Multi-input/multi-output gene switch, and method for producing same
11702763 · 2023-07-18
Assignee
Inventors
Cpc classification
C12Q1/6897
CHEMISTRY; METALLURGY
C12N15/1086
CHEMISTRY; METALLURGY
C07K19/00
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
C12N15/635
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
C12N15/1072
CHEMISTRY; METALLURGY
International classification
C40B30/04
CHEMISTRY; METALLURGY
C12N15/10
CHEMISTRY; METALLURGY
Abstract
[Problem] Provided are a production method for a multi-input/multi-output-type genetic switch or a transcription factor, and a multi-input/multi-output-type genetic switch or a transcription factor. [Solving Means] The inventors of the present invention have completed a production method for a multi-input/multi-output-type genetic switch or a transcription factor, essentially including the steps of “fusing two or more transcription factor genes to each other” and “introducing mutations into the fusion-type transcription factor gene,” and have further succeeded in obtaining a multi-input/multi-output-type genetic switch or a transcription factor by the method.
Claims
1. A production method for a multi-input/multi-output-type genetic switch or a transcription factor for forming the switch, the method comprising one of (A) or (B), the method (A) including the following steps of (1) to (3): (A) (1) a step of introducing, into cells, or adding, to a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a binder B.sub.1 or a transcription factor T.sub.1, which responds to a ligand L.sub.1, and a binder B.sub.2 or a transcription factor T.sub.2, which responds to a ligand L.sub.2, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the binder B.sub.1 or the transcription factor T.sub.1 and a gene sequence encoding the binder B.sub.2 or the transcription factor T.sub.2, and a reporter expression vector carrying a gene sequence encoding a promoter P.sub.1 to be controlled by the transcription factor T.sub.1 and/or a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2, and a gene sequence encoding a reporter Rx functionally linked to the promoter sequence P.sub.1 and/or the promoter sequence P.sub.2, where X represents an integer of 1 or more; (2) a step of adding the ligand L.sub.1 and/or the ligand L.sub.2 to the cells or the cell-free protein synthesis system of (1); and (3) a step of selecting one of the fusion mutants of the binder B.sub.1 or the transcription factor T.sub.1 and the binder B.sub.2 or the transcription factor T.sub.2 as a genetic switch or a transcription factor for forming the switch through use of an expression amount of the reporter as an indicator, or the method (B) including the following steps of (1) to (3): (B) (1) a step of introducing, into cells, or adding, to a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a binder B.sub.1 or a transcription factor T.sub.1, which responds to a ligand L.sub.1, a binder B.sub.2 or a transcription factor T.sub.2, which responds to a ligand L.sub.2, and a binder B.sub.N or a transcription factor T.sub.N, which responds to a ligand L.sub.N, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the binder B.sub.1 or the transcription factor T.sub.1, a gene sequence encoding the binder B.sub.2 or the transcription factor T.sub.2, and a gene sequence encoding the binder B.sub.N or the transcription factor T.sub.N, and a reporter expression vector carrying a gene sequence encoding a promoter P.sub.1 to be controlled by the transcription factor T.sub.1, a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2, and/or a gene sequence encoding a promoter P.sub.N to be controlled by the transcription factor T.sub.N, and a gene sequence encoding a reporter Rx functionally linked to the promoter sequence P.sub.1, the promoter sequence P.sub.2, and/or the promoter sequence P.sub.N, where N represents an integer of 3 or more; (2) a step of introducing the ligand L.sub.1, the ligand L.sub.2, and/or the ligand L.sub.N into the cells or the cell-free protein synthesis system of (1); and (3) a step of selecting one of the fusion mutants of the binder B.sub.1 or the transcription factor T.sub.1, the binder B.sub.2 or the transcription factor T.sub.2, and the binder B.sub.N or the transcription factor T.sub.N as a genetic switch or a transcription factor for forming the switch through use of an expression amount of the reporter as an indicator.
2. The method according to claim 1, wherein in the method (A), the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 and the ligand L.sub.2 to an expression amount of the reporter obtained by introduction of the ligand L.sub.1 or the ligand L.sub.2 as a genetic switch having a 2-input/AND-type output sensor function or a transcription factor for forming the switch.
3. The method according to claim 1, wherein in the method (A), when the promoter P.sub.2 to be controlled by the transcription factor T.sub.2 is used, the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 and the ligand L.sub.2 to an expression amount of the reporter obtained by introduction of the ligand L.sub.2 as a genetic switch having a 2-input/AND-type output sensor function or a transcription factor for forming the switch, or when the promoter P.sub.1 to be controlled by the transcription factor T.sub.1 is used, the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 and the ligand L.sub.2 to an expression amount of the reporter obtained by introduction of the ligand L.sub.1 as a genetic switch having a 2-input/AND-type output sensor function or a transcription factor for forming the switch.
4. The method according to claim 1, wherein in the method (A), when the promoter P.sub.1 to be controlled by the transcription factor T.sub.1 is used, the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.2 to an expression amount of the reporter obtained without any ligand as a genetic switch having a 2-input/OR-type sensor function or a transcription factor for forming the switch, or when the promoter P.sub.2 to be controlled by the transcription factor T.sub.2 is used, the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 to an expression amount of the reporter obtained without any ligand as a genetic switch having a 2-input/OR-type sensor function or a transcription factor for forming the switch.
5. The method according to claim 1, wherein in the method (A), when the promoter P.sub.1 to be controlled by the transcription factor T.sub.1 is used, the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 to an expression amount of the reporter obtained by introduction of the ligand L.sub.2 as a genetic switch for an output-type sensor that specifically responds to the ligand L.sub.1 or a transcription factor for forming the switch, or when the promoter P.sub.2 to be controlled by the transcription factor T.sub.2 is used, the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.2 to an expression amount of the reporter obtained by introduction of the ligand L.sub.1 as a genetic switch for an output-type sensor that specifically responds to the ligand L.sub.2 or a transcription factor for forming the switch.
6. The method according to claim 1, wherein in the method (A), when the promoter P.sub.1 to be controlled by the transcription factor T.sub.1 is used, the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained without any ligand to an expression amount of the reporter obtained by introduction of the ligand L.sub.1, and having a high ratio of the expression amount of the reporter obtained without any ligand to an expression amount of the reporter obtained by introduction of the ligand L.sub.2 as a genetic switch having a NOR-type sensor function or a transcription factor for forming the switch.
7. The method according to claim 1, wherein in the method (A), when the promoter P.sub.1 to be controlled by the transcription factor T.sub.1 is used, the step of selecting comprises selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained without any ligand or obtained by introduction of one of the ligand L.sub.1 or the ligand L.sub.2 to an expression amount of the reporter obtained by introduction of the ligand L.sub.1 and the ligand L.sub.2 as a genetic switch having a NAND-type sensor function or a transcription factor for forming the switch.
8. The method according to claim 1, wherein in the method (A), the step of selecting comprises selecting a fusion mutant in which both of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 and an expression amount of the reporter obtained by introduction of the ligand L.sub.2 are decreased as a genetic switch having a function as a 2-input/3-stage output reduction-type sensor or a transcription factor for forming the switch.
9. The method according to claim 1, wherein in the method (A), the step of selecting comprises selecting a fusion mutant in which both of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 and an expression amount of the reporter obtained by introduction of the ligand L.sub.2 are increased as a genetic switch having a function as a 2-input/3-stage output increase-type sensor or a transcription factor for forming the switch.
10. The method according to claim 1, wherein in the method (B), N=3, and the step of selecting comprises selecting a fusion mutant in which an expression amount of the reporter obtained by introduction of the ligand L.sub.1, an expression amount of the reporter obtained by introduction of the ligand L.sub.2, and an expression amount of the reporter obtained by introduction of a ligand L.sub.3 are decreased as a genetic switch having a function as a 3-input/4-stage output reduction-type sensor or a transcription factor for forming the switch.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DESCRIPTION OF EMBODIMENTS
(22) The present invention relates to “a production method for a multi-input/multi-output-type genetic switch or a transcription factor for forming the switch”, “a multi-input/multi-output-type genetic switch or a transcription factor for forming the switch”, and to “a gene fusion mutant”.
(23) (Multi-Input/Multi-Output-Type)
(24) The term “multi-input/multi-output (gate)-type” in the present invention means being capable of showing a plurality of kinds of responses to a plurality of ligands.
(25) In the present invention, the following terms are used for describing the contents of the invention: “ligand L.sub.1”, “ligand L.sub.2”, “ligand L.sub.N”, “transcription factor T.sub.1”, “transcription factor T.sub.2”, “transcription factor T.sub.N”, “promoter P.sub.1”, “promoter P.sub.2.sup.”, “promoter P.sub.N”, “reporter R.”, “binder B.sub.1”, “binder B.sub.2”, “binder B.sub.N”, and the like. Symbol “x” represents an integer of 1 or more, and “N” represents an integer of 3 or more.
