Multi-input / multi-output gene switch and its manufacturing method

Abstract

The present invention provides a method for producing a multi-input / multi-output gene switch or transcription factor, and a multi-input / multi-output gene switch or transcription factor. [Solution] We have completed a method for producing a multi-input / multi-output gene switch or transcription factor, the essential steps of which are "fusing two or more transcription factor genes" and "introducing mutations into the fused transcription factor gene," and furthermore, we have been able to obtain a multi-input / multi-output gene switch or transcription factor using this method.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a multi-input / multi-output gene switch or a transcription factor that forms the switch, as well as a multi-input / multi-output gene switch or a transcription factor that forms the switch obtained by the production method, and a fusion mutant thereof. This application claims priority from Japanese Patent Application No. 2018-057314, which is incorporated herein by reference. [Background technology]

[0002] (Current status of biosensors) The ability to measure the intracellular concentration of any substance, such as a metabolite or an environmental monitoring substance, in real time would greatly contribute to the development of life sciences. However, it is difficult to adjust the performance (sensitivity) of the sensor.

[0003] (Conventional biosensor configuration) Currently, biosensors are mainly constructed based on the following operating principles. "Gene-inducible" sensors place a reporter, such as a fluorescent protein, downstream of a transcriptional control mechanism that turns on / off its target compound as an inducer. Although transcription factors that respond to various molecules exist in nature, the sensor motifs for the vast majority of metabolites are unknown. Furthermore, even if a suitable sensor is found, it is unlikely to have sufficient performance for practical use in terms of response concentration, S / N ratio, and output intensity. A FRET sensor can be constructed using fluorescence resonance energy transfer (FRET) if it undergoes a significant structural change upon binding to a target molecule. However, the rational design of such a structural change is extremely difficult.

[0004] (prior art) The present inventors have disclosed a method for selecting a gene switch and a gene circuit, characterized in that an expression vector containing at least a gene sequence encoding thymidine kinase, preferably a human herpesvirus-derived thymidine kinase, and a promoter sequence operably linked to the gene sequence upstream of the gene sequence is used as a selector (see Patent Document 1). However, the method for selecting the genetic circuit is based on a different principle from the method for producing the multi-input / multi-output genetic switch of the present invention or the transcription factor that forms the switch.

[0005] The present inventors have disclosed that "ON selection can be performed in a short period of time by using as a selector an expression vector designed to enable the alkylating DNA repair enzyme alkyladenine DNA glycosidase (AAG) to operate on the output side, i.e., downstream, of a genetic circuit, and activating a genetic switch under conditions that induce cell death by DNA alkylation in cells into which the expression vector and an expression vector expressing the genetic circuit have been introduced, and recovering living cells. Furthermore, by combining this ON selection method with an OFF selection method previously developed by the present inventors, i.e., an OFF selection method that uses as a selector an expression vector designed to enable hsvTK to operate on the output side of the genetic circuit, we have discovered a method for selecting genetic switches and genetic circuits that can perform both ON and OFF selection in a short period of time, approximately 5-30 minutes. (See Patent Document 2)" However, the method for selecting the genetic circuit is based on a different principle from the method for producing the multi-input / multi-output genetic switch of the present invention or the transcription factor that forms the switch.

[0006] The present inventors have disclosed "a nucleic acid construct used to modify the genome within a cell, which comprises a gene sequence encoding a nucleoside kinase, preferably thymidine kinase, as a gene sequence for selecting cells whose genomes have been modified by the nucleic acid construct, and a method for modifying the genome within a cell, which is characterized by using the nucleic acid construct (see Patent Document 3)." However, the method for selecting the genetic circuit is based on a different principle from the method for producing the multi-input / multi-output genetic switch of the present invention or the transcription factor that forms the switch. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2011-125333 [Patent Document 2] International Publication WO2012 / 060407 [Patent Document 3] Patent Publication No. 2013-17473 Summary of the Invention [Problem to be solved by the invention]

[0008] If it were possible to integrate a large amount of input information into a single sensor protein and endow it with the ability to make judgments (computing), it would be possible to develop a "variable sensitivity" or "conditional" sensor whose sensitivity or response could be controlled externally by other factors. However, there was no way to create such a sensor protein. The present invention provides a method for producing a multi-input / multi-output gene switch or transcription factor required for the above-mentioned sensor, or a transcription factor that forms the switch, as well as a multi-input / multi-output gene switch or a transcription factor that forms the switch. [Means for solving the problem]

[0009] As a result of extensive research, the inventors have completed a method for producing a multi-input / multi-output gene switch or transcription factor, the essential steps of which are "fusing two or more transcription factor genes or one or more transcription factors with one or more binders" and "introducing a mutation into the fused transcription factor gene." They have also been able to obtain a multi-input / multi-output gene switch or transcription factor using this method, thereby completing the present invention.

