# US 6,544,729 B2 · Bioluminescent biosensor device > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 6,544,729 B2 - Document ID: US6544729B2 - Status: Granted - Kind: B2 - Application number: 09/910,360 - Filed: 2001-07-20 - Published: 2003-02-06 - Granted: 2003-04-08 - Earliest claimed: 2001-07-20 - Assignees: University of Tennessee; Purdue Research Foundation - Inventors: Gary S. Sayler; Steven A. Ripp; Bruce Applegate - Web page: https://patentgrep.com/patent/US6544729B2 ## CPC - C12N 15/86: Viral vectors - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - 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)} - C12Q 1/6897: involving reporter genes operably linked to promoters - G01N 21/763: {Bioluminescence} - G01N 33/56911: {Bacteria} - C12N 2795/10343: viral genome or elements thereof as genetic vector - G01N 35/00069: {whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk} - 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 Disclosed are methods and devices for detection of bacteria based on recognition and infection of one or more selected strains of bacteria with bacteriophage genetically modified to cause production of an inducer molecule in the bacterium following phage infection. The inducer molecule is released from the infected bacterium and is detected by genetically modified bacterial bioreporter cells designed to emit bioluminescence upon stimulation by the inducer. Autoamplification of the bioluminescent signal permits detection of low levels of bacteria without sample enrichment. Also disclosed are methods of detection for select bacteria, and kits for detection of select bacteria based on the described technology. ## Claims ### Claim 1 A biosensor/bioreporter device for detecting a bacterial target cell population comprising: (a) a genetically engineered bacterial bioluminescent bioreporter cell population responsive to an inducer; (b) at least one bacteriophage capable of specifically infecting said bacterial target cell but not the bioreporter cell, said bacteriophage being genetically modified to cause the target cell to express said inducer upon infection of the target cell; and (c) at least one container suitable for housing the bioreporter cell population, target cell population and bacteriophage, said container being operably connected to a light measuring device selected from the group consisting of an integrated circuit, a photomultiplier tube, a photodiode, a microchannel plate, and a charge-coupled device, said light-measuring device being adapted to detect a bioluminescent signal generated in said container upon interaction of the bioreporter cell population and the inducer, wherein detecting the bioluminescent signal is correlated with detecting the presence of the target cell. ### Claim 2 (depends on 1) The biosensor/bioreporter device of claim 1 wherein the genetically engineered bioluminescent bioreporter cell population contains a luxR-luxpro/lux I/luxCDABE gene construct. ### Claim 3 (depends on 1) The biosensor/bioreporter device of claim 1 wherein the genetically modified bacteriophage contains a luxI gene. ### Claim 4 (depends on 1) The biosensor/bioreporter device of claim 1 wherein the infected target cell expresses a luxI gene product that induces the bioreporter cells to produce light. ### Claim 5 (depends on 1) The device of claim 1 wherein the genetically modified bacteriophage comprises a plurality of genetically modified bacteriophage, each specifically infecting a different target bacterium. ### Claim 6 (depends on 1) The device of claim 1 wherein the target bacterium is selected from the group consisting of Salmonella, Vibrio, Pseudomonas, Escherichia, Bacillus, Clostridium, Yersinia, Shigella, Legionella, Burkholderia, Staphylococcus, Streptococcus, Proteus, Enterobacter, Mycobacterium, Campylobacter, and Listeria. ### Claim 7 (depends on 1) The device of claim 1 wherein the genetically engineered bioreporter cells are E. coli. ### Claim 8 (depends on 1) A system comprising the device of claim 1, an automatic sampling device and an analysis component for data processing. ### Claim 9 (depends on 1) The device of claim 1 wherein the genetically engineered bioreporter cells are selected from the group consisting of Escherichia, Pseudomonas, Vibrio, Staphylococcus, Alcaligenes, Acinetobacter, Synechococcus, Aeromonas hydrophila and Ralstonia. ### Claim 10 (depends on 9) The device of claim 9 wherein the bioreporter cell population is held in an encapsulation matrix. ### Claim 11 (depends on 10) The device of claim 10 wherein the encapsulation matrix is alginate, carrageenan, acrylic vinyl acetate copolymer, latex, polyvinyl chloride polymer, sol-gel, agar, agarose, micromachined nanoporous membranes, polydimethylsiloxane (PDMS), polyacrylamide, polyurethane/polycarbomyl sulfonate, or polyvinyl alcohol. ### Claim 12 (depends on 1) The device of claim 1 wherein the light measuring device is an integrated circuit. ### Claim 13 (depends on 12) The device of claim 12 wherein the integrated circuit comprises a photodetector and low-noise electronics. ### Claim 14 (depends on 1, 12) The device of claim 1 or 12 wherein the container and the light measuring device are housed in a single unit. ### Claim 15 A method of detecting a selected target bacterium, comprising: providing a genetically engineered bacterial bioluminescent bioreporter