag gene downstream of the reporter enzyme gene and operably linked to the promoter, the protein affinity tag gene selected from a carbohydrate binding module, a chitin binding protein, glutathione-S-transferase, a His tag, a maltose binding protein, and a Strep-tag.

### Claim 40 (depends on 39)

The expression construct of claim 39, further comprising a ribosome binding site and a periplasm-directing leader sequence.

### Claim 41

A polynucleotide encoding a detectable fusion protein, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1.

### Claim 42 (depends on 41)

An expression construct comprising the polynucleotide of claim 41.