[0010] That is, the present invention is as follows. 1. A method for producing a multi-input / multi-output gene switch or a transcription factor that forms said switch, comprising the steps of: (A) (1) a step of introducing into a cell or adding to a cell-free protein synthesis system a nucleic acid library of fusion mutants of binder B1 or transcription factor T1 and binder B2 or transcription factor T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding binder B1 or transcription factor T1 responsive to ligand L1 and a gene sequence encoding binder B2 or transcription factor T2 responsive to ligand L2, and a reporter expression vector carrying a gene sequence encoding promoter P1 controlled by transcription factor T1 and / or a gene sequence encoding promoter P2 controlled by transcription factor T2 and a gene sequence encoding reporter Rx functionally linked to the promoter sequence P1 and / or P2, wherein X represents an integer of 1 or more; (2) adding ligand L1 and / or ligand L2 to the cell or cell-free protein synthesis system of (1); (3) selecting a fusion mutant of binder B1 or transcription factor T1 and binder B1 or transcription factor T2 as a gene switch or a transcription factor that forms the switch using the expression level of the reporter as an index; Or, (B) (1) A gene sequence encoding a binder B1 or transcription factor T1 that responds to a ligand L1, a gene sequence encoding a binder B2 or transcription factor T2 that responds to a ligand L2, and a gene sequence encoding a ligand L N Binder B responds to N or transcription factor T N Binder B1, or transcription factor T1 and binder B2, or transcription factor T2 and binder B, obtained by introducing mutations into a gene construct carrying a gene sequence encoding N or transcription factor T N a library of nucleic acids of fusion mutants of the above, and a gene sequence encoding a promoter P1 controlled by a transcription factor T1, a gene sequence encoding a promoter P2 controlled by a transcription factor T2, and / or a library of nucleic acids of fusion mutants of the above, N Promoter P controlled byN and the gene sequence encoding the promoter sequence P1, P2 and / or P N a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding a reporter Rx functionally linked to the above, wherein N is an integer of 3 or more; (2) Ligand L1, Ligand L2, and / or Ligand L N (1) into the cell or cell-free protein synthesis system; (3) Using the expression level of the reporter as an indicator, the expression level of binder B1 or transcription factor T1 and binder B2 or transcription factor T2 and binder B N or transcription factor T N a step of selecting the fusion mutant of the above as a gene switch or a transcription factor that forms the switch. 2. In (A) above, A fusion mutant having a high ratio of reporter expression level upon introduction of ligand L1 and ligand L2 to reporter expression level upon introduction of ligand L1 or ligand L2 is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming such a switch. 2. The method according to claim 1, 3. In (A) above, When promoter P2 controlled by transcription factor T2 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of ligand L1 and ligand L2 to reporter expression level upon introduction of ligand L2 is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming said switch. Or, When promoter P1 controlled by transcription factor T1 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of ligands L1 and L2 to reporter expression level upon introduction of ligand L1 is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming said switch. 2. The method according to claim 1, 4. In (A) above, When promoter P1 controlled by transcription factor T1 is used, a fusion mutant that exhibits a high ratio of reporter expression level upon introduction of ligand L2 to reporter expression level without ligand is selected as a genetic switch having a two-input OR-type sensor function or a transcription factor that forms the switch. Or, When promoter P2 controlled by transcription factor T2 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level without ligand is selected as a genetic switch having a two-input OR-type sensor function or a transcription factor forming the switch. 2. The method according to claim 1, 5. In (A) above, When promoter P1 controlled by transcription factor T1 is used, a fusion mutant that has a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand L2 is selected as a genetic switch for an output-type sensor that specifically responds to ligand L1 or a transcription factor that forms the switch. Or, When promoter P2 controlled by transcription factor T2 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of ligand L2 to reporter expression level upon introduction of ligand L1 is selected as a gene switch for an output-type sensor that specifically responds to ligand L2 or a transcription factor that forms the switch. 2. The method according to claim 1, 6. In (A) above, When promoter P1 controlled by transcription factor T1 is used, a fusion mutant that has a high ratio of reporter expression level without a ligand / reporter expression level after introduction of ligand L1 and a high ratio of reporter expression level without a ligand / reporter expression level after introduction of ligand L2 is selected as a genetic switch having a NOR-type sensor function or a transcription factor that forms the switch. 2. The method according to claim 1, 7. In (A) above, When promoter P1 controlled by transcription factor T1 is used, a fusion mutant that has a high ratio of reporter expression level without a ligand or with the introduction of either ligand L1 or ligand L2 to reporter expression level with the introduction of both ligand L1 and ligand L2 is selected as a genetic switch having a NAND-type sensor function or a transcription factor that forms the switch. 2. The method according to claim 1, 8. In (A) above, A fusion mutant in which the expression level of the reporter is reduced by both introduction of ligand L1 and introduction of ligand L2 is selected as a genetic switch that functions as a two-input, three-stage output-decreasing sensor or a transcription factor that forms the switch. 2. The method according to claim 1, 9. In (A) above, A fusion mutant in which the expression level of the reporter is increased by both introduction of ligand L1 and introduction of ligand L2 is selected as a genetic switch that functions as a two-input, three-stage output-enhancing sensor or a transcription factor that forms the switch. 2. The method according to claim 1, 10. In (B), N=3; Fusion mutants in which the expression levels of the reporters are reduced by introduction of ligand L1, by introduction of ligand L2, and by introduction of ligand L3 are selected as genetic switches that function as a three-input, four-stage output-decreasing sensor or transcription factors that form such switches. 2. The method according to claim 1, 11. In (B), N=3; Fusion mutants in which the expression levels of the reporters are increased by introduction of ligand L1, by introduction of ligand L2, and by introduction of ligand L3 are selected as genetic switches that function as a three-input, four-stage output-improving sensor or transcription factors that form such switches. 2. The method according to claim 1, 12. In (B), N=3; A promoter P1 controlled by a transcription factor T1, a reporter R1 operably linked to the promoter sequence P1, a promoter P2 controlled by a transcription factor T2, a reporter R2 operably linked to the promoter sequence P2, and a promoter P3 controlled by a transcription factor T3, a reporter R3 operably linked to the promoter sequence P3 are used, A nucleic acid library of fusion mutants of a ligand L1-binding transcription factor T1 and a binder B2, or a transcription factor T2 and a binder B3, or a transcription factor T3, in which the ligand binding ability has been abolished by mutagenesis, is used, A fusion mutant in which the expression level of the reporter is not improved by introduction of ligand L1 or by introduction of ligand L2, but the expression level of the reporter is improved by introduction of ligand L3, and in which reporters R1, R2, and R3 are expressed, is selected as a genetic switch for a multi-output sensor that specifically responds to ligand L3, or a transcription factor that forms said switch. 2. The method according to claim 1, 13. In (B), N=3; A promoter P1 controlled by a transcription factor T1, a reporter R1 operably linked to the promoter sequence P1, a promoter P2 controlled by a transcription factor T2, a reporter R2 operably linked to the promoter sequence P2, and a promoter P3 controlled by a transcription factor T3, a reporter R3 operably linked to the promoter sequence P3 are used, A nucleic acid library of fusion mutants of binder B1 or transcription factor T1, in which the ligand binding ability has been abolished by mutagenesis, and binder B2 or transcription factor T2, in which the ligand binding ability has been abolished by mutagenesis, and binder B3 or transcription factor T3, is used; A fusion mutant in which the reporter expression level is increased by introduction of ligand L2 and the reporter expression level is increased by introduction of ligand L3, but the reporter expression level is not increased by introduction of ligand L1, and reporter R2 is expressed is selected as a genetic switch for a multi-output sensor that specifically responds to ligand L2 and ligand L3, or a transcription factor that forms the switch. 2. The method according to claim 1, 14. Binder B1, Binder B 2、 Binder B3 and Binder B N 14. The method according to any one of the preceding items 1 to 13, wherein the target molecule is selected from a transcription factor, an enzyme, an antibody, a histone, a chaperone, or a ribosome. 15. Binder B1, Binder B 2、 Binder B3 and Binder B N 14. The method according to any one of the above items 1 to 13, wherein the nucleic acid sequence is a transcription factor or an enzyme. 16. A method for producing a gene switch for detecting ligand L1 or a transcription factor forming said switch, comprising the steps of: (1) a step of introducing a nucleic acid library of fusion mutants of binder B1 or transcription factor T1 and transcription factor T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding binder B1 or transcription factor T1 responsive to ligand L1 and a gene sequence encoding transcription factor T2, and an expression vector carrying a gene sequence encoding promoter P2 controlled by transcription factor T2 and a gene sequence encoding reporter Rx operably linked to promoter P2 into a cell or adding the library to a cell-free protein synthesis system, wherein X represents an integer of 1 or more; (2) adding ligand L1 and / or ligand L2 to the cell or cell-free protein synthesis system of (1); (3) A process of selecting a fusion mutant having a high ratio of reporter expression level upon introduction of ligand L1 and ligand L2 to reporter expression level upon introduction of ligand L2, or a fusion mutant having a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand L2, as a gene switch for detecting ligand L1 or a transcription factor that forms the switch. 17. The method for producing according to the preceding item 16, wherein the ligand L1 is arsenic. 18. A method for producing a transcription factor capable of enhancing the expression of promoter P2 controlled by transcription factor T2 by ligand L1, comprising the steps of: (1) a step of introducing a nucleic acid library of fusion mutants of binder B1 or transcription factor T1 and binder B2 or transcription factor T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding binder B1 or transcription factor T1 responsive to ligand L1 and a gene sequence encoding binder B2 or transcription factor T2 responsive to ligand L2, and a reporter expression vector carrying a gene sequence encoding promoter P2 controlled by transcription factor T2 and a gene sequence encoding reporter Rx operably linked to promoter P2 into a cell or adding the library to a cell-free protein synthesis system, wherein X represents an integer of 1 or more; (2) adding ligand L1 and / or ligand L2 to the cell or cell-free protein synthesis system of (1); (3) A process of selecting fusion mutants with a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand L2 as transcription factors capable of enhancing the expression of promoter P2 controlled by transcription factor T2 through ligand L1. 19. A gene switch or a transcription factor forming said switch obtained by any one or more of the production methods of the preceding items 1 to 18. 20. A gene fusion mutant obtained by any one or more of the production methods of 1 to 15 above, Here, the gene fusion mutant is capable of enhancing or suppressing the expression of promoters P1, P2 and / or Pn controlled by T1, T2 and / or Tn by ligands L1, L2 and / or LN. 21. The gene fusion mutant according to the preceding paragraph 20, which is any one of the following: (1) A gene fusion mutant capable of enhancing the expression of promoter P2, which is controlled by transcription factor T2, by ligand L1. (2) a gene fusion mutant capable of reducing the expression of promoter P2, which is controlled by transcription factor T2, by the ligand L1; (3) a gene fusion mutant capable of enhancing or suppressing the expression of promoters P1, P2, and Pn controlled by transcription factors T1, T2, and Tn, by ligand L1; (4) A gene fusion mutant capable of enhancing or suppressing the expression of promoters P2 and Pn controlled by transcription factors T2 and Tn by ligand L1. (5) gene fusion mutants capable of enhancing or suppressing the expression of promoters P1, P2, and Pn controlled by transcription factors T1, T2, and Tn, respectively, by ligands L1 and L2; (6) A gene fusion mutant capable of enhancing or suppressing the expression of promoters P2 and Pn controlled by transcription factors T2 and Tn, respectively, by ligands L1 and L2. (7) Gene fusion mutants that can enhance the expression of promoters P1, P2, and Pn, which are controlled by T1, T2, and Tn, by ligands L1, L2, and LN. (8) Gene fusion mutants that can suppress the expression of promoters P1, P2, and Pn controlled by T1, T2, and Tn by ligands L1, L2, and LN. (9) A gene fusion mutant in which the expression of promoter P1 controlled by transcription factor T1 is suppressed by ligand L1, the expression of promoter P2 controlled by transcription factor T2 is enhanced, and the expression of promoter P3 controlled by transcription factor T3 is enhanced. 22. A gene switch or a transcription factor forming the switch obtained by any one or more of the production methods of the preceding items 1 to 18, or a gene switch or the switch according to the preceding item 19, which is a ligand L1, a ligand L2, and / or a ligand L N and methods of use as a sensor for detecting protein synthesis, induction of protein secretion, induction of a biosynthetic pathway, flow regulation of a biosynthetic pathway, induction of cell proliferation, induction of a physiological function, or a control mechanism for a physiological function. 23. A gene switch or a transcription factor forming the switch obtained by any one or more of the production methods of the preceding items 1 to 18, or a gene switch or the switch according to the preceding item 19, which is used in combination with ligand L1, ligand L2, and / or ligand L N In the method for using the above as a sensor for detecting the above, the following ligand L1, ligand L2 and / or ligand L N A method of using the method of claim 1, including a step of adjusting the detection sensitivity. (1) To increase the response sensitivity to ligand L1, ligand L2 and / or ligand L N Increase the concentration of (2) When the response sensitivity to the ligand L1 is to be reduced, the ligand L2 and / or the ligand L N Decrease the concentration of (3) To increase the response sensitivity to ligand L2, ligand L1 and / or ligand L N Increase the concentration (4) When the response sensitivity to the ligand L2 is to be reduced, the ligand L1 and / or the ligand L N Decrease the concentration (5) Ligand L N To increase the response sensitivity to the ligand L1, the concentration of the ligand L2 is increased. (6) Ligand L N To decrease the response sensitivity to the ligand L1, decrease the concentration of the ligand L2. 24. (A) A gene switch comprising a fusion mutant of binder B1 or transcription factor T1 and binder B2 or transcription factor T2 obtained by introducing a mutation into a gene construct carrying a gene sequence encoding binder B1 or transcription factor T1 responsive to ligand L1 and a gene sequence encoding binder B2 or transcription factor T2 responsive to ligand L2; or (B) a gene sequence encoding binder B1 or transcription factor T1 that responds to ligand L1, a gene sequence encoding binder B2 or transcription factor T2 that responds to ligand L2, and a gene sequence encoding a ligand L N Binder B responds to N or transcription factor T N Binder B1, or transcription factor T1 and binder B2, or transcription factor T2 and binder B, obtained by introducing mutations into a gene construct carrying a gene sequence encoding N or transcription factor T N A gene switch containing a fusion mutant of 25. In (A) above, 25. The gene switch according to item 24, characterized in that the ratio of the reporter expression level upon introduction of ligand L1 and ligand L2 to the reporter expression level upon introduction of ligand L1 or ligand L2 is high and the gene switch has a two-input, AND-type output sensor function. 26. In (A) above, When promoter P2 controlled by transcription factor T2 is used, the ratio of reporter expression level by introduction of ligands L1 and L2 to reporter expression level by introduction of ligand L2 is high, and the sensor has a two-input, AND-type output function. Or, When promoter P1 controlled by transcription factor T1 is used, the ratio of reporter expression level by introduction of ligands L1 and L2 to reporter expression level by introduction of ligand L1 is high, and the system has a two-input, AND-type output sensor function. 25. The gene switch according to item 24 above, 27. In (A) above, When promoter P1 controlled by transcription factor T1 was used, the ratio of reporter expression level upon introduction of ligand L2 to reporter expression level without ligand was high, and the reporter had two-input OR-type sensor function. Or, When promoter P2 controlled by transcription factor T2 was used, the ratio of reporter expression level upon introduction of ligand L1 to reporter expression level without ligand was high, and the reporter had a two-input OR-type sensor function. 25. The gene switch according to item 24 above, 28. In (A) above, When promoter P1 controlled by transcription factor T1 is used, the ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand L2 is high, and the reporter has the function of an output-type sensor that specifically responds to ligand L1. Or, When promoter P2 controlled by transcription factor T2 is used, the ratio of reporter expression level upon introduction of ligand L2 to reporter expression level upon introduction of ligand L1 is high, and the reporter has the function of an output-type sensor that specifically responds to ligand L2. 25. The gene switch according to item 24 above, 29. In (A) above, 25. The gene switch described in the preceding paragraph 24, characterized in that when promoter P1 controlled by transcription factor T1 is used, the ratio of the expression level of the reporter without a ligand to the expression level of the reporter upon introduction of ligand L1 is high, and the ratio of the expression level of the reporter without a ligand to the expression level of the reporter upon introduction of ligand L2 is high, and the gene switch has a NOR-type sensor function. 30. In (A) above, 25. The gene switch according to item 24, characterized in that, when a promoter P1 controlled by a transcription factor T1 is used, the ratio of the reporter expression level without a ligand or with the introduction of either ligand L1 or ligand L2 to the reporter expression level with the introduction of both ligand L1 and ligand L2 is high, and the gene switch has a NAND-type sensor function. 31. In (A) above, 25. The gene switch according to item 24 above, characterized in that both the expression level of the reporter by introduction of ligand L1 and the expression level of the reporter by introduction of ligand L2 are reduced, and the gene switch functions as a two-input, three-stage output reduction type sensor. 32. In (A) above, 25. The gene switch according to item 24 above, characterized in that both the expression level of the reporter by introduction of ligand L1 and the expression level of the reporter by introduction of ligand L2 increase, and the gene switch functions as a two-input, three-stage output-improving sensor. 33. In (B), N=3; 25. The gene switch according to item 24, characterized in that the expression level of the reporter is reduced by introduction of ligand L1, the expression level of the reporter by introduction of ligand L2, and the expression level of the reporter by introduction of ligand L3, and that the gene switch functions as a three-input, four-stage output reduction sensor. 34. In (B), N=3; 25. The gene switch according to item 24, characterized in that the expression level of the reporter increases upon introduction of ligand L1, the expression level of the reporter increases upon introduction of ligand L2, and the expression level of the reporter increases upon introduction of ligand L3, and the gene switch functions as a three-input, four-stage output-improving sensor. 35. In (B), N=3; 25. The gene switch according to item 24 above, characterized in that the expression level of the reporter is not improved by introduction of ligand L1 or by introduction of ligand L2, but the expression level of the reporter is improved by introduction of ligand L3, reporters R1, R2, and R3 are expressed, and the gene switch has the function of a multi-output sensor that responds specifically to ligand L3. 36. In (B), N=3; 25. The gene switch described in the preceding paragraph 24, characterized in that the expression level of the reporter is improved by introduction of ligand L2 and the expression level of the reporter is improved by introduction of ligand L3, but the expression level of the reporter is not improved by introduction of ligand L1, and the gene switch has the function of a multi-output sensor in which reporter R2 is expressed and which responds specifically to ligands L2 and L3. 37. Binder B1, Binder B2, Binder B3 and Binder B N 37. The gene switch according to any one of the preceding aspects 24 to 36, wherein the target molecule is selected from a transcription factor, an enzyme, an antibody, a histone, a chaperone, or a ribosome. 38. Binder B1, Binder B2, Binder B3 and Binder B N 37. The gene switch according to any one of the preceding items 24 to 36, wherein the gene switch is a transcription factor or an enzyme. 39. A gene switch for detecting ligand L1 obtained by introducing a mutation into a gene construct carrying a gene sequence encoding binder B1 or transcription factor T1 that responds to ligand L1 and a gene sequence encoding transcription factor T2, Here, the gene switch for detecting ligand L1 is characterized in that the ratio of the reporter expression level upon introduction of ligand L1 and ligand L2 to the reporter expression level upon introduction of ligand L2 is high, or the ratio of the reporter expression level upon introduction of ligand L1 to the reporter expression level upon introduction of ligand L2 is high, and the gene switch has the function of detecting ligand L1. 40. The gene switch according to item 39 above, wherein the ligand L1 is arsenic. Alternatively, the following inventions can be exemplified. 1. A method for producing a multi-input / multi-output gene switch or a transcription factor that forms said switch, comprising the steps of: (A) (1) A library of nucleic acids of fusion mutants of transcription factors T1 and T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding transcription factor T1 responsive to ligand L1 and a gene sequence encoding transcription factor T2 responsive to ligand L2, and a gene sequence encoding promoter P1 controlled by transcription factor T1 and / or a gene sequence encoding promoter P2 controlled by transcription factor T2, and a reporter R functionally linked to the promoter sequence P1 and / or P2. x a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding the X means an integer greater than or equal to 1, (2) adding ligand L1 and / or ligand L2 to the cell or cell-free protein synthesis system of (1); (3) selecting a fusion mutant of transcription factor T1 and transcription factor T2 as a gene switch or a transcription factor that forms the switch using the expression level of the reporter as an index; Or, (B) (1) A gene sequence encoding a transcription factor T1 that responds to a ligand L1, a gene sequence encoding a transcription factor T2 that responds to a ligand L2, and a gene sequence encoding a transcription factor T3 that responds to a ligand L3. N Transcription factor T responding to N Transcription factors T1, T2, and T3 were obtained by introducing mutations into a gene construct carrying the gene sequence encodingN a library of nucleic acids of fusion mutants of the above, and a gene sequence encoding a promoter P1 controlled by a transcription factor T1, a gene sequence encoding a promoter P2 controlled by a transcription factor T2, and / or a library of nucleic acids of fusion mutants of the above, N Promoter P controlled by N and the gene sequence encoding the promoter sequence P1, P2 and / or P N and a reporter R operably linked to x a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding the N means an integer equal to or greater than 3, (2) Ligand L1, Ligand L2, and / or Ligand L N (1) into the cell or cell-free protein synthesis system; (3) Using the reporter expression level as an indicator, transcription factors T1, T2, and T N a step of selecting the fusion mutant of the above as a gene switch or a transcription factor that forms the switch. 2. In (A) above, A fusion mutant having a high ratio of reporter expression level upon introduction of ligand L1 and ligand L2 to reporter expression level upon introduction of ligand L1 or ligand L2 is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming such a switch. 2. The method according to claim 1, 3. In (A) above, When promoter P2 controlled by transcription factor T2 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of ligand L1 and ligand L2 to reporter expression level upon introduction of ligand L2 is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming said switch. Or, When promoter P1 controlled by transcription factor T1 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of ligands L1 and L2 to reporter expression level upon introduction of ligand L1 is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming said switch. 2. The method according to claim 1, 4. In (A) above, When promoter P1 controlled by transcription factor T1 is used, a fusion mutant that exhibits a high ratio of reporter expression level upon introduction of ligand L2 to reporter expression level without ligand is selected as a genetic switch having a two-input OR-type sensor function or a transcription factor that forms the switch. Or, When promoter P2 controlled by transcription factor T2 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level without ligand is selected as a genetic switch having a two-input OR-type sensor function or a transcription factor forming the switch. 2. The method according to claim 1, 5. In (A) above, When promoter P1 controlled by transcription factor T1 is used, a fusion mutant that has a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand L2 is selected as a genetic switch for an output-type sensor that specifically responds to ligand L1 or a transcription factor that forms the switch. Or, When promoter P2 controlled by transcription factor T2 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of ligand L2 to reporter expression level upon introduction of ligand L1 is selected as a gene switch for an output-type sensor that specifically responds to ligand L2 or a transcription factor that forms the switch. 2. The method according to claim 1, 6. In (A) above, When a repressor-type promoter P1 controlled by a transcription factor T1 is used, a fusion mutant showing a high ratio of reporter expression level upon introduction of a ligand L1 or reporter expression level upon introduction of a ligand L2 to reporter expression level without a ligand is selected as a genetic switch having a NOR-type sensor function or a transcription factor forming the switch. 2. The method according to claim 1, 7. In (A) above, When a repressor-type promoter P1 controlled by a transcription factor T1 is used, a fusion mutant showing a high ratio of reporter expression level without a ligand or with the introduction of either L1 or L2 to reporter expression level with the introduction of both ligands L1 and L2 is selected as a genetic switch having a NAND-type sensor function or a transcription factor forming the switch. 2. The method according to claim 1, 8. In (A) above, A fusion mutant in which the expression level of the reporter is reduced by both introduction of ligand L1 and introduction of ligand L2 is selected as a genetic switch that functions as a two-input, three-stage output-decreasing sensor or a transcription factor that forms the switch. 2. The method according to claim 1, 9. In (A) above, A fusion mutant in which the expression level of the reporter is increased by both introduction of ligand L1 and introduction of ligand L2 is selected as a genetic switch that functions as a two-input, three-stage output-enhancing sensor or a transcription factor that forms the switch. 2. The method according to claim 1, 10. In (B), N=3; Fusion mutants in which the expression levels of the reporters are reduced by introduction of ligand L1, by introduction of ligand L2, and by introduction of ligand L3 are selected as genetic switches that function as a three-input, four-stage output-decreasing sensor or transcription factors that form such switches. 2. The method according to claim 1, 11. In (B), N=3; Fusion mutants in which the expression levels of the reporters are increased by introduction of ligand L1, by introduction of ligand L2, and by introduction of ligand L3 are selected as genetic switches that function as a three-input, four-stage output-improving sensor or transcription factors that form such switches. 2. The method according to claim 1, 12. In (B), N=3; Using a repressor-type promoter P1 controlled by a transcription factor T1, a reporter R1 functionally linked to the promoter sequence P1, an activator-type promoter P2 controlled by a transcription factor T2, a reporter R2 functionally linked to the promoter sequence P2, and an activator-type promoter P3 controlled by a transcription factor T3, and a reporter R3 functionally linked to the promoter sequence P3, Using a library of nucleic acids of fusion mutants of transcription factors T1, T2, and T3, whose ligand binding ability has been abolished by mutagenesis, A fusion mutant in which the expression level of the reporter is not improved by introduction of ligand L1 or by introduction of ligand L2, but the expression level of the reporter is improved by introduction of ligand L3, and in which reporters R1, R2, and R3 are expressed, is selected as a genetic switch for a multi-output sensor that specifically responds to ligand L3, or a transcription factor that forms said switch. 2. The method according to claim 1, 13. In (B), N=3; Using a repressor-type promoter P1 controlled by a transcription factor T1, a reporter R1 functionally linked to the promoter sequence P1, an activator-type promoter P2 controlled by a transcription factor T2, a reporter R2 functionally linked to the promoter sequence P2, and an activator-type promoter P3 controlled by a transcription factor T3, and a reporter R3 functionally linked to the promoter sequence P3, Using a nucleic acid library of transcription factor T1, which has lost its ligand binding ability due to mutation, and fusion mutants of transcription factor T2 and transcription factor T3, which have lost their ligand binding ability due to mutation, A fusion mutant in which the reporter expression level is increased by introduction of ligand L2 and the reporter expression level is increased by introduction of ligand L3, but the reporter expression level is not increased by introduction of ligand L1, and reporter R2 is expressed is selected as a genetic switch for a multi-output sensor that specifically responds to ligand L2 and ligand L3, or a transcription factor that forms the switch. 2. The method according to claim 1, 14. A method for producing a gene switch for detecting ligand L1 or a transcription factor forming said switch, comprising the steps of: (1) A library of nucleic acids of fusion mutants of transcription factor T1 and transcription factor LuxR obtained by introducing mutations into a gene construct carrying a gene sequence encoding transcription factor T1 that responds to ligand L1 and a gene sequence encoding transcription factor LuxR, and a promoter P controlled by transcription factor LuxR. lux Gene sequence encoding and promoter P lux and a reporter R operably linked to x a step of introducing an expression vector carrying a gene sequence encoding the above into a cell or adding the vector to a cell-free protein synthesis system, wherein X means an integer greater than or equal to 1, (2) adding the ligand L1 and / or the ligand AHL to the cell or cell-free protein synthesis system of (1); (3) A process of selecting a fusion mutant having a high ratio of reporter expression level upon introduction of ligand L1 and ligand AHL to reporter expression level upon introduction of ligand AHL, or a fusion mutant having a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand AHL, as a gene switch for detecting ligand L1 or a transcription factor that forms the switch. 15. The method according to item 14 above, wherein the ligand L1 is arsenic. 16. A method for producing a transcription factor capable of enhancing the expression of promoter P2 controlled by transcription factor T2 by ligand L1, comprising the steps of: (1) A library of nucleic acids of fusion mutants of transcription factors T1 and T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding transcription factor T1 responsive to ligand L1 and a gene sequence encoding transcription factor T2 responsive to ligand L2, and a gene sequence encoding promoter P2 controlled by transcription factor T2 and a reporter R functionally linked to promoter P2. x a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding the X means an integer greater than or equal to 1, (2) adding ligand L1 and / or ligand L2 to the cell or cell-free protein synthesis system of (1); (3) A process of selecting fusion mutants with a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand L2 as transcription factors capable of enhancing the expression of promoter P2 controlled by transcription factor T2 through ligand L1. 17. The gene switch or the transcription factor forming the switch obtained by any one or more of the production methods of the preceding items 1 to 16 is used as a ligand L1, a ligand L2, and / or a ligand L N and a method for using the sensor to detect the 18. The following ligands L1, L2 and / or L N 18. The method for use according to item 17 above, further comprising a step of adjusting the detection sensitivity. (1) To increase the response sensitivity to ligand L1, ligand L2 and / or ligand L N Increase the concentration of (2) When the response sensitivity to the ligand L1 is to be reduced, the ligand L2 and / or the ligand L N Decrease the concentration of (3) To increase the response sensitivity to ligand L2, ligand L1 and / or ligand L N Increase the concentration (4) When the response sensitivity to the ligand L2 is to be reduced, the ligand L1 and / or the ligand L N Decrease the concentration (5) Ligand L N To increase the response sensitivity to the ligand L1, the concentration of the ligand L2 is increased. (6) Ligand L N To decrease the response sensitivity to the ligand L1, decrease the concentration of the ligand L2. 19. AraC-LuxR having an amino acid substitution selected from any one of the following (1) to (5) in the amino acid sequence represented by SEQ ID NO: 15: N86K and C245W Fusion mutant of. (1) F74L, P86T, V249A and N298I (2) P39R, I197N, and N252S (3) E295K (4) M175K and K491E (5) H80F, H81K, Y82L, N393I, Y439H, R523L, and F541L 20. TetR-AraC-LuxR having the following amino acid substitutions in the amino acid sequence represented by SEQ ID NO: 16: N86Kand C245W fusion mutants of K46R, D95G, K108N, I134V, V145A, L204P, I214N, P216T, F217S, L409Q, T545A and S569T. 21. ArsR-LuxR having an amino acid substitution or deletion selected from any one of the following (1) to (2) in the amino acid sequence represented by SEQ ID NO: 17: N86K and C245W Fusion mutant of. (1) E16D, T17- (2)I84N, N102D, F240L, P277A [Effects of the Invention]