(26) In addition, in ligands, when “N” represents 3, three ligands, i.e., a ligand 1, a ligand L.sub.2, and a ligand L.sub.3 are meant; when “N” represents 4, four ligands, i.e., a ligand L.sub.1, a ligand L.sub.2, a ligand L.sub.3, and a ligand L.sub.4 are meant; and when “N” represents 5, five ligands, i.e., a ligand 1, a ligand L.sub.2, a ligand L.sub.3, a ligand L.sub.4, and a ligand L.sub.5 are meant. The same applies to the transcription factor T.sub.N, the promoter P.sub.N, and the binder B.sub.N. Those terms are merely illustrative, and do not limit the contents of the present invention.
(27) (Mode Examples of Multi-Input/Multi-Output-Type Genetic Switch or Transcription Factor)
(28) A multi-output (gate) type that responds to two ligands, i.e., a ligand L.sub.1 and a ligand L.sub.2 may be exemplified by the following (see
(29) (1) An OR gate
(30) (2) An AND gate
(31) (3) A NOR gate
(32) (4) A NAND gate
(33) (5) A gate that responds to the ligand L.sub.1
(34) (6) A gate that responds to the ligand L.sub.2
(35) (7) A 2-input-type 3-stage output reduction-type gate (its output decreases as the number (amount) of ligands increases)
(36) (8) A 2-input-type 3-stage output increase-type gate (its output increases as the number (amount) of ligands increases)
(37) A multi-output (gate) type that responds to three ligands, i.e., a ligand L.sub.1, a ligand L.sub.2, and a ligand L.sub.3 may be exemplified by the following (see
(38) (1) A 3-input-type 4-stage output reduction-type gate (its output decreases as the number (amount) of ligands increases)
(39) (2) A 3-input-type 4-stage output increase-type gate (its output increases as the number (amount) of ligands increases)
(40) (3) A gate that responds to the ligand L.sub.3
(41) (4) A gate that responds to the ligands L.sub.2 and L.sub.3
(42) Output amounts are X1<X2<X3<X4, and X1 represents an output value of substantially 0 or more.
(43) A multi-output (gate) type that responds to N ligands may be exemplified by the following.
(44) (1) An N-input-type N+1-stage output reduction-type gate (its output decreases as the number (amount) of ligands increases)
(45) (2) An N-input-type N+1-stage output increase-type gate (its output increases as the number (amount) of ligands increases)
(46) (3) An N-input-type 2-stage output increase-type gate (its output differs between a case in which the number of ligands is N and any other case.)
(47) (4) An N-input-type 2-stage output reduction-type gate (its output differs between a case in which the number of ligands is N and any other case.)
(48) (Gene Fusion Mutant that Functions as Multi-Input/Multi-Output-Type Genetic Switch)
(49) A gene fusion mutant that functions as a multi-input/multi-output-type genetic switch is capable of enhancing or repressing the expression of a promoter P.sub.1, a promoter P.sub.2, and/or a promoter P.sub.N to be controlled by T.sub.1, T.sub.2, and/or T.sub.N through use of a ligand L.sub.1, a ligand L.sub.2, and/or a ligand L.sub.N.
(50) More specifically, the gene fusion mutant may be exemplified by the following.
(51) (1) A gene fusion mutant capable of enhancing expression of the promoter P.sub.2 to be controlled by the transcription factor T.sub.2 through use of the ligand L.sub.1
(52) (2) A gene fusion mutant capable of decreasing expression of the promoter P.sub.2 to be controlled by the transcription factor T.sub.2 through use of the ligand L.sub.1
(53) (3) A gene fusion mutant capable of enhancing or repressing expression of the promoters P.sub.1, P.sub.2, and P.sub.N to be controlled by the transcription factor T.sub.1, the transcription factor T.sub.2, and the transcription factor T.sub.N through use of the ligand L.sub.1
(54) (4) A gene fusion mutant capable of enhancing or repressing expression of the promoters P.sub.2 and P.sub.N to be controlled by the transcription factor T.sub.2 and the transcription factor T.sub.N through use of the ligand L.sub.1
(55) (5) A gene fusion mutant capable of enhancing or repressing expression of the promoters P.sub.1, P.sub.2, and P.sub.N to be controlled by the transcription factor T.sub.1, the transcription factor T.sub.2, and the transcription factor T.sub.N through use of the ligands L.sub.1 and L.sub.2
(56) (6) A gene fusion mutant capable of enhancing or repressing expression of the promoters P.sub.2 and P.sub.N to be controlled by the transcription factor T.sub.2 and the transcription factor T.sub.N through use of the ligands L.sub.1 and L.sub.2
(57) (7) A gene fusion mutant capable of enhancing expression of the promoters P.sub.1, P.sub.2, and P.sub.N to be controlled by the transcription factors T.sub.1, T.sub.2, and T.sub.N through use of the ligands L.sub.1, L.sub.2, and L.sub.N
(58) (8) A gene fusion mutant capable of repressing expression of the promoters P.sub.1, P.sub.2, and P.sub.N to be controlled by the transcription factors T.sub.1, T.sub.2, and T.sub.N through use of the ligands L.sub.1, L.sub.2, and L.sub.N
(59) (9) A gene fusion mutant capable of repressing the expression of the promoter P.sub.1 to be controlled by the transcription factor T.sub.1, the transcription factor T.sub.2, and a transcription factor T.sub.3, and enhancing the expression of the promoter P.sub.2 and the promoter P.sub.3 through use of the ligand L.sub.1
(60) More specifically, on the basis of Examples to be described below, the following gene fusion mutants may be given as examples.
(61) (1) A transcription factor T.sub.1-T.sub.2 (AraC-LuxR) gene fusion mutant that gives a response of one of AND (AHL/Arabinose), OR (AHL/Arabinose), Ignore-AHL (Arabinose-only), and Ignore-arabinose (AHL-only) types acts on a promoter P.sub.1 and a promoter P.sub.2, and can simultaneously control genes downstream of the promoters.
(2) A transcription factor T.sub.1-T.sub.2-T.sub.3 (TraR-AraC-LuxR) gene fusion mutant responds to a ligand L.sub.1 (aTc), a ligand L.sub.2 (arabinose), and a ligand L.sub.3 (AHL), and can simultaneously control genes (including an operon) downstream of a promoter P.sub.1 (TetP), a promoter P.sub.2 (pBAD), and a promoter P.sub.3 (pLux).
(62) (Production Method for Multi-Input/Multi-Output-Type Genetic Switch)
(63) The “production method for a multi-input/multi-output-type genetic switch or a transcription factor for forming the switch” of the present invention (hereinafter sometimes abbreviated as “production method of the present invention (method of the present invention)”) essentially includes the steps of “fusing two or more transcription factor genes to each other or one or more transcription factors and one or more binders to each other” and “introducing mutations into the fusion-type transcription factor gene,” and other steps are not particularly limited. For example, the production method of the present invention includes the following steps or steps that are substantially the same as the following steps.
(64) In addition, reference may be made to a method described in the literature “PLoS ONE 10 (3):e0120243.” previously published by the inventors of the present invention.