cell population responsive to an inducer; providing a modified bacteriophage capable of specifically infecting said target bacterium but not the biorporter cells, said bacteriophage being genetically modified to cause the target bacterium to express said inducer upon infection of the target bacterium, and contacting a sample suspected of containing said target bacterium with said bacteriophage together with a population of said bioreporter bacterial cells, wherein a detectable bioluminescent signal is induced in said cell population subsequent to infection of the target bacterium with the bacteriophage, said detectable bioluminescent signal being correlated the presence of the target bacterium. ### Claim 16 (depends on 15) The method of claim 15 wherein said genetically modified bioreporter cells are modified to contain a luxR-luxpro/luxl/luxCDABE gene construct and said modified bacteriophage is a luxI-genetically modified bacteriophage. ### Claim 17 (depends on 15) The method of claim 15 wherein the selected target bacterium is selected from The group consisting of Escherichia coli, Listeria monocytogenes, Salmonella spp., Campylobacter spp., B. anthracis, B. thuringiensis and B. subtilis. ### Claim 18 (depends on 15) The method of claim 15 wherein the bioreporter bacterial cells are selected from the group consisting of Escherichia, Pseudomonas, Vibrio, Staphylococcus, Alcaligenes, Acinetobacter, Synechococcus, Aeromonas hydrophila and Ralstonia. ### Claim 19 (depends on 15) The method of claim 15 wherein the bioreporter bacterial cells are Pseudomonas fluorescens. ### Claim 20 A method of determining the amount of a selected bacterium in a sample comprising: contacting a sample suspected of containing a selected bacterium with: (a) a bacterium-specific bacteriophage genetically modified to cause the bacterium to express an inducer upon infection of the bacterium; and (b) a population of bacterial bioreporter cells responsive to the inducer; wherein the amount of the selected bacterium is determined by measuring the time required for production of a selected intensity of bioluminescent signal by the bioreporter cell population in the presence and in the absence of the selected bacterium; and relating the amount of said selected bacterium to the difference in time required for production of the selected intensity of bioluminescent signal by the bioreporter cell population in the presence and in the absence of the bacterium. ### Claim 21 A kit for detection of selected bacteria, comprising in suitable container form: (a) a genetically engineered bacteriophage that harbors a luxI gene and specifically infects a selected bacterium; (b) a bioreporter cell population genetically engineered to include a luxR-luxpro/luxI/luxCDABE gene construct, said bioreporter being incapable of infection by said bacteriophage; and (c) instructions for use. ### Claim 22 A kit for detection of a target bacterial cell comprising in suitable package form: (a) a genetically engineered bacteriophage that harbors a luxI gene and specifically infects a selected bacterium; (b) a bioreporter cell population genetically engineered to include a luxR-luxpro/luxI/luxCDABE gene construct, said bioreporter being incapable of infection by said bacteriophage; and (c) a device for measurement of generated light, said device comprising at least one container suitable for housing the bioreporter cell population, target cell population and bacteriophage, said container being operably connected to an integrated circuit adapted for detecting a luminescent signal produced when a bacteriophage-infected target cell induces a bioluminescent protein in the bioreporter cell population; and (d) instructions for use. ### Claim 23 A composition comprising: a bacterial bioreporter cell population genetically modified to contain a luxR-luxpro/luxI/luxCDABE gene construct; and a luxI-modified bacteriophage selective for an identified bacterium but not said bacterial bioreporter cell population. ### Claim 24 (depends on 23) The composition of claim 23 wherein the bacterial cell population is E. coli. ### Claim 25 (depends on 23) The composition of claim 23 wherein the bacteriophage is M13. ### Claim 26 (depends on 23) The composition of claim 23 wherein the luxI modified bacteriophage comprises a plurality of bacteriophage, each specific for a different bacterial cell host. ### Claim 27 A biosensor/bioreporter device for detecting a bacterial target cell population comprising: (a) a bacterial bioluminescent bioreporter cell population responsive to an acyl-homoserine lactone inducer, said bioreporter containing a luxR-luxpro/luxI/luxCDABE gene construct; (b) at least one bacteriophage capable of specifically infecting the bacterial target cell but not the bioreporter cell, said bacteriophage being genetically modified to cause the target cell to express said acyl-homoserine lactone inducer upon infection of the target cell; and (c) at least one container suitable for housing the bioreporter cell population, target cell population and bacteriophage, said container being operably connected to an integrated circuit adapted to detect a bioluminescent signal generated in said container upon interaction of the bioreporter cell population and the inducer, wherein detecting the bioluminescent signal is correlated with detecting the presence of the target cell.