[0011] The present invention provides a method for producing a multi-input / multi-output gene switch or a transcription factor forming the switch, as well as a multi-input / multi-output gene switch or a transcription factor or gene fusion mutant forming the switch. [Brief explanation of the drawings]

[0012] [Figure 1] Overview of the fabrication method for a multi-input, multi-output gene switch. [Figure 2] (a) AraC-LuxRN86K and C245W fusion construct, (b) ligand evaluation results for AraC-LuxRN86K and C245W fusion construct (before mutagenesis), (c) outline of the construction of the AraC-LuxRN86K and C245W fusion mutant, (d) ligand evaluation results for the AND-gate gene switch. [Figure 3] (a) Ligand evaluation results for AND gate type gene switches, (b) Ligand evaluation results for OR gate type gene switches, (c) Ligand evaluation results for L1 only gate type gene switches, (d) Ligand evaluation results for L2 only gate type gene switches. [Figure 4] Overview of the generation of TetR-AraC-LuxRN86K and C245W fusion mutants. [Figure 5] (a) Ligand evaluation results for the TetR-AraC-LuxRN86Kand C245W fusion protein (before mutagenesis), (b) Ligand evaluation results for the three-input, four-stage output-decreasing gene switch. [Figure 6] (a) Overview of the construction of the ArsR::LuxRN86K-C245W fusion mutant, (b) Ligand evaluation results for the highly stringent AND-type arsenic switch, and (c) Ligand evaluation results for the highly sensitive AND-type arsenic switch. [Figure 7] Graphs plotting the EC50 values ​​of a sensor containing a two-input AND-type arsenic switch against the AHL concentration present in the system {(a) is FIG. 6(c), (b) is FIG. 6(b)}. [Figure 8]Arsenic concentration detection results for each switch {"AND → Plux" is "pUC-ArsR::LuxR" / "pAC-PLux-GFP", "AND → Pars" is "pUC-ArsR::LuxR" / "pAC-Pbad-GFP", "ArsR → Pars" is "pUC-ArsR" / "pAC-Pbad-GFP", and "ArsR(-) → Pars" is "pUC-phi" / "pAC-Pbad-GFP"}. [Figure 9] Overview of the plasmid structures of AraC-LuxRN86K and C245W. [Figure 10] Overview of the plasmid structures of TetR-AraC-LuxRN86K and C245W. [Figure 11] Overview of the plasmid structure of ArsR::LuxRN86K-C245W. [Figure 12] Overview of the plasmid structure of ArsR. [Figure 13] Overview of the plasmid structure of Plux-GFP-TK::APH. [Figure 14] Overview of the plasmid structure of Para-GFP-TK::APH. [Figure 15] Overview of the plasmid structure of Ptet-GFP-TK::APH. [Figure 16] Overview of the Pars-GFP plasmid structure. [Figure 17] An example of a gene switch for two ligands. [Figure 18] An example of a gene switch for three ligands. [Figure 19] Overview of the plasmid structure of pET23d-PrpC::LuxRmut. [Figure 20] Overview of the plasmid structure of pET23d-PrpD::LuxRmut. [Figure 21] Propionic acid responsiveness assessment. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to a "method for producing a multi-input / multi-output gene switch or a transcription factor forming said switch," a "multi-input / multi-output gene switch or a transcription factor forming said switch," and a "gene fusion mutant."

[0014] (Multiple input / multiple output type) The term "multiple-input / multiple-output (gate) type" used in the present invention means that it can exhibit multiple types of responses to multiple ligands. In the present invention, in order to explain the contents of the invention, "ligand L1", "ligand L2", and "ligand L N ", "Transcription factor T1", "Transcription factor T2", "Transcription factor T N ", "Promoter P1", "Promoter P2", "Promoter P N "," Reporter R x ", "Binder B1", "Binder B2", "Binder B N " and so on. X means an integer of 1 or greater, N means an integer of 3 or greater. In addition, in the ligand, N " means 3, which means three ligands, Ligand 1, Ligand L2, and Ligand L3. N " means 4, there are four ligands, ligand L1, ligand L 2、 Ligand L3 and Ligand L4 are referred to as " N " means 5, where five ligands are ligand 1, ligand L 2、 Ligand L 3、 Ligand L4 and Ligand L5. Transcription factor T N Promoter P N , Binder B N The same applies to the above. Note that these terms are merely examples and do not limit the content of the present invention.

[0015] (Examples of multi-input / multi-output gene switches or transcription factors) Examples of the multi-output (gate) type that responds to two ligands L1 and L2 are as follows (see Fig. 17). Note that the multi-input / multi-output type gene switch of the present invention or the transcription factor that forms the switch can, as is clear from the results of the following examples, not only output simply "0" or "1", but also change the output amount depending on the amount of ligand L (particularly, the concentration of ligand L in the environment). The output amounts are X1 < X2 < X3, and X1 means an output value of substantially 0 or more. (1) OR gate (2) AND gate (3) NOR gate (4) NAND gate (5) Gate that responds to ligand L1 (6) Gate that responds to ligand L2 (7) 2-input type 3-step output decrease type gate (the output decreases as the number (amount) of ligands increases) (8) 2-input type 3-step output increase type gate (the output increases as the number (amount) of ligands increases)

[0016] Examples of the multi-output (gate) type that responds to three ligands L1, L2, and L3 are as follows (see Fig. 18). (1) 3-input type 4-step output decrease type gate (the output decreases as the number (amount) of ligands increases) (2) 3-input type 4-step output increase type gate (the output increases as the number (amount) of ligands increases) (3) Gate that responds to ligand L3 (4) Gate that responds to ligands L2 and L3 The output amounts are X1 < X2 < X3 < X4, and X1 means an output value of substantially 0 or more.

[0017] Examples of the multi-output (gate) type that responds to N ligands are as follows. (1) N-input type N+1-step output decrease type gate (the output decreases as the number (amount) of ligands increases) (2) N-input N+1-stage output-enhancing gate (the more ligands there are, the higher the output) (3) N-input two-stage output enhancement gate (the output differs depending on whether there are N or not). (4) N-input two-stage output reduction gate (the output differs depending on whether there are N or not).

[0018] (Gene fusion mutants functioning as multi-input / multi-output gene switches) The gene fusion mutants functioning as multi-input / multi-output gene switches are characterized by the ligands L1, L2 and / or L3. N T1, T2 and / or T N promoters P1, P2 and / or P N The expression of the gene can be enhanced or suppressed. More specifically, the following can be exemplified. (1) A gene fusion mutant that can enhance the expression of promoter P2, which is controlled by transcription factor T2, by the ligand L1. (2) A gene fusion mutant that can reduce the expression of promoter P2, which is controlled by transcription factor T2, by the ligand L1. (3) Ligand L1 induces transcription factors T1, T2, and T3 N Promoters P1, P2 and P N A gene fusion mutant capable of enhancing or suppressing the expression of (4) Ligand L1 induces transcription factor T2 and transcription factor T N Promoters P2 and P N A gene fusion mutant capable of enhancing or suppressing the expression of (5) Ligands L1 and L2 induce transcription factors T1, T2, and T3. N Promoters P1, P2 and P N A gene fusion mutant capable of enhancing or suppressing the expression of (6) Ligands L1 and L2 induce transcription factors T2 and T N Promoters P2 and PN A gene fusion mutant capable of enhancing or suppressing the expression of (7) Ligands L1, L2 and L N by transcription factors T1, T2 and T N Promoters P1, P2 and P N A gene fusion mutant that can enhance the expression of (8) Ligands L1, L2 and L N by transcription factors T1, T2 and T N Promoters P1, P2 and P N A gene fusion mutant that can suppress the expression of (9) A gene fusion mutant in which the expression of promoter P1, which is controlled by transcription factors T1, T2, and T3, is suppressed by ligand L1, and the expression of promoters P2 and P3 is enhanced. More specifically, the following gene fusion mutants can be exemplified by the examples below. (1) The transcription factor T1-T2 (AraC-LuxR) gene fusion mutants, which respond to AND (AHL / Arabinose), OR (AHL / Arabinose), Ignore-AHL (Arabinose-only), and Ignore-arabinose (AHL-only) types, act on promoters P1 and P2 and can simultaneously regulate genes downstream of the promoters. (2) The transcription factor T1-T2-T3 (TraR-AraC-LuxR) gene fusion mutant can simultaneously regulate genes (including operans) downstream of promoters P1 (TetP), P2 (pBAD), and P3 (pLux) in response to ligands L1 (aTc), L2 (arabinose), and L3 (AHL).

[0019] (Method of manufacturing a multi-input / multi-output gene switch) The "method for producing a multi-input / multi-output gene switch or a transcription factor that forms said switch" of the present invention (hereinafter sometimes abbreviated as "the production method of the present invention (method of the present invention)") requires the essential steps of "fusing two or more transcription factor genes or one or more transcription factors with one or more binders" and "introducing a mutation into the fused transcription factor gene," but other steps are not particularly limited. For example, the method may include the following steps or steps substantially the same as the following steps: Further, the method described in the publication already published by the present inventors, "PLoS ONE 10(3):e0120243," can be referred to.