(65) (Steps of Production Method of the Present Invention)
(66) A production method for a multi-input/multi-output-type genetic switch or a transcription factor for forming the switch, the method including one of (A) or (B), the method (A) including the following steps of (1) to (3):
(67) (A)
(68) (1) a step of introducing, into cells, or adding, to a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a binder B.sub.1 or a transcription factor T.sub.1, which responds to a ligand L.sub.1, and a binder B.sub.2 or a transcription factor T.sub.2, which responds to a ligand L.sub.2, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the binder B.sub.1 or the transcription factor T.sub.1 and a gene sequence encoding the binder B.sub.2 or the transcription factor T.sub.2, and a reporter expression vector carrying a gene sequence encoding a promoter P.sub.1 to be controlled by the transcription factor T.sub.1 and/or a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2, and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter sequence P.sub.1 and/or the promoter sequence P.sub.2, where “x” represents an integer of 1 or more;
(69) (2) a step of adding the ligand L.sub.1 and/or the ligand L.sub.2 to the cells or the cell-free protein synthesis system of (1); and
(70) (3) a step of selecting one of the fusion mutants of the binder B.sub.1 or the transcription factor T.sub.1 and the binder B.sub.1 or the transcription factor T.sub.2 as a genetic switch or a transcription factor for forming the switch through use of an expression amount of the reporter as an indicator, or
(71) (B)
(72) (1) a step of introducing, into cells, or adding, to a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a binder B.sub.1 or a transcription factor T.sub.1, which responds to a ligand L.sub.1, a binder B.sub.2 or a transcription factor T.sub.2, which responds to a ligand L.sub.2, and a binder B.sub.N or a transcription factor T.sub.N, which responds to a ligand L.sub.N, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the binder B.sub.1 or the transcription factor T.sub.1, a gene sequence encoding the binder B.sub.2 or the transcription factor T.sub.2, and a gene sequence encoding the binder B.sub.N or the transcription factor T.sub.N, and a reporter expression vector carrying a gene sequence encoding a promoter P.sub.1 to be controlled by the transcription factor T.sub.1, a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2, and/or a gene sequence encoding a promoter P.sub.N to be controlled by the transcription factor T.sub.N, and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter sequence P.sub.1, the promoter sequence P.sub.2, and/or the promoter sequence P.sub.N, where “N” represents an integer of 3 or more;
(73) (2) a step of introducing the ligand L.sub.1, the ligand L.sub.2, and/or the ligand L.sub.N into the cells or the cell-free protein synthesis system of (1); and
(74) (3) a step of selecting one of the fusion mutants of the binder B.sub.1 or the transcription factor T.sub.1, the binder B.sub.2 or the transcription factor T.sub.2, and the binder B.sub.N or the transcription factor T.sub.N as a genetic switch or a transcription factor for forming the switch through use of an expression amount of the reporter as an indicator.
(75) More specifically, the steps are as described below.
(76) (A)
(77) (1) A step of introducing, into cells, or adding, to a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a transcription factor T.sub.1, which responds to a ligand L.sub.1, and a transcription factor T.sub.2, which responds to a ligand L.sub.2, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the transcription factor T.sub.1 and a gene sequence encoding the transcription factor T.sub.2, and a reporter expression vector carrying a gene sequence encoding a promoter P.sub.1 to be controlled by the transcription factor T.sub.1 and/or a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2, and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter sequence P.sub.1 and/or the promoter sequence P.sub.2, where “x” represents an integer of 1 or more
(78) (2) A step of adding the ligand L.sub.1 and/or the ligand L.sub.2 to the cells or the cell-free protein synthesis system of (1)
(79) (3) A step of selecting one of the fusion mutants of the transcription factor T.sub.1 and the transcription factor T.sub.2 as a genetic switch or a transcription factor for forming the switch through use of an expression amount of the reporter as an indicator
(80) (B)
(81) (1) A step of introducing, into cells, or adding, to a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a transcription factor T.sub.1, which responds to a ligand L.sub.1, a transcription factor T.sub.2, which responds to a ligand L.sub.2, and a transcription factor T.sub.N, which responds to a ligand L.sub.N, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the transcription factor T.sub.1, a gene sequence encoding the transcription factor T.sub.2, and a gene sequence encoding the transcription factor T.sub.N, and a reporter expression vector carrying a gene sequence encoding a promoter P.sub.1 to be controlled by the transcription factor T.sub.1, a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2, and/or a gene sequence encoding a promoter P.sub.N to be controlled by the transcription factor T.sub.N, and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter sequence P.sub.1, the promoter sequence P.sub.2, and/or the promoter sequence P.sub.N, where “N” represents an integer of 3 or more
(82) (2) A step of introducing the ligand L.sub.1, the ligand L.sub.2, and/or the ligand L.sub.N into the cells or the cell-free protein synthesis system of (1)
(83) (3) A step of selecting one of the fusion mutants of the transcription factor T.sub.1, the transcription factor T.sub.2, and the transcription factor T.sub.N as a genetic switch or a transcription factor for forming the switch through use of an expression amount of the reporter as an indicator
(84) (Genetic Switch)
(85) The “genetic switch” in the present invention means a “gene obtained by fusing two or more transcription factors to each other or one or more transcription factors and one or more binders to each other (fusion-type transcription factor gene)” having a switch function when expressed as a protein, and in particular, means a “fusion-type transcription factor gene having a mutation introduced therein” or a “gene fusion mutant” having a switch function when expressed as a protein.
(86) A spacer may be introduced between the transcription factors or between the transcription factor and the binder as required. A recognition sequence of a restriction enzyme may be preferably inserted. An example thereof in the case of using a restriction enzyme XbaI may be an amino acid sequence SR (base sequence TCTAGA) derived from the recognition sequence of the enzyme, and an example thereof in the case of using a restriction enzyme SpeI may be an amino acid sequence TS (base sequence ACTAGT) derived from the recognition sequence of the enzyme.
(87) The genetic switch of the present invention has any of various gate functions as described above.
(88) (Ligand)
(89) The “ligand L” in the present invention means a substance, such as a compound, which changes the function of a genetic switch by binding to a transcription factor, and as a result, induces direct or indirect regulation of the expression of a gene or a plurality of genes. The “ligand” may also be said to be a “compound that activates a genetic switch.” The activating substance varies for each genetic switch.
(90) (Binder)
(91) The “binder B” in the present invention is selected from a transcription factor, an enzyme, an antibody, a histone, a chaperone, or a ribosome, but is preferably a transcription factor or an enzyme.
(92) (Promoter)
(93) The “promoter P” in the present invention means a nucleic acid sequence having promoter activity to be controlled by a transcription factor. The promoter means a nucleic acid sequence that is located 5′-upstream of translation initiation of a gene encoding a reporter R gene or an active portion thereof, and that controls the transcription of the reporter R.
(94) The promoter is appropriately selected and used depending on the kind of host cells to be used. When bacteria are used as the hosts, any promoter may be used without any particular limitation as long as the promoter enables expression in the host cells, such as E. coli. Examples thereof may include promoters derived from E. coli and a phage, such as a λPR promoter, a PL promoter, a trp promoter, and a lac promoter. An artificially designed and modified promoter, such as a tac promoter, may be used. When yeast is used as the host, any promoter may be used without any particular limitation as long as the promoter enables expression in the yeast. Examples thereof may include a gal1 promoter, a gal10 promoter, a heat shock protein promoter, an MFα1 promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and an AOX1 promoter. When animal cells are used as the hosts, it is preferred that a recombinant vector be autonomously replicable in the cells, and at the same time, be constituted of the promoter, an RNA splice site, a gene of interest, a polyadenylation site, and a transcription termination sequence. In addition, an origin of replication may be contained as desired. As the promoter, there may be used an SRα promoter, an SV40 promoter, an LTR promoter, a CMV promoter, and the like. In addition, an early gene promoter of cytomegalovirus or the like may be used.
(95) (Combination of Ligand L, Transcription Factor T, and Promoter P)
(96) Combinations of a ligand L, a transcription factor T that responds to the ligand L, and a promoter P to be controlled by the transcription factor T may be exemplified by the following.
(97) Activator-Type Promoter
(98) Arabinose, AraC, and P.sub.BAD (Arabinose Operon)
(99) Transcription does not occur unless an activator protein is bound to operator DNA. When arabinose is present in the environment, the conformation of the arabinose activator changes. The arabinose activator whose conformation has changed binds to the operator. Consequently, an RNA polymerase can transcribe the operon, with the result that a reporter R gene downstream of P.sub.BAD is expressed.
(100) AHL, LuxR, and P.sub.lux
(101) When AHL is present in the environment, AHL binds to the transcription factor (transcription regulatory factor) LuxR. Then, the AHL-LUxR complex activates the pluX promoter, with the result that a reporter gene R, which is a downstream gene, is expressed.
(102) Xylose, XylR, and P.sub.xyl
(103) Repressor-Type Promoter
(104) aTc, TetR, and Ptet
(105) Arsenic, ArsR, and P.sub.ars
(106) IPTG, LacI, and P.sub.lac
(107) (Library of Nucleic Acids of Fusion Mutants)
(108) The “library of nucleic acids of fusion mutants” in the present invention is fusion mutants of a transcription factor T.sub.1 or a binder B.sub.1, which responds to a ligand L.sub.1, and a transcription factor T.sub.2 or a binder B.sub.2, which responds to a ligand L.sub.2 (fusion mutants of a transcription factor T.sub.1 or a binder B.sub.1, which responds to a ligand L.sub.1, the transcription factor T.sub.2 or the binder B.sub.2, which responds to a ligand L.sub.2, and a transcription factor T.sub.N or a binder B.sub.N, which responds to a ligand L.sub.N), having a plurality of kinds of mutations obtained by introducing mutations known per se (e.g., random mutations, or site-directed mutations using stability prediction software, such as Fold-X) into a genetic construct (including an expression vector) carrying a gene sequence encoding the transcription factor T.sub.1 or the binder B.sub.1 and a gene sequence encoding the transcription factor T.sub.2 or the binder B.sub.2 or a genetic construct (including an expression vector) carrying a gene sequence encoding the transcription factor T.sub.1 or the binder B.sub.1, a gene sequence encoding the transcription factor T.sub.2 or the binder B.sub.2, and a gene sequence encoding the transcription factor T.sub.N or the binder B.sub.N.
