# US 2020/0399679 A1 · Methods and kits for using recombinant microorganisms as direct reagents in biological applications > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399679 A1 - Document ID: US20200399679A1 - Status: Published application - Kind: A1 - Application number: 16/764,642 - Filed: 2018-11-15 - Published: 2020-12-24 - Earliest claimed: 2017-11-15 - Assignees: Board of Regents, The University of Texas System - Applicants: Board of Regents, the University of Texas System - Inventors: Andrew ELLINGTON; Sanchita BHADRA; Jared ELLEFSON; Jimmy GOLLIHAR; Arti POTHUKUCHY; Michelle BYROM; Raghav SHROFF - Web page: https://patentgrep.com/patent/US20200399679A1 ## CPC - C12M 1/34: Measuring or testing with condition measuring or sensing means, e.g. colony counters - C12Q 1/686: Polymerase chain reaction [PCR] - C12N 1/06: Lysis of microorganisms - C12N 9/1007: {Methyltransferases (general) (2.1.1.)} - C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7) - C12N 9/16: acting on ester bonds (3.1) - C12N 9/24: acting on glycosyl compounds (3.2) - C12N 9/90: Isomerases (5.) - C12N 9/93: {Ligases (6)} - C12N 11/12: Cellulose or derivatives thereof - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - 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 - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Disclosed herein is a method of utilizing an enzyme in a nucleic acid manipulation process, the method comprising: a) transforming a microorganism with a non-native enzyme; b) inducing expression of the enzyme in the microorganism, thereby producing the non-native enzyme; c) adding the microorganism of step b) directly to a non-naturally occurring nucleic acid manipulation process, wherein the non-native enzyme is not purified from the microorganism prior to addition to the nucleic acid manipulation process; and carrying out the nucleic acid manipulation process using the enzyme. Importantly, this method can be carried out without the need to purify the enzyme from the cell producing it before it is used in the nucleic acid manipulation method. Also disclosed herein is a kit for carrying out a nucleic acid manipulation process, the kit comprising a) a microorganism expressing a non-native enzyme; b) nucleic acids of interest; and c) reagents for use in the nucleic acid manipulation process. ## Claims ### Claim 1 A method of utilizing an enzyme in a nucleic acid manipulation process, the method comprising: a. transforming a microorganism with a non-native enzyme; b. inducing expression of the enzyme in the microorganism, thereby producing the non-native enzyme; c. adding the microorganism of step b) directly to a non-naturally occurring nucleic acid manipulation process, wherein the non-native enzyme is not purified from the microorganism prior to addition to the nucleic acid manipulation process; d. carrying out the nucleic acid manipulation process using the enzyme. ### Claim 2 (depends on 1) The method of claim 1, wherein the enzyme is necessary for the nucleic acid manipulation process. ### Claim 3 (depends on 1) The method of claim 1, wherein the enzyme comprises polymerase, reverse transcriptase, methylase, nuclease, cleavase, phosphatase, kinase, nickase, pyrophosphatase, DNA glycosylase, recombinase, helicase, topoisomerase, methyltransferase, capping enzyme, deadenylase, or ligase. ### Claim 4 (depends on 1) The method of claim 1, wherein the process is nucleic acid amplification. ### Claim 5 (depends on 4) The method of claim 4, wherein the nucleic acid amplification is thermostable amplification. ### Claim 6 (depends on 4) The method of claim 4, wherein the nucleic acid amplification is isothermal amplification. ### Claim 7 (depends on 1) The method of claim 1, wherein more than one enzyme is transformed into the microorganism. ### Claim 8 (depends on 1) The method of claim 1, wherein multiple enzymes are transformed into multiple microorganisms, and multiple microorganisms are added to the non-naturally occurring molecular process. ### Claim 9 (depends on 1) The method of claim 1, wherein the nucleic acid manipulation process comprises further components needed to carry out the nucleic acid manipulation process. ### Claim 10 (depends on 9) The method of claim 9, wherein the further components are provided exogenously during the nucleic acid manipulation process. ### Claim 11 (depends on 9) The method of claim 9, wherein the further components are naturally occurring products produced by the microorganism. ### Claim 12 (depends on 1) The method of claim 1, wherein the microorganism is lysed prior to addition to the molecular process. ### Claim 13 (depends on 1) The method of claim 1, wherein the microorganism is lyophilized prior to addition to the molecular process. ### Claim 14 (depends on 13) The method of claim 13, wherein the microorganism is lyophilized onto a solid support. ### Claim 15 (depends on 14) The method of claim 14, wherein the solid support is paper. ### Claim 16 (depends on 1) The method of claim 1, wherein the microorganism is prokaryotic. ### Claim 17 (depends on 1) The method of claim 1, wherein the microorganism is eukaryotic. ### Claim 18 A kit for a nucleic acid manipulation process, the kit comprising a) a microorganism expressing a non-native enzyme; b) nucleic acids of interest; c) reagents for use in the nucleic acid manipulation process. ### Claim 19 (depends on 18) The kit of claim 18, wherein the nucleic acid manipulation process is nucleic acid amplification. ### Claim 20 (depends on 18) The kit of claim 18, wherein the microorganism is prokaryotic. ### Claim at position 21 21-28. (canceled)