[0020] (Steps of the manufacturing method of the present invention) A method for producing a multi-input / multi-output gene switch or a transcription factor forming said switch, comprising the steps of: (A) (1) a step of introducing into a cell or adding to a cell-free protein synthesis system a nucleic acid library of fusion mutants of binder B1 or transcription factor T1 and binder B2 or transcription factor T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding binder B1 or transcription factor T1 responsive to ligand L1 and a gene sequence encoding binder B2 or transcription factor T2 responsive to ligand L2, and a reporter expression vector carrying a gene sequence encoding promoter P1 controlled by transcription factor T1 and / or a gene sequence encoding promoter P2 controlled by transcription factor T2 and a gene sequence encoding reporter Rx functionally linked to the promoter sequence P1 and / or P2, wherein X represents an integer of 1 or more; (2) adding ligand L1 and / or ligand L2 to the cell or cell-free protein synthesis system of (1); (3) selecting a fusion mutant of binder B1 or transcription factor T1 and binder B1 or transcription factor T2 as a gene switch or a transcription factor that forms the switch using the expression level of the reporter as an index; Or, (B) (1) A gene sequence encoding a binder B1 or transcription factor T1 that responds to a ligand L1, a gene sequence encoding a binder B2 or transcription factor T2 that responds to a ligand L2, and a gene sequence encoding a ligand L N Binder B responds to N or transcription factor T N Binder B1, or transcription factor T1 and binder B2, or transcription factor T2 and binder B, obtained by introducing mutations into a gene construct carrying a gene sequence encoding N or transcription factor T N a library of nucleic acids of fusion mutants of the above, and a gene sequence encoding a promoter P1 controlled by a transcription factor T1, a gene sequence encoding a promoter P2 controlled by a transcription factor T2, and / or a library of nucleic acids of fusion mutants of the above, N Promoter P controlled by N and the gene sequence encoding the promoter sequence P1, P2 and / or P N a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding a reporter Rx functionally linked to the above, wherein N is an integer of 3 or more; (2) Ligand L1, Ligand L2, and / or Ligand L N (1) into the cell or cell-free protein synthesis system; (3) Using the expression level of the reporter as an indicator, the expression level of binder B1 or transcription factor T1 and binder B2 or transcription factor T2 and binder B N or transcription factor T N a step of selecting the fusion mutant of the above as a gene switch or a transcription factor that forms the switch. More details are as follows: (A) (1) A library of nucleic acids of fusion mutants of transcription factors T1 and T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding transcription factor T1 responsive to ligand L1 and a gene sequence encoding transcription factor T2 responsive to ligand L2, and a gene sequence encoding promoter P1 controlled by transcription factor T1 and / or a gene sequence encoding promoter P2 controlled by transcription factor T2, and a reporter R functionally linked to the promoter sequence P1 and / or P2. x a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding the X means an integer of 1 or greater. (2) adding the ligand L1 and / or the ligand L2 to the cells or cell-free protein synthesis system of (1). (3) A step of selecting a fusion mutant of transcription factor T1 and transcription factor T2 as a gene switch or a transcription factor that forms the switch, using the expression level of the reporter as an index. (B) (1) A gene sequence encoding a transcription factor T1 that responds to a ligand L1, a gene sequence encoding a transcription factor T2 that responds to a ligand L2, and a gene sequence encoding a transcription factor T3 that responds to a ligand L3. N Transcription factor T responding to N Transcription factors T1, T2, and T3 were obtained by introducing mutations into a gene construct carrying the gene sequence encoding N a library of nucleic acids of fusion mutants of the above, and a gene sequence encoding a promoter P1 controlled by a transcription factor T1, a gene sequence encoding a promoter P2 controlled by a transcription factor T2, and / or a library of nucleic acids of fusion mutants of the above, N Promoter P controlled by N and the gene sequence encoding the promoter sequence P1, P2 and / or P N and a reporter R operably linked to x a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding the N means an integer of 3 or greater. (2) Ligand L1, Ligand L2, and / or Ligand L N (1) into the cell or cell-free protein synthesis system. (3) Using the reporter expression level as an indicator, transcription factors T1, T2, and T N a step of selecting the fusion mutant of the above as a gene switch or a transcription factor that forms the switch.

[0021] (gene switch) In the present invention, a "gene switch" means a "gene in which two or more transcription factors or one or more transcription factors and one or more binders are fused (a fusion transcription factor gene)" that has a switching function when expressed as a protein, and in particular means a "fusion transcription factor gene into which a mutation has been introduced" or a "gene fusion mutant" that has a switching function when expressed as a protein. If necessary, a spacer may be introduced between each transcription factor or between a transcription factor and a binder. Preferably, a restriction enzyme recognition sequence may be inserted. For example, when the restriction enzyme XbaI is used, the amino acid sequence SR (nucleotide sequence TCTAGA) derived from the enzyme recognition sequence can be used, and when the restriction enzyme SpeI is used, the amino acid sequence TS (nucleotide sequence ACTAGT) derived from the enzyme recognition sequence can be used. The gene switch of the present invention has each gate function as described above.

[0022] (ligand) In the present invention, "ligand L" refers to a substance, e.g., a compound, that changes the function of a gene switch by binding to a transcription factor, thereby inducing direct or indirect regulation of the expression of a gene or multiple genes. A "ligand" can also be referred to as a "compound that activates a gene switch." Activating substances vary depending on the gene switch.

[0023] (binder) "Binder B" in the present invention is selected from a transcription factor, an enzyme, an antibody, a histone, a chaperone, or a ribosome, and is preferably a transcription factor or an enzyme.

[0024] (Promoter) In the present invention, "promoter P" refers to a nucleic acid sequence having promoter activity controlled by a transcription factor. A promoter refers to a nucleic acid sequence located 5' upstream of the translation initiation site of a gene encoding the reporter R gene or an active portion thereof, and controls the transcription of the reporter R. The promoter to be used is selected appropriately depending on the species of host cell used. When a bacterium is used as the host, any promoter that can be expressed in host cells such as E. coli may be used, without particular limitation. Examples include promoters derived from E. coli or phages, such as the λPR promoter, PL promoter, trp promoter, and lac promoter. Artificially engineered promoters, such as the tac promoter, may also be used. When a yeast is used as the host, any promoter that can be expressed in yeast may be used, without particular limitation. Examples include the gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, and AOX1 promoter. When an animal cell is used as the host, it is preferable that the recombinant vector be capable of autonomous replication in the cell and comprise a promoter, RNA splice site, target gene, polyadenylation site, and transcription termination sequence. An origin of replication may also be included, if desired. As the promoter, an SRα promoter, an SV40 promoter, an LTR promoter, a CMV promoter, or the like can be used, and also an early gene promoter of a cytomegalovirus, or the like may be used.

[0025] (Combination of ligand L, transcription factor T and promoter P) Examples of combinations of a ligand L, a transcription factor T responsive to the ligand L, and a promoter P controlled by the transcription factor T include the following: Activator-type promoter Arabinose, AraC, P BAD( arabinose operon) Transcription does not occur unless an activator protein binds to the operator DNA. When arabinose is present in the environment, the conformation of the arabinose activator changes. The conformationally changed arabinose activator binds to the operator. This allows RNA polymerase to transcribe the operon, and P BAD The downstream reporter R gene is expressed. AHL, LuxR, P lux When AHL is present in the environment, it binds to the transcription factor LuxR, and the AHL-LUxR complex activates the pluX promoter, resulting in the expression of the downstream reporter gene R. Xylose, XylR, P xyl * Repressor-type promoter aTc, TetR, Ptet Arsenic, ArsR, P ars IPTG, LacI, P lac

[0026] (library of nucleic acids of fusion mutants) The "library of nucleic acids of fusion mutants" in the present invention refers to a gene construct (including an expression vector) carrying a gene sequence encoding a transcription factor T1 or binder B1 responsive to a ligand L1 and a gene sequence encoding a transcription factor T2 or binder B2 responsive to a ligand L2, or a gene construct (including an expression vector) carrying a gene sequence encoding a transcription factor T1 or binder B1 responsive to a ligand L1, a gene sequence encoding a transcription factor T2 or binder B2 responsive to a ligand L2, and a gene sequence encoding a transcription factor T3 or binder B4 responsive to a ligand L3. N Transcription factor T responding to N Or Binder B NMutations known per se (e.g., random mutations, site-specific mutations using stability prediction software such as Fold-X) are introduced into a gene construct (including an expression vector) carrying a gene sequence encoding the transcription factor T1 or binder B1, and a fusion mutant of the transcription factor T2 or binder B2 (a fusion mutant of the transcription factor T1 or binder B1 and the transcription factor T2, or a fusion mutant of the binder B2 and the transcription factor T1) having multiple types of mutations. N Or Binder B N It is a fusion mutant of The method for fusing transcription factor T1 and transcription factor T2 is not particularly limited, and may be tandem in-frame fusion or a fusion method in which the other gene is inserted into the loop portion of the transcription factor. Additionally, a preferred example of a mutation of the present invention is one that causes destabilization of a protein (fusion mutant). First, protein stability refers to the stability of the folded state, i.e., the free energy change (ΔGfold) that occurs when the polypeptide chain that constitutes the protein forms a functional structure (folds). Furthermore, "destabilization" refers to reducing and ultimately canceling the free energy change (ΔGfold) associated with the folding energy. For example, if a certain amino acid substitution reduces the stability of the functional structure (fold), that amino acid substitution is a "destabilizing mutation (destabilizing mutation)." Specifically, if a mutation can induce appropriate destabilization, the folded state of the fusion mutant can be maintained only in the presence of a ligand (i.e., ΔGfold<0).

[0027] (Reporter) The "reporter Rx" in the present invention is not particularly limited as long as it serves as an indicator for selecting fusion mutants, and examples thereof include fluorescent protein (GFP), thymidine kinase (see JP 2013-17473 A), alkyladenine DNA glycosidase (see International Publication WO2012 / 060407 A), pigment synthesis protein (see JP 2014-223038 A), pigment protein (amilCP), etc. Specifically, when fluorescent proteins or chromoproteins are used, fusion mutants are selected based on the color change of the culture medium when ligand L is added or not added. When thymidine kinase, alkyladenine DNA glycosylase, various drug transporters, drug resistance markers, toxin-antitoxin pairs, etc. are used, fusion mutants are selected using cell viability and proliferation as indicators. Furthermore, the reporter R is not particularly limited as long as it is functionally linked to each promoter P, but the type of reporter R can be different for each promoter P. For example, combinations such as promoter P1-reporter R1, promoter P2-reporter R2, and promoter P3-reporter R3 can be exemplified.

[0028] (Reporter expression vector) The "reporter expression vector" of the present invention is a vector comprising a gene sequence encoding a promoter P1 controlled by a transcription factor T1 and / or a gene sequence encoding a promoter P2 controlled by a transcription factor T2, and a gene sequence encoding a reporter Rx operably linked to the promoter P1 sequence and / or a gene sequence encoding a reporter Rx operably linked to the promoter P2 sequence, or a gene sequence encoding a promoter P1 controlled by a transcription factor T1, a gene sequence encoding a promoter P2 controlled by a transcription factor T2, and / or a gene sequence encoding a reporter Rx operably linked to the promoter P2 sequence. N Promoter P controlled by N and a gene sequence encoding a reporter Rx functionally linked to a promoter P1 sequence, a gene sequence encoding a reporter Rx functionally linked to a promoter P2 sequence, and / or a gene sequence encoding a reporter Rx functionally linked to a promoter P3 sequence. X There may be multiple types of promoters, and one or more may be present downstream of each promoter.

[0029] An expression vector is DNA that delivers a foreign gene to a host cell, in other words, vector DNA, capable of expressing a gene of interest in the host cell. Vector DNA is not particularly limited as long as it is replicable in the host, and can be appropriately selected depending on the type of host and intended use. Vector DNA may be obtained by extracting naturally occurring DNA, or it may be vector DNA lacking portions of DNA other than those necessary for replication. Representative examples of vector DNA include vector DNA derived from plasmids, bacteriophages, and viruses. Examples of plasmid DNA include plasmids derived from Escherichia coli, Bacillus subtilis, and yeast. Examples of bacteriophage DNA include λ phage. Examples of virus-derived vector DNA include vectors derived from animal viruses such as retroviruses, vaccinia viruses, adenoviruses, papovaviruses, SV40, fowlpox viruses, and pseudorabies viruses, as well as vectors derived from insect viruses such as baculoviruses. Other examples include vector DNA derived from transposons, insertion elements, and yeast chromosomal elements. Alternatively, vector DNAs created by combining these elements, such as vector DNAs (cosmids, phagemids, etc.) created by combining genetic elements of plasmids and bacteriophages, can be used. The vector DNA must incorporate a gene of interest so that it can be expressed, and its components include at least the gene of interest and a regulatory DNA element, such as a promoter. In addition to these elements, if desired, gene sequences carrying information related to replication and control can be combined and incorporated into the vector DNA by methods known per se. Examples of such gene sequences include cis elements such as ribosome binding sequences, terminators, signal sequences, and enhancers, splicing signals, and selectable markers (selectors: dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, kanamycin resistance gene, etc.). One or more gene sequences selected from these can be incorporated into the vector DNA. A gene of interest can be incorporated into vector DNA using known genetic engineering techniques. For example, the gene of interest can be digested at specific sites with an appropriate restriction enzyme, mixed with similarly digested vector DNA, and religated with a ligase. Alternatively, the desired vector DNA can be obtained by ligating an appropriate linker to the gene of interest and inserting it into the multicloning site of a vector suitable for the purpose. The method for introducing an expression vector into a host cell is not particularly limited as long as it is a method that can introduce vector DNA into the host cell and cause expression of a target gene in the host cell, and any known method appropriately selected depending on the species of the host cell may be used, such as electroporation, calcium phosphate method, and lipofection.

[0030] (Cells or cell-free protein synthesis systems) The "cell or cell-free protein synthesis system" in the present invention is a system that contains a transcription factor T, a promoter P, and a reporter R. X The cell-free protein synthesis system is not particularly limited as long as it allows protein expression. For example, the cells may be either prokaryotic cells or isolated eukaryotic cells, but prokaryotic cells with a short cell cycle and a fast proliferation rate are preferred. Cells with such properties are useful for rapid production of gene switches. For example, the cell-free protein synthesis system may be a known cell-free protein synthesis system (wheat, E. coli, etc.) containing components essential for protein synthesis. The "step of adding ligand L1 and / or ligand L2 to cells or a cell-free protein synthesis system" means that the addition of ligand L1 and / or ligand L2 to cells or a cell-free protein synthesis system may be before, after, or substantially simultaneously with the addition of the library of nucleic acids of fusion mutants and / or the expression vector for a reporter to the cells.

[0031] (Selected as a gene switch or a transcription factor that forms the switch) In the present invention, the "step of selecting a gene switch or a transcription factor forming the switch" is carried out by using a reporter R XUsing the expression level as an indicator, fusion mutants that exhibit the desired properties of a multi-input / multi-output gene switch are selected. The gene sequence and / or amino acid sequence of the selected fusion product are analyzed by a method known per se, thereby obtaining information (nucleotide sequence, amino acid sequence) about the multi-input / multi-output gene switch or transcription factor. Furthermore, based on this information, a multi-input / multi-output gene switch or transcription factor can be easily obtained by using a protein synthesis system known per se.

[0032] (gene circuit) A "gene circuit" according to the present invention has at least the following: - Gene sequence encoding transcription factor T1, which responds to the ligand L1 The gene sequence encoding the transcription factor T2, which responds to the ligand L2 A gene sequence encoding a promoter P1 controlled by the transcription factor T1 and / or a gene sequence encoding a promoter P2 controlled by the transcription factor T2 〇Reporter R operably linked to promoter P1 sequence X and / or a reporter R operably linked to the promoter P2 sequence. X All of the above may be contained in the same genetic construct, or may be contained separately in multiple genetic constructs. Furthermore, if necessary, it may contain cis elements such as a ribosome binding sequence, a terminator, a signal sequence, and an enhancer, a splicing signal, and a selection marker (selector: dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, kanamycin resistance gene, etc.). Furthermore, it may also contain an operator sequence (a DNA region to which a repressor or activator binds) or a regulatory gene sequence.