(109) A fusion method for the transcription factor T.sub.1 and the transcription factor T.sub.2 is not particularly limited, and may be in-frame fusion in a tandem form or a fusion mode involving inserting one of the genes into a loop portion of the other transcription factor.
(110) In addition, as an example of the mutations of the present invention, it is preferred to cause destabilization of a protein (fusion mutant). First, the stability of a protein refers to the stability of its folding state, i.e., a free energy change (ΔGfold) occurring when a polypeptide chain forming the protein forms a functional structure (is folded). In addition, the “destabilization” means that the free energy change (ΔGfold) associated with folding energy is reduced and ultimately canceled. For example, when a given amino acid substitution reduces the stability of the functional structure (fold), the amino acid substitution is a “mutation that causes destabilization (destabilizing mutation).” Specifically, if moderate destabilization can be induced with a mutation, the folded state of the fusion mutant can be retained (i.e., ΔGfold<0) only when a ligand is present.
(111) (Reporter)
(112) The “reporter Rx” in the present invention is not particularly limited as long as the reporter R.sub.x serves as an indicator for selecting a fusion mutant, but examples thereof may include a fluorescent protein (GFP), a thymidine kinase (see JP 2013-17473 A), alkyladenine DNA glycosidase (see WO 2012/060407 A1), a pigment synthesis protein (see JP 2014-223038 A), and a pigment protein (amilCP).
(113) Specifically, in the case where a fluorescent protein or a pigment protein is used, a fusion mutant is selected on the basis of the coloration of a culture solution when a ligand L is added or not added into the medium. In the case where a thymidine kinase, alkyladenine DNA glycosylase, any of various drug transporters or drug resistance markers, a toxin-antitoxin pair, or the like is used, a fusion mutant is selected through use of the following indicator: the survival or death of cells or whether or not cells can proliferate.
(114) Further, the reporter R is not particularly limited as long as the reporter R is functionally linked to its corresponding promoter P, but the kind of the reporter R may be changed for each promoter P. Examples thereof may include combinations such as promoter P1-reporter R1, promoter P2-reporter R2, and promoter P3-reporter R3.
(115) (Reporter Expression Vector)
(116) The “reporter expression vector” in the present invention carries: a gene sequence encoding a promoter P.sub.1 to be controlled by a transcription factor T.sub.1 and/or a gene sequence encoding a promoter P.sub.2 to be controlled by a transcription factor T.sub.2, and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter P.sub.1 sequence and/or a gene sequence encoding the reporter R.sub.x functionally linked to the promoter P.sub.2 sequence; or a gene sequence encoding a promoter P.sub.1 to be controlled by a transcription factor T.sub.1, a gene sequence encoding a promoter P.sub.2 to be controlled by a transcription factor T.sub.2, and/or a gene sequence encoding a promoter P.sub.N to be controlled by a transcription factor T.sub.N, and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter P.sub.1 sequence, a gene sequence encoding the reporter R.sub.x functionally linked to the promoter P.sub.2 sequence, and/or a gene sequence encoding the reporter R.sub.x functionally linked to the promoter P.sub.3 sequence. A plurality of kinds of the reporters R.sub.x may be present, or one or a plurality thereof may be present downstream of each promoter.
(117) The expression vector refers to DNA that transfers an exogenous gene to host cells, in other words, vector DNA, the DNA allowing a gene of interest to be expressed in the host cells. The vector DNA is not particularly limited as long as the vector DNA is replicable in the host, and is appropriately selected depending on the kind of the host and intended use. The vector DNA may be vector DNA lacking a part of DNA except a part needed for replication as well as vector DNA obtained by extracting naturally occurring DNA. Typical examples of the vector DNA may include vector DNAs derived from a plasmid, a bacteriophage, and a virus. Examples of the plasmid DNA may include an E. coli-derived plasmid, a Bacillus subtilis-derived plasmid, and a yeast-derived plasmid. An example of the bacteriophage DNA is A phage. Examples of the virus-derived vector DNA may include vectors derived from animal viruses, such as a retrovirus, a vaccinia virus, an adenovirus, a papovavirus, SV40, a fowlpox virus, and a pseudorabies virus, or vectors derived from insect viruses, such as a baculovirus. Other examples of the vector DNA may include transposon-derived, insertion element-derived, and yeast chromosome element-derived vector DNAs. Alternatively, for example, there may be given vector DNA prepared by combining the above-mentioned materials, such as vector DNA prepared by combining genetic elements of a plasmid and a bacteriophage (e.g., a cosmid or a phagemid). It is required to incorporate a gene of interest into the vector DNA so that the gene of interest may be expressed, and at least the gene of interest and a regulatory DNA element, such as a promoter, are included as the constituent elements of the vector DNA. In addition to those elements, as desired, gene sequences carrying information on replication and control may be further incorporated in combination into the vector DNA by a method known per se. Examples of such gene sequences may include: cis-elements, such as a ribosome binding sequence, a terminator, a signal sequence, and an enhancer; a splicing signal; and selection markers (selectors: a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, a kanamycin resistance gene, and the like). One or more kinds of gene sequences selected therefrom may be incorporated into the vector DNA.
(118) A genetic engineering technology known per se may be applied as a method of incorporating the gene of interest into the vector DNA. For example, there may be employed a method involving treating the gene of interest with an appropriate restriction enzyme to cleave the gene at a specific site, then mixing the resultant with vector DNA treated in the same manner, and recombining them with a ligase. Alternatively, desired vector DNA may be obtained by ligating the gene of interest with an appropriate linker, and inserting the resultant into a multiple cloning site of a vector suited for the purpose.
(119) A method of introducing the expression vector into host cells is not particularly limited as long as the method is an introduction method by which the vector DNA can be introduced into the host cells and the gene of interest can be expressed in the host cells, and any known method appropriately selected depending on the kind of the host cells may be used. Examples thereof may include an electroporation method, a calcium phosphate method, and a lipofection method.
(120) (Cells or Cell-Free Protein Synthesis System)
(121) The “cells or cell-free protein synthesis system” in the present invention is not particularly limited as long as the environment allows the transcription factor T, the promoter P, and the reporter R.sub.x to express a protein. For example, the cells may be any of prokaryotic cells and isolated eukaryotic cells, but are preferably prokaryotic cells having short cell cycles and high proliferation rates. Cells having such properties are useful for a rapid production method for a genetic switch. An example of the cell-free protein synthesis system may be a known cell-free protein synthesis system (e.g., wheat or E. coli) containing components essential for protein synthesis.
(122) In the “step of adding the ligand L.sub.1 and/or the ligand L.sub.2 to the cells or the cell-free protein synthesis system,” the addition of the ligand L.sub.1 and/or the ligand L.sub.2 to the cells or the cell-free protein synthesis system may be before, after, or substantially simultaneous with the addition of the library of nucleic acids of fusion mutants and/or the reporter expression vector to the cells.
(123) (Selecting as Genetic Switch or Transcription Factor for Forming the Switch)
(124) In the “step of selecting as a genetic switch or a transcription factor for forming the switch” in the present invention, a fusion mutant showing the properties of a multi-input/multi-output-type genetic switch type of interest is selected through use of the expression amount of the reporter R.sub.x as an indicator.
(125) The gene sequence and/or amino acid sequence of the selected fusion is analyzed by a method known per se. Thus, information (base sequence, amino acid sequence) on the multi-input/multi-output-type genetic switch or the transcription factor can be obtained.
(126) Further, with the information, the multi-input/multi-output-type genetic switch or the transcription factor can be easily obtained by using a protein synthesis system known per se.
(127) (Genetic Circuit)
(128) The “genetic circuit” in the present invention has at least the following. A gene sequence encoding a transcription factor T.sub.1 that responds to a ligand L.sub.1 gene sequence encoding a transcription factor T.sub.2 that responds to a ligand L.sub.2 A gene sequence encoding a promoter P.sub.1 to be controlled by the transcription factor T.sub.1 and/or a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2 A reporter R.sub.x functionally linked to the promoter P.sub.1 sequence and/or a reporter R.sub.x functionally linked to the promoter P.sub.2 sequence
(129) All of the foregoing may be contained in the same genetic construct, or may be separately contained in a plurality of genetic constructs.
(130) As required, the genetic circuit may further contain cis-elements, such as a ribosome binding sequence, a terminator, a signal sequence, and an enhancer, a splicing signal, selection markers (selectors: a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, a kanamycin resistance gene, and the like), and the like.
(131) The genetic circuit may also further contain an operator (DNA region to which a repressor or an activator binds) sequence or a regulatory gene sequence.
(132) (Biosensor)
(133) The configuration of a “biosensor” in the present invention is not particularly limited as long as the biosensor can show a plurality of kinds of responses to a plurality of ligands, but an example thereof may be cells serving as a system in which transcription factors, promoters, and reporters can be expressed as proteins (serving as a system capable of expressing a genetic circuit) or a cell-free protein synthesis system containing a genetic circuit.
Examples of Production Method for Multi-Input/Multi-Output-Type Genetic Switch or Transcription Factor
(134) In the multi-input/multi-output-type genetic switch of the present invention, a multi-input/multi-output-type genetic switch or transcription factor of interest may be selected by a method described in
Example 1 of Production Method for Multi-Input/Multi-Output-Type Genetic Switch or Transcription Factor
(135) As an example of the production method of the present invention in which mutation introduction is performed 2 or more times, a production method for a genetic switch or transcription factor for an output-type sensor that specifically responds to the ligand L.sub.2 is described:
(136) (i) when the promoter P.sub.1 to be controlled by the transcription factor T.sub.1 is used, a step of obtaining a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.2 to an expression amount of the reporter obtained by introduction of the ligand L.sub.1 as a parent of a second generation;
(137) (ii) a step of introducing random mutations into the parent of the second generation to obtain a second generation library of nucleic acids or proteins of fusion mutants;
(138) (iii) a step of introducing, into cells, the second generation library and an expression vector carrying a gene sequence encoding the promoter P.sub.1 to be controlled by the transcription factor T.sub.1 and a gene sequence encoding a reporter functionally linked to the promoter sequence;
(139) (iV) a step of introducing the ligand L.sub.1 and/or the ligand L.sub.2 into the cells of (iii);
(140) (V) a step of selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.2 to an expression amount of the reporter obtained without any ligand as a genetic switch for a 2-input/l-way output-type sensor that specifically responds to the ligand L.sub.2.
(141) As required, the steps (i) to (V) may be repeated to increase the number of times of mutation introduction and the number of times of selection.
Example 2 of Production Method for Multi-Input/Multi-Output-Type Genetic Switch or Transcription Factor
(142) When the number of kinds of transcription factors included in the mutant fusion is increased, it is also appropriate to partially mutate only a certain transcription factor in advance (see Example 3). With this, the ligand binding site of the transcription factor T may be made dysfunctional.
(143) (Production Method for Genetic Switch for Detection of Ligand or Transcription Factor)
(144) In the production method of the present invention, a genetic switch for detection of the ligand L.sub.1 or a transcription factor for forming the switch may be obtained through the following steps.
(145) (1) A step of introducing, into cells, or adding, to a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a binder B.sub.1 or a transcription factor T.sub.1, which responds to a ligand L.sub.1, and a transcription factor T.sub.2, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the binder B.sub.1 or the transcription factor T.sub.1 and a gene sequence encoding the transcription factor T.sub.2, and an expression vector carrying a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2 and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter P.sub.2, where X represents an integer of 1 or more
(146) (2) A step of adding the ligand L.sub.1 and/or the ligand L.sub.2 to the cells or the cell-free protein synthesis system of (1)
(147) (3) A step of selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 and the ligand L.sub.2 to an expression amount of the reporter obtained by introduction of the ligand L.sub.2 or a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 to an expression amount of the reporter obtained by introduction of the ligand L.sub.2 as a genetic switch for detection of the ligand L.sub.1 or a transcription factor for forming the switch
(148) More specifically, on the basis of Example 6 to be described below, a genetic switch for high-sensitivity ligand detection or a transcription factor therefor can be obtained by using AHL as a ligand, LuxR as a transcription factor, and P.sub.lux as a promoter. A production example is as described below.
(149) (1) A step of introducing, into cells or a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a transcription factor T.sub.1, which responds to a ligand and a transcription factor LuxR, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the transcription factor T.sub.1 and a gene sequence encoding the transcription factor LuxR, and an expression vector carrying a gene sequence encoding a promoter P.sub.lux to be controlled by the transcription factor LuxR and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter sequence
(150) (2) A step of adding the ligand L.sub.1 and/or a ligand AHL to the cells or the cell-free protein synthesis system of (1)
(151) (3) A step of selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 and the ligand AHL to an expression amount of the reporter obtained by introduction of the ligand AHL or a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 to an expression amount of the reporter obtained by introduction of the ligand AHL as a genetic switch for detection of the ligand L.sub.1 or a transcription factor therefor
(152) An example of the ligand may be arsenic.
(153) (Method of Adjusting Detection Sensitivity of Ligand)
(154) In the biosensor of the present invention, the detection sensitivity of the ligand L.sub.1, the ligand L.sub.2, and/or the ligand L.sub.N can be easily adjusted as described below.
(155) (1) When response sensitivity to the ligand L.sub.1 is to be increased, an addition concentration of the ligand L.sub.2 and/or the ligand L.sub.N is increased.
(156) (2) When response sensitivity to the ligand L.sub.1 is to be decreased, an addition concentration of the ligand L.sub.2 and/or the ligand L.sub.N is decreased.
(157) (3) When response sensitivity to the ligand L.sub.2 is to be increased, an addition concentration of the ligand L.sub.1 and/or the ligand L.sub.N is increased.
(158) (4) When response sensitivity to the ligand L.sub.2 is to be decreased, an addition concentration of the ligand L.sub.1 and/or the ligand L.sub.N is decreased.
(159) (5) When response sensitivity to the ligand L.sub.N is to be increased, an addition concentration of the ligand L.sub.1 and/or the ligand L.sub.2 is increased.
(160) (6) When response sensitivity to the ligand L.sub.N is to be decreased, an addition concentration of the ligand L.sub.1 and/or the ligand L.sub.2 is decreased.
(161) (Production Method for Transcription Factor Capable of Increasing Expression of Promoter P.sub.2 to be Controlled by Transcription Factor T.sub.2 Through Use of Ligand L.sub.1)
(162) In the production method of the present invention, there may be obtained such a fusion mutant that a combination of the ligand L.sub.1 and the transcription factor T.sub.1 can enhance/repress (ON/OFF) the expression of the promoter P.sub.2 gene under the control sequence of the transcription factor T.sub.2 serving as a fusion partner. For example, the following method may be given.
(163) (1) A step of introducing, into cells or a cell-free protein synthesis system, a library of nucleic acids of fusion mutants of a transcription factor T.sub.1, which responds to a ligand L.sub.1, and a transcription factor T.sub.2, which responds to a ligand L.sub.2, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the transcription factor T.sub.1 and a gene sequence encoding the transcription factor T.sub.2, and a reporter expression vector carrying a gene sequence encoding a promoter P.sub.2 to be controlled by the transcription factor T.sub.2 and a gene sequence encoding a reporter R.sub.x functionally linked to the promoter sequence
(164) (2) A step of adding the ligand L.sub.1 and/or the ligand L.sub.2 to the cells or the cell-free protein synthesis system of (1)
(165) (3) A step of selecting a fusion mutant having a high ratio of an expression amount of the reporter obtained by introduction of the ligand L.sub.1 to an expression amount of the reporter obtained by introduction of the ligand L.sub.2 as a transcription factor capable of increasing expression of the promoter P.sub.2 to be controlled by the transcription factor T.sub.2 through use of the ligand L.sub.1
(166) The multi-input/multi-output-type genetic switch, the transcription factor for forming the switch, and the fusion mutant of the present invention may be used for protein synthesis, induction of protein secretion, induction of a biosynthetic pathway, regulation of a flow rate of a biosynthetic pathway, induction of cell proliferation, induction of a physiological function, or a control mechanism for a physiological function.
(167) The present invention also relates to a “fusion mutant of AraC-LuxR.sub.N86K and C245W”, a “fusion mutant of TetR-AraC-LuxR.sub.N86K and C245W”, and a “fusion mutant of ArsR-LuxR.sub.N86K and C245W”.
(168) The fusion mutant of AraC-LuxR.sub.N86K and C245W of the present invention has an amino acid substitution selected from any one of the following groups (1) to (5) in an amino acid sequence set forth in SEQ ID NO: 15 (see Table 5).