[0033] (biosensor) The "biosensor" of the present invention is not particularly limited in configuration as long as it can show multiple types of responses to multiple ligands, but an example of such a biosensor is a cell-free protein synthesis system including cells or gene circuits in which transcription factors, promoters, and reporters can be expressed as proteins (a system in which a gene circuit can be expressed).

[0034] (Example of a method for producing a multi-input / multi-output gene switch or transcription factor) In the multi-input / multi-output gene switch of the present invention, the desired multi-input / multi-output gene switch or transcription factor can be selected by the methods described in FIGS.

[0035] (Example 1 of manufacturing a multi-input / multi-output gene switch or transcription factor) As an example of the production method of the present invention in which two or more mutations are introduced, a method for producing a gene switch or transcription factor of an output-type sensor that specifically responds to the ligand L2 will be described. (i) obtaining, as a parent of a second generation, a fusion mutant that exhibits a high ratio of reporter expression level upon introduction of ligand L2 to reporter expression level upon introduction of ligand L1 when promoter P1 controlled by transcription factor T1 is used; (ii) introducing random mutations into the second generation parent to obtain a second generation library of nucleic acids or proteins of fusion variants; (iii) introducing into cells the second-generation library and an expression vector carrying a gene sequence encoding a promoter P1 controlled by a transcription factor T1 and a gene sequence encoding a reporter operably linked to the promoter sequence; (iv) introducing ligand L1 and / or ligand L2 into the cells of (iii); (V) A process of selecting fusion mutants with a high ratio of reporter expression level upon introduction of ligand L2 to reporter expression level without ligand as gene switches of two-input, one-output type sensors that respond specifically to ligand L2. If necessary, the number of times of mutagenesis and selection can be increased by repeating steps (i) to (V).

[0036] (Example 2 of manufacturing a multi-input / multi-output gene switch or transcription factor) When the number of transcription factors included in the mutant fusion is increased, partial mutations can be performed in advance on only specific transcription factors (see Example 3). This makes it possible to disable the ligand-binding site of transcription factor T.

[0037] (Method of manufacturing a gene switch or transcription factor for detecting a ligand) In the production method of the present invention, the gene switch for detecting ligand L1 or the transcription factor that forms the switch can be obtained by the following steps. (1) a step of introducing a nucleic acid library of fusion mutants of binder B1 or transcription factor T1 and transcription factor T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding binder B1 or transcription factor T1 responsive to ligand L1 and a gene sequence encoding transcription factor T2, and an expression vector carrying a gene sequence encoding promoter P2 controlled by transcription factor T2 and a gene sequence encoding reporter Rx operably linked to promoter P2 into a cell or adding the library to a cell-free protein synthesis system, wherein X represents an integer of 1 or more; (2) adding ligand L1 and / or ligand L2 to the cell or cell-free protein synthesis system of (1); (3) A process of selecting a fusion mutant having a high ratio of reporter expression level upon introduction of ligand L1 and ligand L2 to reporter expression level upon introduction of ligand L2, or a fusion mutant having a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand L2, as a gene switch for detecting ligand L1 or a transcription factor that forms the switch. More specifically, in Example 6 below, the ligand AHL, the transcription factor LuxR, and the promoter P lux By using the above, a gene switch or transcription factor for highly sensitive ligand detection can be obtained. A production example is as follows. (1) A library of nucleic acids of fusion mutants of transcription factor T1 and transcription factor LuxR obtained by introducing mutations into a gene construct carrying a gene sequence encoding transcription factor T1 that responds to ligand L1 and a gene sequence encoding transcription factor LuxR, and a promoter P controlled by transcription factor LuxR. lux a gene sequence encoding the reporter R operably linked to the promoter sequence; X introducing an expression vector carrying a gene sequence encoding the (2) adding the ligand L1 and / or the ligand AHL to the cell or cell-free protein synthesis system of (1); (3) A process of selecting fusion mutants with a high ratio of reporter expression level upon introduction of ligand L1 and ligand AHL to reporter expression level upon introduction of ligand AHL, or fusion mutants with a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand AHL, as gene switches or transcription factors for detecting ligand L1. An example of the ligand is arsenic.

[0038] (Method for adjusting ligand detection sensitivity) In the biosensor of the present invention, the ligand L1, the ligand L2 and / or the ligand L N The detection sensitivity can be easily adjusted as described below. (1) To increase the response sensitivity to ligand L1, ligand L2 and / or ligand L N Increase the concentration of added (2) When the response sensitivity to the ligand L1 is to be reduced, the ligand L2 and / or the ligand L N Reduce the concentration of added (3) To increase the response sensitivity to ligand L2, ligand L1 and / or ligand L N Increase the concentration of the additive. (4) When the response sensitivity to the ligand L2 is to be reduced, the ligand L1 and / or the ligand L N Decrease the concentration of the additive. (5) Ligand L NTo increase the response sensitivity to the ligand L1, the concentration of the ligand L1 and / or the ligand L2 is increased. (6) Ligand L N To decrease the response sensitivity to the ligand L1, the concentration of the ligand L1 and / or the ligand L2 is decreased.

[0039] (Method for producing a transcription factor capable of enhancing the expression of promoter P2 controlled by transcription factor T2 by ligand L1) The production method of the present invention can obtain a fusion mutant in which the combination of ligand L1 and transcription factor T1 can enhance or suppress (ON / OFF) the expression of a gene under the control of promoter P2, which is under the regulatory sequence of the fusion partner transcription factor T2. For example, the following method can be used. (1) A library of nucleic acids of fusion mutants of transcription factors T1 and T2 obtained by introducing mutations into a gene construct carrying a gene sequence encoding transcription factor T1 responsive to ligand L1 and a gene sequence encoding transcription factor T2 responsive to ligand L2, and a gene sequence encoding promoter P2 controlled by transcription factor T2 and a reporter R functionally linked to the promoter sequence. X A step of introducing a reporter expression vector carrying a gene sequence encoding the above into cells or a cell-free protein synthesis system. (2) adding the ligand L1 and / or the ligand L2 to the cells or cell-free protein synthesis system of (1). (3) A process of selecting fusion mutants with a high ratio of reporter expression level upon introduction of ligand L1 to reporter expression level upon introduction of ligand L2 as transcription factors capable of improving the expression of promoter P2 controlled by transcription factor T2 through ligand L1.

[0040] The multi-input / multi-output gene switch of the present invention, the transcription factors forming the switch, and the fusion mutants thereof can be used for inducing protein synthesis, protein secretion, inducing a biosynthetic pathway, regulating the flow rate of a biosynthetic pathway, inducing cell proliferation, inducing a physiological function, or as a control mechanism for a physiological function.

[0041] Furthermore, the present invention provides "AraC-LuxR N86K and C245W fusion mutants of "TetR-AraC-LuxR" and "TetR-AraC-LuxR N86K and C245W fusion mutants of ArsR and LuxR N86K and C245W This relates to "fusion mutants of

[0042] AraC-LuxR of the present invention N86K and C245W The fusion mutant of this invention has an amino acid substitution selected from any one of the following (1) to (5) in the amino acid sequence represented by SEQ ID NO: 15 (see Table 5). (1) F74L, P86T, V249A and N298I (2) P39R, I197N, and N252S (3) E295K (4) M175K and K491E (5) H80F, H81K, Y82L, N393I, Y439H, R523L, and F541L

[0043] AraC-LuxR (1) above N86K and C245W The fusion mutant of this gene has AND-gate type transcriptional activity (it responds to the presence of both arabinose and homoserine lactone). AraC-LuxR (2) above N86K and C245W The fusion mutant of this gene has AND-gate type transcriptional activity (it responds to the presence of both arabinose and homoserine lactone). AraC-LuxR (3) above N86K and C245W The fusion mutant of this gene has OR-gated transcriptional activity (responds to the presence of arabinose or homoserine lactone). AraC-LuxR (4) above N86K and C245W The fusion mutant of this gene has L1-only gated transcriptional activity (specifically responds to arabinose). AraC-LuxR (5) above N86Kand C245W The fusion mutant of this gene has L2-only gated transcriptional activity (specifically responds to AHL). AraC-LuxR of the present invention N86K and C245WThe fusion mutants of the present invention include amino acid sequences in which 1 to 20, preferably 1 to 15, more preferably 1 to 10, and most preferably 1 to 5 amino acids have been substituted, deleted, inserted, and / or added in the amino acid substitution sequences described in (1) to (5) above, and which have substantially the same transcription activity as the substitution products of (1) to (5) above, as well as amino acid sequences which have 90% or more (or 92% or more, 94% or more, 96% or more, 98% or more, 99% or more) homology with the amino acid substitution sequences described in (1) to (5) above and have substantially the same transcription activity as the substitution products of (1) to (5) above. In addition, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations into the peptide, it is easily conceivable to substitute, for example, mutually homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) with each other.

[0044] TetR-AraC-LuxR of the present invention N86K and C245W The fusion mutant of has the following amino acid substitutions in the amino acid sequence of SEQ ID NO: 16: K46R, D95G, K108N, I134V, V145A, L204P, I214N, P216T, F217S, L409Q, T545A, and S569T. In addition, the TetR-AraC-LuxR of the present invention N86Kand C245W The fusion mutant of this gene has a three-input, four-stage, output-decreasing transcriptional activity (the response decreases with increasing types and amounts of the three ligands). TetR-AraC-LuxR of the present invention N86Kand C245WThe fusion mutants of the present invention include an amino acid sequence in which 1 to 20, preferably 1 to 15, more preferably 1 to 10, and most preferably 1 to 5 amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of the amino acid substitution product described above, and which has substantially the same transcription activity as the substitution product, as well as an amino acid sequence which has 90% or more (or 92% or more, 94% or more, 96% or more, 98% or more, 99% or more) homology with the amino acid sequence of the amino acid substitution product described above and has substantially the same transcription activity as the substitution product.

[0045] The ArsR-LuxR of the present invention N86K and C245W The fusion mutant of the present invention has an amino acid substitution or deletion selected from any one of the following (1) to (2) in the amino acid sequence represented by SEQ ID NO: 17: (1) E16D, T17- ("-" means missing). (2)I84N, N102D, F240L, P277A ArsR-LuxR in (1) above N86K and C245W The fusion mutant of is a highly stringent AND-type arsenic switch. ArsR-LuxR (2) above N86K and C245W The fusion mutant of is a highly sensitive AND-type arsenic switch. The ArsR-LuxR of the present invention N86Kand C245W The fusion mutants of the present invention include amino acid sequences in which 1 to 20, preferably 1 to 15, more preferably 1 to 10, and most preferably 1 to 5 amino acids have been substituted, deleted, inserted, and / or added in the amino acid substitution / deletion sequences described in (1) and (2) above, and which have substantially the same transcription activity as the substitution / deletion mutants of the present invention (1) and (2) above, as well as amino acid sequences which have 90% or more (or 92% or more, 94% or more, 96% or more, 98% or more, 99% or more) homology with the amino acid substitution / deletion sequences described in (1) and (2) above and which have substantially the same transcription activity as the substitution / deletion mutants of the present invention (1) and (2).

[0046] The present invention will be described below with reference to examples, but the present invention is not limited to these examples in any way. [Example]

[0047] (reagent) The reagents used in Examples 2 and 3 below are as follows:

[0048] (Pre-PCR) Based on the composition table below, PCR was performed once using the plasmid as a template to linearize the DNA.

[0049] [Table 1]

[0050] {Error-prone PCR (EP-PCR)} EP-PCR was performed using linear DNA as a template based on the composition table below. The amplification ratio was 100-fold, 1,000-fold, or 10,000-fold, and the MnCl2 concentration was 10 μM or 50 μM. MnCl2 was used by diluting a 10-fold concentrated stock solution.

[0051] [Table 2]

[0052] (Digestion reaction) Based on the composition table below, AL (AraC-LuxR N86K and C245W , see Figure 9) and TAL (TetR-AraC-LuxR N86K and C245W Both vectors (see Figure 10) were digested with NcoI-HF (New England Biolabs) and BamHI-HF (New England Biolabs). For vector digestion, rSAP (Shrimp Alkaline Phosphatase, New England Biolabs) was also added. Approximately 1 μg of template DNA was prepared per 50 μL reaction system. The reaction time was 37°C for 3 hours.

[0053] [Table 3]

[0054] (Ligation reaction) Based on the composition table below, AL reactions were performed using 100 ng of vector and approximately 100 ng of insert, and TAL reactions were performed using 100 ng of vector and approximately 150 ng of insert. The reaction conditions were overnight at 16°C.

[0055] [Table 4] [Example]

[0056] (Development of multi-input / multi-output sensors) In this example, as an example of the production of a multi-input / multi-output sensor, we used the arabinose-responsive transcription factor AraC (obtained by PCR from Escherichia coli MG1655) and the AHL (homoserine lactone)-responsive transcription factor LuxR mutants {N86K, C245W: Kimura et al., J. Gen. Appl. Microbiol., 62, 240-247 (2016)}.

[0057] (AraC-LuxR N86K and C245W (Confirmation of characteristics) As shown in FIG. 2(a), AraC-LuxR was synthesized based on a method known per se. N86K and C245W A fusion construct (SEQ ID NO: 15) was prepared. The results of adding L-arabinose and / or AHL to the medium containing the fusion construct are shown in Figure 2(b). AraC-LuxR N86K and C245WThis fusion protein, which consists of two transcription factor proteins fused in tandem, confirmed that the functions of each transcription factor were not affected. More specifically, the fusion protein enhanced the expression of genes downstream of the arabinose promoter in response to arabinose alone, without any interference from AHL. Similarly, the expression of genes downstream of the Lux promoter was enhanced by this fusion protein in an AHL-dependent manner, but not by arabinose.

[0058] (AraC-LuxR N86K and C245W (Creation of mutants) As shown in Figures 2(c) and (d), the AraC-LuxR gene was isolated by the known EP-PCR method. N86K and C245W Random mutations were introduced into AraC-LuxR N86K and C245W The details are as follows.