(169) (1) F74L, P86T, V249A, and N298I
(170) (2) P39R, I197N, and N252S
(171) (3) E295K
(172) (4) M175K and K491E
(173) (5) H80F, H81K, Y82L, N393I, Y439H, R523L, and F541L
(174) The fusion mutant of AraC-LuxR.sub.N86K and C245W of the above-mentioned item (1) has AND gate-type transcriptional activity (responds when both of arabinose and homoserine lactone are present).
(175) The fusion mutant of AraC-LuxR.sub.N86K and C245W of the above-mentioned item (2) has AND gate-type transcriptional activity (responds when both of arabinose and homoserine lactone are present).
(176) The fusion mutant of AraC-LuxR.sub.N86K and C245W of the above-mentioned item (3) has OR gate-type transcriptional activity (responds when arabinose or homoserine lactone is present).
(177) The fusion mutant of AraC-LuxR.sub.N86K and C245W of the above-mentioned item (4) has L1 only gate-type transcriptional activity (specifically responds to arabinose).
(178) The fusion mutant of AraC-LuxR.sub.N86K and C245W of the above-mentioned item (5) has L2 only gate-type transcriptional activity (specifically responds to AHL).
(179) The fusion mutant of AraC-LuxR.sub.N86K and C245W of the present invention contains: an amino acid sequence having 1 to 20, preferably 1 to 15, more preferably 1 to 10, most preferably 1 to 5 amino acids substituted, truncated, inserted, and/or added in the amino acid-substituted sequence described in any one of the above-mentioned items (1) to (5), and having substantially equivalent activity to the transcriptional activity of the substitution mutant of any one of the above-mentioned items (1) to (5); and an amino acid sequence having 90% or more (or 92% or more, 94% or more, 96% or more, 98% or more, 99% or more) homology to the amino acid-substituted sequence described in any one of the above-mentioned items (1) to (5), and having substantially equivalent activity to the transcriptional activity of the substitution mutant of the above-mentioned items (1) to (5).
(180) In the introduction of a mutation into a peptide, for example, a substitution between homologous amino acids (e.g., polar amino acids, non-polar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, and aromatic amino acids) is easily conceivable from the viewpoint of preventing basic properties (e.g., physical properties, function, physiological activity, or immunological activity) of the peptide from being changed.
(181) The fusion mutant of TetR-AraC-LuxR.sub.N86K and C245W of the present invention has amino acid substitutions of K46R, D95G, K108N, I134V, V145A, L204P, I214N, P216T, F217S, L409Q, T545A, and S569T in an amino acid sequence set forth in SEQ ID NO: 16.
(182) In addition, the fusion mutant of TetR-AraC-LuxR.sub.N86K and C245W of the present invention has 3-input-type 4-stage output reduction-type transcriptional activity (its response reduces as the kinds and amounts of three ligands increase).
(183) The fusion mutant of TetR-AraC-LuxR.sub.N86K and C245W of the present invention contains: an amino acid sequence having 1 to 20, preferably 1 to 15, more preferably 1 to 10, most preferably 1 to 5 amino acids substituted, truncated, inserted, and/or added in the amino acid sequence of the amino acid substitution mutant described above, and having substantially equivalent activity to the transcriptional activity of the substitution mutant; and an amino acid sequence having 90% or more (or 92% or more, 94% or more, 96% or more, 98% or more, or 99% or more) homology to the amino acid sequence of the amino acid substitution mutant described above, and having substantially equivalent activity to the transcriptional activity of the above-mentioned substitution mutant.
(184) The fusion mutant of ArsR-LuxR.sub.N86K and C245W of the present invention has one amino acid substitution or deletion selected from one of the following groups (1) and (2) in an amino acid sequence set forth in SEQ ID NO: 17.
(185) (1) E16D and T17-(“-” means truncation).
(186) (2) I84N, N102D, F240L, and P277A
(187) The fusion mutant of ArsR-LuxR.sub.N86K and C245W of the above-mentioned item (1) is an AND-type arsenic switch having high stringency.
(188) The fusion mutant of ArsR-LuxR.sub.N86K and C245W of the above-mentioned item (2) is a high-sensitivity AND-type arsenic switch.
(189) The fusion mutant of ArsR-LuxR.sub.N86K and C245W of the present invention contains: an amino acid sequence having 1 to 20, preferably 1 to 15, more preferably 1 to 10, most preferably 1 to 5 amino acids substituted, truncated, inserted, and/or added in the amino acid-substituted/deleted sequence described in the above-mentioned item (1) or (2), and having substantially equivalent activity to the transcriptional activity of the substitution/deletion mutant of the above-mentioned item (1) or (2); and an amino acid sequence having 90% or more (or 92% or more, 94% or more, 96% or more, 98% or more, or 99% or more) homology to the amino acid-substituted/deleted sequence described in the above-mentioned item (1) or (2), and having substantially equivalent activity to the transcriptional activity of the substitution/deletion mutant of the above-mentioned item (1) or (2).
(190) The present invention is described below by way of Examples, but the present invention is by no means limited to Examples.
Example 1
(191) (Reagents)
(192) Reagents and the like used in Example 2 and Example 3 described below are as described below.
(193) (Pre-PCR)
(194) On the basis of the following composition table, a plasmid was used as a template and subjected to PCR once to make DNA linear.
(195) TABLE-US-00001 TABLE 1 Volume [μL] Final Concentration Template DNA 1 — 5 μM Fwd primer 3 0.3 μM 5 μM Rev primer 3 0.3 μM 10× KOD plus buffer 2 5 1× (product of Toyobo Life Science) 2 mM each dNTPs 5 0.2 mM each 25 mM MgSO.sub.4 3 1.5 mM 1 U/μL KOD plus 1 0.02 U/μL (product of Toyobo Life Science) NFW (Nuclease-free Water) 29 — Total amount 50
(196) {Error-Prone PCR (EP-PCR)}
(197) On the basis of the following composition table, EP-PCR was performed using linear DNA as a template. An amplification factor was set to 100 times, 1,000 times, or 10,000 times, and a MnCl.sub.2 concentration was set to 10 μM or 50 μM. For MnCl.sub.2, a stock solution concentrated 10-fold was diluted before use.
(198) TABLE-US-00002 TABLE 2 Volume [μL] Final Concentration Template DNA 1 — 5 μM Fwd primex 5 0.5 μM 5 μM Rev primer 5 0.5 μM 10× ThermoPol buffer 5 1× 2 mM each dNTPs 5 0.2 mM each 10 μM, 50 μM MnCl.sub.2 5 10 μM, 50 μM/10 μM 5 U/μl Taq 1 0.1 U/μL NFW 23 — Total amount 50
(199) (Digestion Reaction)
(200) On the basis of the following composition table, both of AL (AraC-LuxR.sub.N86K and C245W, see
(201) TABLE-US-00003 TABLE 3 Volume [μL] Final Concentration Template DNA x 20 ng/μL 10× CutSmart 5 1× (product manufactured by New England Biolabs) 20 U/μL NcoI-HF 1 0.4 U/μL 20 U/μL BamHI-HF 1 0.4 U/μL 1 U/μL rSAP (or NFW) 1 0.02 U/μL NFW 42 − x — Total amount 50
(202) (Ligation Reaction)
(203) On the basis of the following composition table, for AL, a reaction was performed using 100 ng of the vector and about 100 ng of the insert, and for TAL, a reaction was performed using 100 ng of the vector and about 150 ng of the insert. Reaction conditions were set to 16° C. overnight.
(204) TABLE-US-00004 TABLE 4 Volume [μL] Final Concentration Insert DNA x Vector DNA y 10× T4 DNA ligase buffer 1 1× (product manufactured by New England Biolabs) 400 U/μL T4 DNA ligase 1 40 U/μL NFW 8 − x − y Total amount 10
Example 2
(205) (Development of Multi-Input/Multi-Output Sensor)
(206) In this Example, as an example of the production of a multi-input/multi-output sensor, an arabinose-responsive transcription factor AraC (obtained by PCR from E. coli MG1655) and a mutant of an AHL (homoserine lactone)-responsive transcription factor LuxR {N86K, C245W: Kimura et al., J. Gen. Appl. Microbiol., 62, 240-247 (2016)} were used.
(207) (Confirmation of Characteristics of AraC-LuxR.sub.N86K and C245W)
(208) As illustrated in
(209) AraC-LuxR.sub.N86K and C245W has a structure in which two transcription factor proteins are fused in tandem. Therefore, it was confirmed that the function of each transcription factor was unchanged. More specifically, the fusion protein enhanced the gene downstream of the arabinose promoter by responding only to arabinose, but AHL did not interfere therewith at all. Similarly, the expression of the gene downstream of the Lux promoter was enhanced by the fusion protein in an AHL-dependent manner, but was not enhanced with arabinose.