[0059] (Example 2-1: Development example 1 of a two-input AND-type sensor) AraC-LuxR N86K and C245W The full-length plasmid (Figure 9) was subjected to EP-PCR (50 μM MnCl2, amplification factor 10,000-fold) to generate a mutant pool with random mutations (library size 3 x 10 6 (It was). P lux Using -GFP as a reporter, 90 colonies that showed fluorescence on solid medium containing 10 μM AHL and 13 mM L-arabinose were picked. Their ligand responsiveness was evaluated (see Figure 2). Specifically, 15 mutants were cultured in either "10 μM AHL only" or "10 μM AHL + 13 mM L-arabinose" and showed a 2-fold or greater improvement in the BOTH (L-arabinose + AHL) / AHL ratio. Among these mutants, one showed high stringency when only AHL was added, and the other showed a reporter P BAD7 Even when the GFP was changed to -GFP, a mutant that functioned as an AND (logical product) gate (two-input AND-type sensor) was obtained (see Figure 2(d)). AraC-LuxR was detected with just one round of EP-PCR / screening. N86Kand C245WThe following mutants (see Table 5: F74L, P86T, V249A and N298I) could be obtained. For more information, see P lux We confirmed that the regulated GFP expression acts as an AND gate, responding only when both arabinose and AHL are present. Furthermore, we confirmed that the Lux promoter and the arabinose promoter {araP(P BAD )} was confirmed to have the same effect. N86Kand C245W The mutants (see Table 5: F74L, P86T, V249A, and N298I) were able to independently induce an AND response to two different promoters. Therefore, we confirmed that this mutant can function as a "two-input AND-type" sensor, capable of independently generating an AND-type output of arabinose / AHL from the arabinose promoter and the Lux promoter. The method for evaluating the above-mentioned ligand responsiveness is described in detail below. A 200 μL sample prepared by diluting the culture medium 10-fold with saline was placed in a microwell plate (manufactured by Nunc). Cell density was measured at 595 nm with light irradiation and fluorescence at 535 nm with excitation at 485 nm using a FilterMaxF5 (a commercially available absorbance / fluorescence detector, manufactured by Molecular Devices). A 200 μL blank sample prepared by diluting a bacteria-free liquid medium 10-fold with saline was also measured. The blank sample values ​​were subtracted from the sample measurements, and data analysis was performed using values ​​corrected for the dilution rate. The same applies to Examples 2-2, 2-3, 2-4, and 2-5 below.

[0060] (Example 2-2: Development example 2 of a two-input AND-type output sensor) AraC-LuxR N86K and C245W The full-length fragments were subjected to EP-PCR (50 μM MnCl2, amplification factor 10,000-fold) to generate a mutant pool with random mutations (library size 3 x 10 6 Unlike Example 2-1, P BAD7Using -GFP as a reporter, 90 colonies were randomly picked (see Figure 3). Their ligand responsiveness was evaluated. Specifically, they were cultured in either "13 mM L-arabinose only" or "13 mM L-arabinose + 100 μM AHL," and seven mutants were obtained that showed a more than two-fold increase in the BOTH (L-arabinose + AHL) / L-arabinose ratio. Figure 3(a) shows the fluorescence intensity measurement results for the mutant with the highest BOTH / L-arabinose ratio. AraC-LuxR was detected with just one round of EP-PCR / screening. N86K and C245W The following mutants (see Table 5: P39R, I197N and N252S) could be obtained.

[0061] (Example 2-3: Development example of a two-input OR-type output sensor) The library shown in Figure 2(c) contains AraC-LuxR with various mutations. N86K and C245W In this example, P BAD7 Screening was performed using a downstream GFP reporter. Unlike the two-input AND type (Figure 2(d)), the presence of either arabinose or AHL resulted in P BAD We confirmed that we could obtain an "OR type (logical sum type)" biosensor that enhances the reporter gene below. Details are as follows. AraC-LuxR N86K and C245W The full-length fragments were subjected to EP-PCR (50 μM MnCl2, amplification factor 10,000-fold) to generate a mutant pool with random mutations (library size 3 x 10 6 (It was). P BAD7 Using -GFP as a reporter, 90 colonies that showed fluorescence on solid medium containing 10 μM AHL were picked. Their ligand responsiveness was evaluated. Specifically, they were cultured in either "no ligand" or "10 μM AHL" media, and two mutants were obtained that showed a more than three-fold increase in the AHL / none ratio. Figure 3(b) shows the fluorescence intensity measurement results for the mutant with the highest AHL / none ratio. AraC-LuxR was detected with just one round of EP-PCR / screening. N86Kand C245W The mutant (see Table 5: E295K) was obtained. In other words, we were able to develop a two-input OR-type sensor.

[0062] (Example 2-4: Development example 1 of a two-input, one-output type sensor) In this example, P was used as an "arabinose-only" gate, which is an example of a two-input, one-output sensor that does not respond to AHL but is active only with arabinose (responding in a concentration-dependent manner). BAD We have developed a gene switch using the following: AraC-LuxR N86K and C245W The full-length fragments were subjected to EP-PCR (50 μM MnCl2, amplification factor 10,000-fold) to generate a mutant pool with random mutations (library size 3 x 10 6 (It was). P BAD7 Using -GFP as a reporter, 90 colonies that showed fluorescence on solid medium containing 13 mM L-arabinose were picked. Their ligand responsiveness was evaluated. Specifically, they were cultured in either 13 mM L-arabinose or 10 μM AHL, and 46 mutants were obtained that showed a more than two-fold increase in the L-arabinose / AHL ratio. Figure 3(c) shows the fluorescence intensity measurements of mutants that showed a high L-arabinose / AHL ratio and low leakage expression with AHL alone. AraC-LuxR was detected with just one round of EP-PCR / screening. N86Kand C245W The following mutants (see Table 5: M175K and K491E) were obtained. In other words, we have developed a two-input, one-output sensor.

[0063] (Example 2-5: Development example 2 of a two-input, one-output type sensor) In this example, P was used as an "AHL-Only" gate, which is an example of a two-input, one-output sensor that does not respond to arabinose but is active only with AHL (responding in a concentration-dependent manner). BAD developed a gene switch using First, we reduced the affinity of AraC-LuxR for arabinose by introducing mutations into the amino acid residues required for arabinose recognition in the AraC ligand (arabinose) binding site. Then, we performed EP-PCR / screening using the reduced library. Details are as follows. First generation: A library was prepared by randomizing the L-arabinose binding residues (H80, H81, and Y82) of AraC-LuxR using a known method (library size 3 x 10 6 (It was). P BAD7 Using -GFP as a reporter, 48 colonies that showed fluorescence on solid medium containing 10 μM AHL were selected. Their ligand responsiveness was evaluated. Specifically, they were cultured in "no ligand," "13 mM L-arabinose," and "10 μM AHL." Five mutants were obtained that did not respond to L-arabinose but showed fluorescence upon AHL addition. Among these, mutants with AHL / none and L-arabinose / none ratios of 1.0 and 1.6, respectively (H80F, H81K, and Y82L) were selected as parents for the second generation. Second generation: The full-length mutants obtained in the first generation were subjected to EP-PCR (50 μM MnCl2, amplification factor 10,000-fold) to generate a mutant pool with random mutations (library size 5 x 10 5 (It was). P BAD7 Using -GFP as a reporter, 41 colonies showing moderate to strong fluorescence were selected from a total of 1,600 colonies formed on solid medium containing 10 μM AHL. These colonies were then evaluated for their ligand responsiveness. Specifically, they were cultured in "no ligand," "13 mM L-arabinose," and "10 μM AHL," and five mutants were obtained that did not respond to L-arabinose and had an AHL / none ratio of 2-fold or more. The fluorescence intensity measurement results of the mutant with the highest AHL / none ratio are shown in Figure 3(d). After two rounds of screening (evolutionary engineering), AraC-LuxR N86K and C245WThe following mutants (see Table 5: H80F, H81K, Y82L, N393I, Y439H, R523L and F541L) were obtained. That is, a two-input, one-output type sensor different from that of Example 2-4 was developed. [Example]

[0064] (Example 3: Development example of a three-input sensor) TetR-AraC-LuxR N86K and C245W Preparation of fusion A three-input sensor was developed based on the two-input sensor prepared in Example 2. AraC-LuxR prepared in Example 2 N86K and C245W Another transcription factor, TetR (a tetracycline-responsive transcription factor), was fused to the N-terminus of the fusion product, resulting in TetR-AraC-LuxR. N86Kand C245W We created a three-transcription factor fusion protein (SEQ ID NO: 16) (see Figure 4). We confirmed that this three-transcription factor fusion protein (SEQ ID NO: 16) retained its function for all target promoters. Furthermore, we confirmed that each transcription factor responded only to its own target ligand (see Figure 5(a)). Specifically, for TetP (Tet promoter), it acted only in response to the TetR target substance aTc (anhydrotetracycline). In other words, it did not respond to arabinose or AHL.

[0065] TetR-AraC-LuxR N86K and C245W Generation of mutants 1st generation: TetR-AraC-LuxR N86K and C245W The full-length fragments of the clones (see Figure 10) were subjected to EP-PCR (50 μM MnCl2, amplification factor 1,000) to generate a mutant pool with random mutations (library size 1 x 10 5 (It was). P tetUsing Ptet-GFP-TK::APH as a reporter (see Figure 15), 35 weakly fluorescent colonies were picked from a total of 3,700 colonies formed on solid medium containing 216 nM aTc, 13 mM L-arabinose, and 10 μM AHL. Their ligand responsiveness was qualitatively evaluated using the spot method. In eight solid medium combinations with and without each ligand, it was confirmed that one mutant exhibited transcriptional repression when bound to all ligands. To remove the base deletion introduced in this mutant, the TetR domain was replaced with AraC-LuxR. N86Kand C245W The re-fused product was adopted as the parent for the second generation. Second generation: The full-length mutants obtained in the first generation were subjected to EP-PCR (10 μM MnCl2, amplification factor 1,000-fold) to generate a mutant pool with random mutations (library size 7 x 10 5 (It was). P lux -APH( A minoglycoside ph To eliminate mutants with base deletions or stop codons, selection was performed using the selector α-phosphotransferase (α-phosphotransferase: kanamycin resistance gene) (216 nM aTc, 13 mM L-arabinose, 10 μM AHL, and 30 μg / mL kanamycin for 3 hours). Next, P tet Using -GFP as a reporter, eight weakly fluorescent colonies were selected from a total of 2,000 colonies formed on solid medium containing 216 nM aTc, 13 mM L-arabinose, and 10 μM AHL. Their ligand responsiveness was qualitatively evaluated using the spot method. In eight solid medium crosses with and without each ligand, three mutants showed gradual transcriptional repression without base deletion or stop codons. Among these, mutants that showed suppressed leaky expression under all ligand-containing conditions were selected as the third-generation parents. Third generation: The full-length mutants obtained in the second generation were subjected to EP-PCR (10 μM MnCl2, amplification factor 1,000-fold) to generate a mutant pool with random mutations (library size 3 x 104 (It was). P lux To remove mutants with base deletions or stop codons, selection was performed using -APH as a selector (216 nM aTc, 13 mM L-arabinose, 10 μM AHL, and 60 μg / mL kanamycin for 3 hours). tet Using -APH as a selector, selection was performed to enrich for mutants with reduced transcriptional repression activity in the presence of aTc alone (216 nM aTc and 30 μg / mL kanamycin for 3 hours). tet Using -GFP as a reporter, 184 weakly fluorescent colonies were selected from a total of 384 colonies formed on solid medium containing 216 nM aTc, 13 mM L-arabinose, and 10 μM AHL. Their ligand responsiveness was evaluated. Specifically, they were cultured in "no ligand," "216 nM aTc only," or "216 nM aTc, 13 mM L-arabinose, and 10 μM AHL." Five mutants showed weak transcriptional repression with aTc alone, but strong transcriptional repression with all ligands. Because the functions of these five mutants were similar but the mutation sites were different, all were used as parents for the fourth generation. Fourth generation: The five mutants obtained in the third generation were mixed and subjected to full-length PCR (10 μM MnCl2, amplification factor 1,000-fold) to generate a mutant pool with random mutations (library size 2 x 10 4 (It was). P lux Using Ptet-APH as a selector, selection was performed to remove mutants with base deletions or stop codons (216 nM aTc, 13 mM L-arabinose, 10 μM AHL, and 30 μg / mL Kan for 3 hours). Furthermore, using Ptet-APH as a selector, selection was performed to enrich for mutants with reduced transcriptional repression activity in the presence of aTc alone (216 nM aTc and 30 μg / mL Kan for 3 hours). tetUsing -GFP as a reporter, 45 weakly fluorescent colonies were picked from a total of 900 colonies formed on solid medium containing 216 nM aTc, 13 mM L-arabinose, and 10 μM AHL. Their ligand responsiveness was qualitatively evaluated using the spot method. In eight solid medium combinations with and without each ligand, the transcriptional repression activity of three mutants increased with increasing ligand concentration. The mutations introduced into these three mutants were identical, and the fluorescence intensity measurement results of one of the mutants are shown in Figure 5(b). After four rounds of screening (evolutionary engineering), TetR-AraC-LuxR N86Kand C245W Mutants (see Table 5: K46R, D95G, K108N, I134V, V145A, L204P, I214N, P216T, F217S, L409Q, T545A and S569T) were obtained. In other words, we were able to develop a three-input sensor (especially a three-input three-stage output reduction sensor).

[0066] [Table 5] [Example]

[0067] In this example, we used ArsR, which is known as an arsenic sensor, instead of the arabinose sensor (AraC) to confirm that the two-input sensor developed above can function as a gene switch in combinations other than AraC and LuxR. Details are as follows (see Figure 6(a)). The arsenic standard solution (hereinafter referred to as As(III)) used in this example was purchased from Wako Pure Chemical Industries, Ltd. as an arsenic standard solution (As 1000).

[0068] (1-1)ArsR::LuxR N86K and C245W (SEQ ID NO: 17) (See Figure 11) (1-1-1)ArsR::LuxR N86K and C245W Preparation of vectors for Plasmid 1 (see Table 7) was subjected to PCR (KOD plus) using Primers 1 and 2 (see Table 6: SEQ ID NOs: 1 and 2) to obtain a PCR product. The PCR product was then digested with DpnI and gel extracted. The extract was digested with XhoI (commercially available restriction enzyme) and SpeI-HF (commercially available restriction enzyme) and further gel extracted. (1-1-2) Preparation of ArsR domain (see Figure 12) PCR (KOD plus) was performed using the genome of E. coli strain MG1655 as a template and Primers 3 and 4 (see Table 6: SEQ ID NOs: 3 and 4) to obtain a PCR product. The PCR product was then digested with DpnI and gel extracted. The extract was digested with XhoI and SpeI-HF and further gel extracted. (1-1-3)ArsR::LuxR N86K and C245W Collection of The vector fragment (1-1-1) and the insert fragment (1-1-2) were ligated. The ligation product was then transformed into XL10-Gold (commercially available competent cells) and clones were recovered.

[0069] (1-2) Construction of ArsR (1-2-1) Preparation of the vector Plasmid 1 (see Table 7) was subjected to PCR (KOD plus) using Primers 1 and 2 (see Table 6) to obtain a PCR product. The PCR product was then digested with DpnI and gel extracted. The extract was then digested with XhoI and SpeI-HF and further gel extracted. (1-2-2) Preparation of the insert side PCR (KOD plus) was performed using the genome of E. coli strain MG1655 as a template and Primers 3 and 5 (see Table 6: SEQ ID NOs: 3 and 5) to obtain a PCR product. The PCR product was then digested with DpnI and gel extracted. The extract was digested with XhoI and SpeI-HF and further gel extracted. (1-2-3) Collection of ArsR Ligation was performed using the vector fragment from "1-2-1" and the insert fragment from "1-2-2." Next, XL10-Gold was transformed with the ligation product, and clones were recovered.

[0070] (1-4)P ars- Preparation of GFP (1-4-1)P ars - [rbs] GFP library generation Using Plasmid 2 (see Table 7) as a template, PCR (KOD plus) was performed with Primers 6 and 7 (see Table 6: SEQ ID NOs: 6 and 7) to obtain a PCR product. The PCR product was then gel extracted. DNA fragments were assembled using the Golden Gate method using commercially available gel extracts, followed by column purification. XL10-Gold was transformed with the purified Golden Gate product, followed by liquid culture to recover the RBS library. (1-4-2) P with moderate RBS ars -GFP (Pars-GFP with RBS that shows high fluorescence in the absence of ArsR expression plasmid) E. coli MG1655 was co-transformed with the RBS library from "1-4-1". Next, one colony with strong fluorescence was selected from the colonies isolated on the solid medium and cultured overnight. Next, P ars The P-GFP plasmid was then recovered. ars E. coli MG1655 was co-transformed with -GFP and ArsR (Plasmid 4; see Table 7). Colonies were then isolated on solid medium and confirmed to have weaker fluorescence than the strongly fluorescent colonies obtained from the isolated colonies.

[0071] [Table 6]

[0072] [Table 7]

[0073] (2-1) Construction of ArsR::LuxR library (2-1-1) Preparation of vector fragment Using Plasmid 3 (see Table 7) as a template, PCR (KOD plus) was performed with Primers 8 and 9 (see Table 6: SEQ ID NOs: 8 and 9) to obtain a PCR product. The PCR product was then digested with DpnI and gel extracted. The extract was digested with XhoI, BamHI-HF, and rSAP, and further gel extracted. (2-1-2) Preparation of ArsR::LuxR fragment with random mutations introduced throughout the gene Using Plasmid 3 (see Table 7) as a template, PCR (KOD plus) was performed with Primers 10 and 11 (see Table 6: SEQ ID NOs: 10 and 11) to obtain a PCR product. The PCR product was then digested with DpnI and gel extracted. Using the gel extract as a template, PCR (Taq, 10 or 50 μM MnCl2) was performed with Primers 10 and 11 (see Table 6) to obtain a PCR product. The PCR product was then gel extracted. The extract was digested with XhoI and BamHI-HF and further gel extracted. (2-1-3) Recovery of the ArsR::LuxR library Ligation was performed using the vector fragment (2-1-1) and the insert fragment (2-1-2). The ligation product was then purified using a column. The purified ligation product was then transformed into E. coli strain BW25113, and the library was recovered from the liquid culture medium.

[0074] (2-2) Evaluation of reporter (GFP) expression level MG1655 was transformed with a reporter plasmid of your choice. One transformant was then selected to prepare competent cells (E. coli). The E. coli was transformed with the library (plasmid). An arbitrary number of isolated colonies were selected and cultured overnight in liquid. A 1 / 100 volume of the pre-culture solution was inoculated into a liquid medium containing As(III) or AHL at an arbitrary concentration and cultured for 12 hours. Next, the culture solution was diluted 10-fold with saline to prepare a 200 μL measurement sample. The cell density (OD ) was measured using a FilterMax F5 (a commercially available absorbance detector). 595 ) and fluorescence at 535 nm when excited at 485 nm were measured. A blank sample (200 μL) prepared by diluting the liquid medium 10-fold with saline was also measured as a blank sample (control), and data analysis was performed by subtracting the measurement value of the blank sample from the measurement value of the sample.

[0075] (2-3) Selection of ArsR::LuxR library Reporter P lux MG1655 was transformed with ArsR::LuxR (Plasmid 5: see Table 7). One transformant was selected and used to prepare competent cells (E. coli). The E. coli was transformed with the ArsR::LuxR library from "2-1" above, inoculated into 10 mL of liquid medium, and cultured overnight. Next, 10 mL of the pre-culture solution was inoculated into 10 mL of liquid medium containing 1 μM AHL and 5000 ppb (67 μM) As(III). 7 The cells were inoculated and cultured for 1 hour. Next, 100 μg / mL kanamycin was added and cultured for 3 hours. The culture was then centrifuged and the supernatant removed. The cells were resuspended in 10 mL of liquid medium containing 1 μM AHL and 5000 ppb (67 μM) As(III), and the culture was centrifuged again to remove the supernatant (removing the kanamycin). The kanamycin was removed again. The cells were then resuspended in 10 mL of liquid medium containing 1 μM AHL and 5000 ppb (67 μM) As(III) and cultured overnight. Finally, the culture was harvested, and the enriched library plasmids were recovered.

[0076] (3-1) Fabrication of a highly sensitive two-input AND-type arsenic switch (ArsR-LuxR) Reporter plasmid P lux MG1655 was transformed with ArsR-GFP (Plasmid 5: see Table 7). One transformant was then selected to prepare competent cells (E. coli). The competent cells (E. coli) were transformed with the ArsR-LuxR library described in "2-1" above. 89 colonies formed on solid medium were randomly selected. Using the method described in "2-2" above, we evaluated the fluorescence intensity per cell density when the cells were cultured in a medium containing 1 μM AHL and a medium containing 1 μM AHL and 5000 ppb As(III). We recovered mutants that showed weak fluorescence when only AHL was added, but showed strong fluorescence when both AHL and As(III) were added. Using the recovered mutants as a template, a library was constructed using the method described in "2-1" above. The constructed library was then used to transform the competent cells (Escherichia coli). 59 colonies formed on the solid medium were randomly selected. The fluorescence intensity per cell density was evaluated using the method described in "2-2" above when the cells were cultured in a medium containing 1 μM AHL and a medium containing 1 μM AHL and 5 ppb As(III). Mutants that exhibited weak fluorescence when only AHL was added, but exhibited strong fluorescence when both AHL and As(III) were added, were recovered. We confirmed that a highly sensitive AND-type arsenic switch was fabricated using the method described in "2-2" above (see Figure 6(c)).

[0077] (3-2) Fabrication of a highly stringent two-input AND-type arsenic switch (ArsR-LuxR) Reporter plasmid P lux MG1655 was transformed with ArsR-GFP (Plasmid 5: see Table 7). One transformant was then selected and used to prepare competent cells (E. coli). The competent cells (E. coli) were transformed with the ArsR-LuxR library described in "2-1" above. Using the method described in "2-3" above, mutants that exhibited fluorescence when 1 μM AHL and 5000 ppb As(III) were added were enriched. The enriched library was then transformed into the competent cells (E. coli). Eighty colonies with weak fluorescence were then selected on solid medium containing 1 μM AHL. Using the method described in "2-2" above, we evaluated the fluorescence intensity per cell density when culturing cells in three media: "medium containing 1 μM AHL," "medium containing 1 μM AHL and 5 ppb As(III)," and "medium containing 1 μM AHL and 500 ppb As(III)." We identified mutants that exhibited weak fluorescence when only AHL was added, but exhibited strong fluorescence when AHL and 5 ppb As(III) were added, and when AHL and 500 ppb As(III) were added. We confirmed that a highly stringent AND-type arsenic switch was fabricated using the method described in "2-2" above (see Figure 6(b)).

[0078] (ArsR / P ars (Assay of arsenic response of In this example, the reporter plasmid was P lux We evaluated the performance of a two-input AND-type arsenic switch without using .

[0079] (4-1) Change to pUC vector (4-1-1) Vector preparation pUC-TAL(Plasmid 8: see Table 7, TetR-AraC-LuxR N86Kand C245W ) as a template and primers 12 and 13 (see Table 6: SEQ ID NOs: 12 and 13) were used to perform PCR (KOD plus) to obtain a PCR product. The PCR product was then gel extracted. The extract was digested with KpnI-HF (a known restriction enzyme), HindIII-HF, and rSAP, and further gel extracted. (4-1-2) Preparation of inserts PCR (KOD plus) was performed using ArsR and ArsR::LuxR (Plasmids 4 and 7; see Table 7) as templates and Primers 11 & 14 (see Table 6; SEQ ID NOS: 11 and 14) to obtain PCR products. The PCR products were then gel-extracted. The extracts were digested with KpnI-HF and HindIII-HF and further gel-extracted. (4-1-3) Recovery of pUC products The vector fragment of "1-2-1" and the insert fragment of "1-2-2" were ligated. Next, XL10-Gold was transformed with the ligation product, and clones were recovered.

[0080] (4-2)P ars -Assessment of arsenic response using GFP (see Figure 16) Reporter plasmid P ars MG1655 was transformed with .gamma.-GFP (Plasmid 6: see Table 7). One transformant was selected and competent cells (Escherichia coli) were prepared. Competent cells (Escherichia coli) were transformed with pUC-ArsR, pUC-ArsR::LuxR, or pUC-phi (Plasmids 9-11: see Table 7). Three colonies formed on solid medium were randomly selected. Using the method described in "2-2" above, the fluorescence intensity per cell density was evaluated when cells were cultured containing 100 μM AHL and with various As(III) concentrations (see FIG. 8). [Example]

[0081] (Method for selecting a sensitivity-variable gene switch) From the results shown in Figures 2 and 6 above, the inventors confirmed that the response sensitivity of a sensor containing a gene switch obtained by the gene switch manufacturing method of the present invention to each ligand depends on the concentration of the other ligand. Specifically, when the EC50 value of the sensor containing the two-input AND-type arsenic switch prepared in Example 4 was plotted against the AHL concentration present in the system, it was confirmed that it decreased as the AHL concentration increased (see Figure 7). This phenomenon is a heterogeneous effect of sensors containing gene switches obtained by the method of the present invention. AND-type gene switches maintain their functional structure depending on stabilization by binding to both AHL and arsenic. In the case of gene switches with this property, the higher the AHL concentration in the system, the higher the effective concentration of ArsR, which acts as an arsenic sensor, and therefore the apparent sensitivity of "arsenic + ArsR ⇔ arsenic · ArsR." That is, in a sensor including the gene switch of the present invention, the sensitivity of the gene switch to a ligand can be easily adjusted by the following method. If it is desired to increase the sensitivity of the ligand L1, the concentration of the added ligand L2 is increased. If it is desired to reduce the sensitivity of the ligand L1, the concentration of the added ligand L2 is reduced. If it is desired to increase the sensitivity of the ligand L2, the concentration of the added ligand L1 is increased. If it is desired to reduce the sensitivity of the ligand L2, the concentration of the added ligand L1 is reduced. [Example]

[0082] (Method for selecting highly sensitive gene switches) From the results of the above examples, the inventors have confirmed that the manufacturing method of the present invention can improve the sensitivity, output intensity, stringency, etc. of the sensor. For example, when using the transcription factor LuxR, the arsenic detection switch is intentionally set to P lux Detection based on the expression of a reporter gene downstream of a promoter has the following advantages. (1) Output strength and stringency: LuxR is the most stringent transcription factor (low expression leakage in the non-induced state) and has a high maximum output (transcriptional enhancement efficiency) upon induction. ars ) system has low output intensity and low stringency. Therefore, the signal-to-noise ratio during the arsenic response was very low (see "ArsR→P" in Figure 8). arsFurthermore, the AND of ArsR-LuxR resulted in even lower stringency due to the influence of the fusion (see Figure 8, "AND → P"). ars On the other hand, when the reporter gene was placed under LuxP, a larger change in fluorescence intensity was observed in response to the arsenic concentration due to the superior SN ratio of LuxR / LuxP (see "ArsR→P" in Figure 8). lux , AHL). That is, by selecting a transcription factor (e.g., LuxR) that can be detected with high sensitivity as a fusion partner and further selecting a promoter (e.g., LuxP) of the transcription factor, a gene switch that can detect a ligand with high sensitivity can be selected. (2) Highly sensitive detection of ligands: For example, in the case of the combination of the ArsR-LuxR transcription factor and promoter ArsP, the expression profile of the reporter gene downstream of ArsP had an inflection point at around 50 ppb. This detection sensitivity is almost the same as that when ArsR alone acts on ArsP. However, in the case of the combination of the ArsR-LuxR transcription factor and promoter P, lux In the case of the combination of P lux The expression profile of the downstream reporter gene had an inflection point around 5 ppb. This sensitivity is the highest in the world for a biosensor. The WHO standard for arsenic concentration is 10 ppb. When using the ArsR / ArsP system, the inflection point is at a concentration one order of magnitude higher than that, making it unusable. However, the transcription factor and promoter P of ArsR-LuxR lux The combination of these two can be used directly for environmental monitoring. This increased sensitivity is due to the release of the As-ArsR / ArsP from the "depressor" mechanism. ArsR can naturally bind to As with high affinity, but in the absence of As, it tightly binds to ArsP. In other words, the inhibited ArsR structure is greatly stabilized by binding to ArsP. Arsenic cancels this stabilized structure, causing ArsR to desorb from ArsP. In other words, the equilibrium between As + ArsR and As-ArsR is significantly shifted to the left by the presence of ArsP (i.e., the sensitivity is reduced). In the absence of ArsP, the As-ArsR binding detected by LuxP is free from this sensitivity-reducing effect, resulting in an order of magnitude increase in sensitivity. [Example]

[0083] In this example, to confirm that the two-input sensor developed above can also be made using enzymes as materials, the enzymes PrpC and PrpD were used instead of the arabinose sensor (AraC) and the arsenic sensor (ArsR). Details are as follows: PrpC and PrpD are enzymes derived from Escherichia coli called 2-methylcitrate synthase (EC:2.3.3.5) and 2-methylcitrate dehydratase (EC:4.2.1.3), respectively.

[0084] (1-1)PrpC::LuxR N86K-C245W , PrpD::LuxR N86K-C245W Preparation of (1-1-1)PrpC::LuxR N86K-C245W , PrpD::LuxR N86K-C245W Preparation of vectors for Plasmid-12 (see Table 7) was digested with NcoI-HF (commercially available restriction enzymes) and SpeI-HF (commercially available restriction enzymes), followed by gel extraction. (1-1-2) Preparation of PrpC and PrpD domains PCR products were obtained by PCR (Q5 primerase) using Primers 15 and 16 or Primers 17 and 18 (see Table 6) with the genome of E. coli strain MG1655 as a template. The PCR products were then digested with DpnI and gel extracted. (1-1-3)PrpC::LuxR N86K-C245W , PrpC::LuxR N86K-C245W Collection of The vector fragment (1-1-1) and the insert fragment (1-1-2) were assembled using commercially available enzymes by the Gibson assembly method. The Gibson assembly product was then transformed into XL10-Gold (commercially available competent cells), and clones were recovered (Plasmids 14 and 15).