(210) (Generation of Mutants of AraC-LuxR.sub.N86K and C245W)
(211) As illustrated in
Example 2-1: Development Example 1 of 2-Input/AND-Type Sensor
(212) The full length of AraC-LuxR.sub.N86K and C245W (plasmid:
(213) Through only one round of EP-PCR/screening, a mutant of AraC-LuxR.sub.N86K and C245W (see Table 5: F74L, P86T, V249A, and N298I) was able to be obtained.
(214) More specifically, it was confirmed that GFP expression under P.sub.lux control acted as an AND gate that responded only when both of arabinose and AHL were present. Further, it was confirmed that a similar action was obtained for not only the Lux promoter, but also the arabinose promoter {araP (P.sub.BAD)}. That is, the obtained mutant of AraC-LuxR.sub.N86K and C245W (see Table 5: F74L, P86T, V249A, and N298I) was able to be caused to independently AND-respond to each of two different promoters. Accordingly, it was confirmed that this mutant was capable of functioning as a “2-input/AND-type” sensor capable of making an “arabinose/AHL” AND-type output to each of the arabinose promoter and the Lux promoter independently.
(215) A method for the evaluation of the above-mentioned ligand responsiveness is specifically as described below.
(216) 200 μL of a measurement sample obtained by diluting the culture solution 10-fold with physiological saline was injected into a microwell plate (product manufactured by Nunc), and was measured for cell density when irradiated at 595 nm and fluorescence at 535 nm when excited at 485 nm, through use of FilterMaxF5 (commercially available absorbance/fluorescence detection apparatus, product manufactured by Molecular Devices). In addition, as a blank sample, 200 μL of a blank sample obtained by diluting cell-free liquid medium 10-fold with physiological saline was also subjected to measurement simultaneously. Data analysis was performed with a value obtained by subtracting the value of the blank sample from the measured value of the sample and performing correction for the dilution factor. The same applies to Examples 2-2, 2-3, 2-4, and 2-5 described below.
Example 2-2: Development Example 2 of 2-Input/AND-Type Output Sensor
(217) The full length of AraC-LuxR.sub.N86K and C245W was subjected to EP-PCR (50 μM MnCl.sub.2, amplification factor: 10,000 times) to generate a mutant pool having random mutations introduced therein (the library size was 3×10.sup.6). Unlike Example 2-1 described above, through use of P.sub.BAD7-GFP as a reporter, 90 colonies were randomly picked (see
(218) Through only one round of EP-PCR/screening, a mutant of AraC-LuxR.sub.N86K and C245W (see Table 5: P39R, I197N, and N252S) was able to be obtained.
Example 2-3: Development Example of 2-Input/OR-Type Output Sensor
(219) The library illustrated in
(220) The full length of AraC-LuxR.sub.N86K and C245W was subjected to EP-PCR (50 μM MnCl.sub.2, amplification factor: 10,000 times) to generate a mutant pool having random mutations introduced therein (the library size was 3×10.sup.6). Through use of P.sub.BAD7-GFP as a reporter, 90 colonies showing fluorescence on solid medium containing 10 μM AHL were picked. The ligand responsiveness of those colonies was evaluated. Specifically, culture was performed under separate conditions of “no ligand” and “10 μM AHL”, and 2 mutants each having an improved AHL/none ratio of 3 times or more were obtained. Fluorescence intensity measurement results of the mutant showing the highest AHL/none ratio among those mutants are shown in
(221) Through only one round of EP-PCR/screening, a mutant of AraC-LuxR.sub.N86K and C245W (see Table 5: E295K) was obtained.
(222) That is, a 2-input/OR-type sensor was able to be developed.
Example 2-4: Development Example 1 of 2-Input/l-Way Output-Type Sensor
(223) In this Example, as an example of a 2-input/l-way output-type sensor, a genetic switch using P.sub.BAD as an “arabinose-only” gate that did not respond to AHL and was activated only with arabinose (showed a response thereto in a concentration-dependent manner) was developed. The details are as described below.
(224) The full length of AraC-LuxR.sub.N86K and C245W was subjected to EP-PCR (50 μM MnCl.sub.2, amplification factor: 10,000 times) to generate a mutant pool having random mutations introduced therein (the library size was 3×10.sup.6). Through use of P.sub.BAD7-GFP as a reporter, 90 colonies showing fluorescence on solid medium containing 13 mM L-arabinose were picked. The ligand responsiveness of those colonies was evaluated. Specifically, culture was performed under separate conditions of “13 mM L-arabinose” and “10 μM AHL”, and 46 mutants each having an improved L-arabinose/AHL ratio of 2 times or more were obtained. Fluorescence intensity measurement results of a mutant having a high L-arabinose/AHL ratio and having low leaky expression in the presence of AHL alone among those mutants are shown in
(225) Through only one round of EP-PCR/screening, a mutant of AraC-LuxR.sub.N86K and C245W (see Table 5: M175K and K491E) was obtained.
(226) That is, a 2-input/l-way output-type sensor was able to be developed.
Example 2-5: Development Example 2 of 2-Input/l-Way Output-Type Sensor
(227) In this Example, as an example of a 2-input/l-way output-type sensor, a genetic switch using P.sub.BAD as an “AHL-only” gate that did not respond to arabinose and was activated only with AHL (showed a response thereto in a concentration-dependent manner) was developed.
(228) First, mutations were introduced into amino acid residues required for arabinose recognition by the ligand (arabinose) binding site of AraC, to thereby reduce the affinity of AraC-LuxR for arabinose. Then, EP-PCR/screening was performed using a library having the thus reduced affinity. The details are as described below.
(229) First generation: The L-arabinose binding residues of AraC-LuxR (H80, H81, and Y82) were randomized by a known method to generate a library (the library size was 3×10.sup.6). Through use of P.sub.BAD7-GFP as a reporter, 48 colonies showing fluorescence on solid medium containing 10 μM AHL were selected. The ligand responsiveness of those colonies was evaluated. Specifically, culture was performed under separate conditions of “no ligand”, “13 mM L-arabinose”, and “10 μM AHL”, and 5 mutants each of which did not respond to L-arabinose and showed fluorescence when AHL was added were obtained. Of those, a mutant (H80F, H81K, and Y82L) having an AHL/none ratio and an L-arabinose/none ratio of 1.0 times and 1.6 times, respectively was adopted as a parent of a second generation.
(230) Second generation: The full length of the mutant obtained in the first generation was subjected to EP-PCR (50 μM MnCl.sub.2, amplification factor: 10,000 times) to generate a mutant pool having random mutations introduced therein (the library size was 5×10.sup.5). Through use of P.sub.BAD7-GFP as a reporter, 41 colonies showing intermediate to high levels of fluorescence were picked from a total of 1,600 colonies formed on solid medium containing 10 μM AHL. The ligand responsiveness of those colonies was evaluated. Specifically, culture was performed under separate conditions of “no ligand”, “13 mM L-arabinose”, and “10 μM AHL”, and 5 mutants each of which did not respond to L-arabinose and had an AHL/none ratio of 2 times or more were obtained. Fluorescence intensity measurement results of the mutant having the highest AHL/none ratio among those mutants are shown in
(231) Through two rounds of screening (evolutionary engineering), a mutant of AraC-LuxR.sub.N86K and C245W (see Table 5: H80F, H81K, Y82L, N393I, Y439H, R523L, and F541L) was obtained.
(232) That is, a 2-input/l-way output-type sensor different from that of Example 2-4 was able to be developed.
Example 3
Example 3: Development Example of 3-Input-Type Sensor
(233) Generation of TetR-AraC-LuxR.sub.N86K and C245W Fusion
(234) A 3-input-type sensor was developed on the basis of the 2-input-type sensor generated in Example 2. Another transcription factor, TetR (tetracycline-responsive transcription factor), was further fused to the N-terminus of the AraC-LuxR.sub.N86K and C245W fusion generated in Example 2 to generate TetR-AraC-LuxR.sub.N86K and C245W (see
(235) Generation of Mutants of TetR-AraC-LuxR.sub.N86K and C245W
(236) First generation: The full length of TetR-AraC-LuxR.sub.N86K and C245W (see
(237) Second generation: The full length of the mutant obtained in the first generation was subjected to EP-PCR (10 μM MnCl.sub.2, amplification factor: 1,000 times) to generate a mutant pool having random mutations introduced therein (the library size was 7×10.sup.5). Through use of P.sub.lux-APH (Aminoglycoside phosphotransferase: kanamycin resistance gene) as a selector, selection was performed in order to remove mutants each having base truncation or a stop codon occurring therein (3 hours with 216 nM aTc, 13 mM L-arabinose, 10 μM AHL, and 30 μg/mL Kanamycin).