[0085] (1-2)pMC-P lux Preparation of -GFP (1-2-1)pMC-P lux Preparation of vector for -GFP Plasmid-16 (see Table 7) was subjected to PCR (KOD plus) with Primers-19 and 20 (see Table 6) to obtain a PCR product. The PCR product was then digested with DpnI and gel-extracted. The extract was subjected to PCR (KOD plus) with Primers-19 and 21 (see Table 6) to obtain a PCR product. The PCR product was then gel-extracted. The extract was digested with ApaI (a commercially available restriction enzyme), HindIII-HF (a commercially available restriction enzyme), and rSAP (a commercially available phosphatase), and then gel-extracted. (1-2-2) Preparation of GFP-hsvTK::APH domain with randomized RBS Using Plasmid-5 (see Table 7) as a template, PCR (KOD plus) was performed with Primers-22 and 23 (see Table 6) to obtain a PCR product. The PCR product was then digested with DpnI and gel extracted. The extract was then digested with ApaI and HindIII-HF and further gel extracted. (1-2-3)pMC-P lux Recovery of the -GFP RBS library The vector fragment (1-2-1) and the insert fragment (1-2-2) were ligated. The ligation product was then transformed into XL10-Gold (commercially available competent cells), and clones were recovered. (1-2-4)pMC-P lux -GFP recovery The E. coli strain BW25113 was transformed with the library plasmids and Plasmid-17 described in "1-2-3" above. Thirty isolated colonies were randomly selected and cultured overnight in liquid. A 1 / 100 volume of the cells was inoculated into a medium containing or not containing 1 μM AHL and cultured for 12 hours. Then, the culture medium was diluted 10-fold with saline to prepare a 200 μL measurement sample. The cell density (OD ) was measured using FilterMax F5 (a commercially available absorbance detection system). 595 ) and fluorescence at 535 nm when excited at 482 nm. A 200 μL blank sample (control) prepared by diluting the liquid medium 10-fold with saline was also measured simultaneously, and data analysis was performed by subtracting the blank sample measurement value from the sample measurement value. From the data obtained, the RBS mutant with the greatest change in fluorescence intensity between the presence and absence of AHL was isolated and recovered.

[0086] (1-3)PrpC K318R ::LuxR N86K-C245W , PrpD S163T-A225G-I285V ::LuxR N86K-C245W Preparation of (1-3-1)PrpC::LuxR N86K-C245W , PrpD::LuxR N86K-C245W Preparation of library vectors Plasmid 14 or 15 (see Table 7) was subjected to inverse PCR (Q5 primerase) using Primer 24 or 25 (see Table 6) to obtain a PCR product, which was then digested with DpnI and gel extracted. (1-3-2)PrpC-LuxR N86K-C245W , PrpD-LuxR N86K-C245W Preparation of domain libraries Using Plasmid-14 or 15 (see Table 7) as a template, error-prone PCR (Taq PCR enzyme) was performed with Primers-26 and 27 (see Table 6) to obtain PCR products, which were then digested with DpnI and gel extracted. (1-3-3)PrpC::LuxR N86K-C245W , PrpD::LuxR N86K-C245WRecovery of library plasmids The vector fragment (1-3-1) and the insert fragment (1-3-2) were assembled into DNA fragments by the Gibson assembly method using commercially available enzymes. The Gibson assembly product was then transformed into BW25113 (commercially available competent cells) to recover the library plasmids. (1-3-4)pET23d-PrpC K318R ::LuxR N86K-C245W , pET23d-PrpD S163T-A225G-I285V ::LuxR N86K-C245W Collection of The E. coli strain BW25113 was transformed with the library plasmids and Plasmid-23 described in "1-3-3" above, and the resulting mixture was spread onto a screening plate containing 10 μM AHL and 50 mM propionic acid. 48 colonies emitting GFP fluorescence were selected from the isolated colonies and cultured overnight in liquid medium. A 1 / 100 volume of the bacteria was inoculated into liquid medium containing or not containing 50 mM propionic acid, and cultured for 12 hours. Next, the culture medium was diluted 10-fold with saline to prepare a 200 μL measurement sample. The cell density (OD ) was measured using a FilterMax F5 (a commercially available absorbance detection system). 595 ) and 535 nm fluorescence when excited at 482 nm. 200 μL of a blank sample (control) prepared by diluting the liquid medium 10-fold with saline was also measured at the same time, and data analysis was performed by subtracting the measurement value of the blank sample from the measurement value of the sample. From the obtained data, the mutant with the greatest change in fluorescence intensity between the presence and absence of propionic acid was isolated and recovered, and its sequence was analyzed to identify the mutation site (PrpC K318R -LuxR N86K and C245W Full length amino acid sequence of: SEQ ID NO: 31, PrpD S163T-A225G-I285V -LuxR N86Kand C245W Full-length amino acid sequence: SEQ ID NO: 32).

[0087] (2) pMC-P lux -Propionate response evaluation using GFP Reporter plasmid pMC-P luxBW25113 was transformed with -GFP (Plasmid 13: see Table 7). One transformant was selected and competent cells (Escherichia coli) were prepared. pET23d-PrpC::LuxR mut (See Figure 19), pET23d-PrpD::LuxR mut Competent cells (Escherichia coli) were transformed with Plasmid 18-19 (see Table 7) (see Figure 20). Three colonies formed on the solid medium were randomly selected and cultured overnight in liquid. 1 / 100 of the pre-culture solution was inoculated into liquid media containing 10 μM sodium propionate at random concentrations and cultured for 12 hours. Next, the culture solution was diluted 10-fold with saline to prepare a 200 μL measurement sample. The cell density (OD ) was measured using a FilterMax F5 (a commercially available absorbance detector). 595 ) and fluorescence at 535 nm when excited at 485 nm were measured. 200 μL of a blank sample (control) prepared by diluting the liquid medium 10-fold with saline was also measured at the same time, and data analysis was performed by subtracting the blank sample measurement value from the sample measurement value. Fluorescence intensity per cell density during culture was evaluated. The results are shown in Figure 21. In E. coli, propionate becomes propionyl-CoA, a substrate for PrpC, and then 2-methylcitrate, a substrate for the enzyme PrpD. When PrpC and PrpD in PrpC::LuxR and PrpD::LuxR bind to their respective substrates (propionyl-CoA and 2-methylcitrate), PrpC::LuxR and PrpD::LuxR are stabilized, increasing the transcriptional activity of LuxR. LuxR then binds to Plux in Plux-GFP, transcribing GFP. Therefore, in Figure 21, the fluorescence intensity increased in a propionate concentration-dependent manner.

[0088] (General remarks) As shown in the above examples, the method for manufacturing a gene switch of the present invention not only produces a functionally integrated, highly sophisticated, and variable sensor, but also compensates for the shortcomings of sensor function with low basic performance and can output a superior transcription factor function. [Industrial Applicability]

[0089] It is possible to provide a method for producing a multi-input / multi-output gene switch or transcription factor, and a multi-input / multi-output gene switch or transcription factor.

Claims

1. A method for producing a gene switch or a transcription factor that forms said switch, comprising the steps of: (A) (1) Ligand L １ Binder B responds to 1 or transcription factor T １ Gene sequence encoding the ligand L ２ Binder B responds to 2 or transcription factor T ２ Binder B obtained by introducing a mutation into a gene construct carrying a gene sequence encoding 1 or transcription factor T １ and Binder B 2 or transcription factor T ２ a library of nucleic acids of fusion mutants of transcription factor T １ Promoter P controlled by １ and / or a gene sequence encoding transcription factor T ２ Promoter P controlled by ２ and the promoter sequence P １ and / or P ２ a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding a reporter Rx functionally linked to the reporter Rx, wherein x is an integer of 1 or more; (2) Ligand L １ and / or ligand L ２ (1) to the cell or cell-free protein synthesis system; (3) Using the reporter expression level as an indicator, 1 or transcription factor T １ and Binder B 2 or transcription factor T ２ selecting the fusion mutant as a gene switch or a transcription factor forming the switch; Or, (B) (1) Ligand L １ Binder B responds to 1 or transcription factor T １ a gene sequence encoding the ligand L ２ Binder B responds to 2 or transcription factor T ２ Gene sequence encoding the ligand L Ｎ Binder B responds to N or transcription factor T Ｎ Binder B obtained by introducing a mutation into a gene construct carrying a gene sequence encoding 1 or transcription factor T １ and Binder B 2 or transcription factor T ２ and Binder B N or transcription factor T Ｎ a library of nucleic acids of fusion mutants of transcription factor T １ Promoter P controlled by １ The gene sequence encoding the transcription factor T ２ Promoter P controlled by ２ and / or a gene sequence encoding the transcription factor T Ｎ Promoter P controlled by Ｎ and the promoter sequence P １ , P ２ and / or P Ｎ a step of introducing into a cell or adding to a cell-free protein synthesis system a reporter expression vector carrying a gene sequence encoding a reporter Rx functionally linked to the reporter Rx, wherein N is an integer of 3 or more; (2) Ligand L1, Ligand L ２ and / or ligand L Ｎ (1) into the cell or cell-free protein synthesis system; (3) Using the reporter expression level as an indicator, 1 or transcription factor T １ and Binder B 2 or transcription factor T ２ and Binder B N or transcription factor T Ｎ a step of selecting the fusion mutant of the above as a gene switch or a transcription factor that forms the switch.

2. In the above (A), Ligand L １ and ligand L ２ Expression level of reporter by introduction of ligand L １ or ligand L ２ A fusion mutant exhibiting a high reporter expression ratio as a result of the introduction of the above is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming the switch.

2. The method of claim 1.

3. In the above (A), transcription factor T ２ Promoter P controlled by ２ When using the ligand L １ and ligand L ２ Expression level of reporter by introduction of ligand L ２ A fusion mutant exhibiting a high reporter expression ratio as a result of the introduction of the above is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming the switch. Or, transcription factor T １ Promoter P controlled by １ When using the ligand L １ and ligand L ２ Expression level of reporter by introduction of ligand L １ A fusion mutant exhibiting a high reporter expression ratio as a result of the introduction of the above is selected as a genetic switch having a two-input, AND-type output sensor function or a transcription factor forming the switch.

2. The method of claim 1.

4. In the above (A), transcription factor T １ Promoter P controlled by １ When using the ligand L ２ A fusion mutant having a high ratio of reporter expression level upon introduction of the above to reporter expression level without a ligand is selected as a genetic switch having a two-input OR-type sensor function or a transcription factor that forms the switch. Or, transcription factor T ２ Promoter P controlled by ２ When using the ligand L １ A fusion mutant having a high ratio of reporter expression level upon introduction of the above to reporter expression level without a ligand is selected as a genetic switch having a two-input OR-type sensor function or a transcription factor that forms the switch.

2. The method of claim 1.

5. In the above (A), transcription factor T １ Promoter P controlled by １ When using the ligand L １ Expression level of reporter by introduction of ligand L ２ The fusion mutant with a high reporter expression ratio due to the introduction of ligand L １ and selecting a gene switch for an output-type sensor that specifically responds to the gene or a transcription factor that forms the switch. Or, transcription factor T ２ When promoter P2 controlled by ２ Expression level of reporter by introduction of ligand L １ The fusion mutant with a high reporter expression ratio due to the introduction of ligand L ２ and selecting a gene switch for an output-type sensor that specifically responds to the gene or a transcription factor that forms the switch.

2. The method of claim 1.

6. In the above (A), transcription factor T １ Promoter P controlled by １ When using the ligand-free reporter, the expression level of the reporter / ligand L １ The ratio of reporter expression by introduction of the ligand is high, and the ratio of reporter expression without the ligand / ligand L ２ A fusion mutant having a high reporter expression ratio due to the introduction of the above is selected as a gene switch having a NOR-type sensor function or a transcription factor that forms the switch.

2. The method of claim 1.

7. In the above (A), Promoter P controlled by transcription factor T1 １ When using the ligand-free or ligand L １ or ligand L ２ Expression level of reporter by introduction of one of the ligands / ligand L １ and ligand L ２ A fusion mutant having a high reporter expression ratio due to the introduction of the above is selected as a gene switch having a NAND-type sensor function or a transcription factor that forms the switch.

2. The method of claim 1.

8. In the above (A), Ligand L １ Expression level of reporter and ligand L ２ A fusion mutant in which the expression levels of both reporters are reduced by the introduction is selected as a genetic switch that functions as a two-input, three-stage output-decreasing sensor or a transcription factor that forms the switch.

2. The method of claim 1.

9. In the above (A), Ligand L １ Expression level of reporter and ligand L ２ A fusion mutant in which the expression levels of both reporters are increased by the introduction is selected as a genetic switch that functions as a two-input, three-stage output-enhancing sensor or a transcription factor that forms the switch.

2. The method of claim 1.

10. In (B), N=3, Ligand L １ The expression level of the reporter by introduction of the ligand L ２ Expression level of reporter and ligand L ３ A fusion mutant that reduces the expression level of the reporter upon introduction is selected as a genetic switch that functions as a three-input, four-stage output-decreasing sensor or a transcription factor that forms the switch.

2. The method of claim 1.

11. In (B), N=3, Ligand L １ The expression level of the reporter by introduction of the ligand L ２ Expression level of reporter and ligand L ３ A fusion mutant in which the expression level of the reporter increases upon introduction is selected as a genetic switch that functions as a three-input, four-stage output-improving sensor or a transcription factor that forms the switch.

2. The method of claim 1.

12. In (B), N=3, transcription factor T １ Promoter P controlled by １ , promoter sequence P １ and a reporter R operably linked to １ , transcription factor T ２ Promoter P controlled by ２ , promoter sequence P ２ and a reporter R operably linked to ２ , and transcription factor T ３ Promoter P controlled by ３ , promoter sequence P ３ and a reporter R operably linked to ３ Use Ligand L whose ligand binding ability has been eliminated by mutation introduction １ Transcription factor T binding １ and Binder B 2 or transcription factor T ２ and Binder B 3 or transcription factor T ３ using a nucleic acid library of fusion variants of Ligand L １ Expression level of reporter and ligand L ２ The expression level of the reporter was not improved by the introduction of the ligand L ３ The expression level of the reporter was improved by introducing the reporter R １ , Reporter R ２ and Reporter R ３ The fusion mutant expressed by the ligand L ３ a gene switch for a multi-output sensor that specifically responds to the gene or a transcription factor that forms the switch; 2. The method of claim 1.

13. In (B), N=3, transcription factor T １ Promoter P controlled by １ , promoter sequence P １ and a reporter R operably linked to １ , transcription factor T ２ Promoter P controlled by ２ , promoter sequence P ２ and a reporter R operably linked to ２ , and transcription factor T ３ Promoter P controlled by ３ , promoter sequence P ３ and a reporter R operably linked to ３ Use Binder B, whose ligand-binding ability has been abolished by mutation 1 or transcription factor T １ and binder B, which has lost its ligand-binding ability due to mutation 2 or transcription factor T ２ and Binder B 3 or transcription factor T ３ using a nucleic acid library of fusion variants of Ligand L ２ Expression level of reporter and ligand L ３ The expression level of the reporter was improved by introducing the ligand L １ The reporter expression level was not improved by the introduction of reporter R. ２ The fusion mutant expressed by the ligand L ２ and ligand L ３ a gene switch for a multi-output sensor that specifically responds to the gene or a transcription factor that forms the switch; 2. The method of claim 1.

14. The method according to any one of claims 1 to 13, wherein binder B1, binder B2, binder B3 and binder BN are selected from the group consisting of transcription factors, enzymes, antibodies, histones, chaperones and ribosomes.

15. The method according to any one of claims 1 to 13, wherein Binder B1, Binder B2, Binder B3 and Binder BN are transcription factors or enzymes.

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