(238) Next, through use of Ptet-GFP as a reporter, 8 colonies showing weak fluorescence were picked from a total of 2,000 colonies formed on solid medium containing 216 nM aTc, 13 mM L-arabinose, and 10 μM AHL. The ligand responsiveness of those colonies was qualitatively evaluated by a spot method. In eight kinds of solid media obtained by combining the presence or absence of each ligand, 3 mutants showed staged transcription repression and had no base truncation or stop codon occurring therein. Of those, a mutant suppressed in leaky expression under the condition of including all ligands was used as a parent of a third generation.
(239) Third generation: The full length of the mutant obtained in the second generation was subjected to EP-PCR (10 μM MnCl.sub.2, amplification factor: 1,000 times) to generate a mutant pool having random mutations introduced therein (the library size was 3×10.sup.4). Through use of P.sub.lux-APH as a selector, selection was performed in order to remove mutants each having base truncation or a stop codon occurring therein (3 hours with 216 nM aTc, 13 mM L-arabinose, 10 μM AHL, and 60 μg/mL kanamycin). Next, through use of Ptet-APH as a selector, selection was performed in order to concentrate mutants showing weakened transcription repression activity with the addition of aTc alone (3 hours with 216 nM aTc and 30 μg/mL kanamycin). Further, through use of Ptet-GFP as a reporter, 184 colonies showing weak fluorescence were picked from a total of 384 colonies formed on solid medium containing 216 nM aTc, 13 mM L-arabinose, and 10 μM AHL. The ligand responsiveness of those colonies was evaluated. Specifically, culture was performed under separate conditions of “no ligand”, “216 nM aTc alone”, and “216 nM aTc, 13 mM L-arabinose, and 10 μM AHL”, and 5 mutants showed high transcription repression when all ligands were added, while showing weak transcription repression with aTc alone. The 5 mutants all had similar functions, but had different mutation sites, and hence all of these mutants were used as parents of a fourth generation.
(240) Fourth generation: the five mutants obtained in the third generation were mixed and the full length was subjected to EP-PCR (10 μM MnCl.sub.2, amplification factor: 1,000 times) to generate a mutant pool having random mutations introduced therein (the library size was 2×10.sup.4). Through use of P.sub.lux-APH as a selector, selection was performed in order to remove mutants each having base truncation or a stop codon occurring therein (3 hours with 216 nM aTc, 13 mM L-arabinose, 10 μM AHL, and 30 μg/mL Kan). Further, through use of Ptet-APH as a selector, selection was performed in order to concentrate mutants showing weakened transcription repression activity with the addition of aTc alone (3 hours with 216 nM aTc and 30 μg/mL Kan). Next, through use of Ptet-GFP as a reporter, 45 colonies showing weak fluorescence were picked from a total of 900 colonies formed on solid medium containing 216 nM aTc, 13 mM L-arabinose, and 10 μM AHL. The ligand responsiveness of those colonies was qualitatively evaluated by a spot method. In eight kinds of solid media obtained by combining the presence or absence of each ligand, 3 mutants were improved in transcription repression activity as the number of ligands increased. Those 3 mutants have the same mutations introduced therein, and fluorescence intensity measurement results of one of the mutants are shown in
(241) Through four rounds of screening (evolutionary engineering), a mutant of TetR-AraC-LuxR.sub.N86K and C245W (see Table 5: K46R, D95G, K108N, I134V, V145A, L204P, I214N, P216T, F217S, L409Q, T545A, and S569T) was obtained.
(242) That is, a 3-input-type sensor (in particular, 3-input-type 3-stage output reduction sensor) was able to be developed.
(243) TABLE-US-00005 TABLE 5 Mutation sites of mutants Synonymous Name See Non-Synonymous Mutation Mutation pluxAND FIG. 2(d) T220C (F74L), C256A (P86T) A1221G (K407K), A1536G (T521T) 4-9G T746C (V249A), A893T (N298I) AND FIG. 3(a) C116G (P39R), T590A (I197N), A1104G (L368L), A1200T (P400P) 4-2A A755G (N252S) OR FIG. 3(b) G883A (E295K) T192C (F64F) A801G (K267K) 4-9G ARA FIG. 3(c) T524A (M175K), A1471G (K491E) T1449C (C483C), T1518C (T506T) 4-5H AHL FIG. 3(d) TTT (H80F), AAG (H81K), C864T (A288A), A1167T (I389I) Gen2-4-8B TTG (Y82L), A1178T (N393I), T1315C (Y439H), G1568T (R523L), T1621C (F541L) TAL FIG. 5(b) A137G (K46R), A284G (D95G), G1263A (Q421Q), T2160C (G720G) Gen 4-1E A324T (K108N), A400G (I134V), T434C (V145A), T611C (L204P), T641A (I214N), C646A (P216T), T650C (F217S), T1226A (L409Q), A1633G (T545A), T1705A (S569T)
Example 4
(244) In this Example, in order to ascertain that the 2-input-type sensor developed in the foregoing was capable of functioning as a genetic switch in a combination other than AraC and LuxR, ArsR, which was known as an arsenic sensor, was used in place of the arabinose sensor (AraC). The details are as described below (see
(245) As an arsenic standard solution (hereinafter As(III)), an arsenic standard solution (As 1000) was purchased from Wako Pure Chemical Industries, Ltd. and used in this Example.
(246) (1-1) Generation of ArsR::LuxR.sub.N86K and C245W (SEQ ID NO: 17) (see
(247) (1-1-1) Preparation of Vector for ArsR::LuxR.sub.N86K and C245W
(248) Plasmid 1 (see Table 7) was subjected to PCR (KOD plus) treatment with Primers 1 and 2 (see Table 6: SEQ ID NOS: 1 and 2) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction. The extract was subjected to digestion with XhoI (commercially available restriction enzyme) and SpeI-HF (commercially available restriction enzyme), and further subjected to gel extraction.
(249) (1-1-2) Preparation of ArsR Domain (see
(250) The genome of the E. coli strain MG1655 was used as a template and subjected to PCR (KOD plus) treatment with Primers 3 and 4 (see Table 6: SEQ ID NOS: 3 and 4) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction. The extract was subjected to digestion with XhoI and SpeI-HF, and further subjected to gel extraction.
(251) (1-1-3) Recovery of ArsR::LuxR.sub.N86K and C245W
(252) The vector fragment of the section “1-1-1” and the insert fragment of the section “1-1-2” were subjected to ligation. Next, XL10-Gold (commercially available competent cells) was transformed with the ligation product, and clones were recovered.
(253) (1-2) Generation of ArsR
(254) (1-2-1) Preparation of Vector Side
(255) Plasmid 1 (see Table 7) was subjected to PCR (KOD plus) treatment with Primers 1 and 2 (see Table 6) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction. The extract was subjected to digestion with XhoI and SpeI-HF, and further subjected to gel extraction.
(256) (1-2-2) Preparation of Insert Side
(257) The genome of the E. coli strain MG1655 was used as a template and subjected to PCR (KOD plus) treatment with Primers 3 and 5 (see Table 6: SEQ ID NOS: 3 and 5) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction. The extract was subjected to digestion with XhoI and SpeI-HF, and further subjected to gel extraction.
(258) (1-2-3) Recovery of ArsR
(259) The vector fragment of the section “1-2-1” and the insert fragment of the section “1-2-2” were subjected to ligation. Next, XL10-Gold was transformed with the ligation product, and clones were recovered.
(260) (1-4) Generation of P.sub.ars-GFP
(261) (1-4-1) Generation of P.sub.ars.sup.[rbs]GFP Library
(262) Plasmid 2 (see Table 7) was used as a template and subjected to PCR (KOD plus) treatment with Primers 6 and 7 (see Table 6: SEQ ID NOS: 6 and 7) to give a PCR product. Next, the PCR product was subjected to gel extraction. The gel extraction product was subjected to the Golden Gate method using a commercially available product to assemble DNA fragments, followed by column purification. XL10-Gold was transformed with the purified Golden Gate product and then subjected to liquid culture, and an RBS library was recovered.
(263) (1-4-2) Recovery of P.sub.ars-GFP Having Appropriate RBS (Pars-GFP Having Such RBS as to Show High Fluorescence Under ArsR Expression Plasmid-Free Condition)
(264) E. coli MG1655 was cotransformed with the RBS library of the section “1-4-1”. Next, from colonies isolated on solid medium, one showing strong fluorescence was selected, and cultured overnight. Next, a P.sub.ars-GFP plasmid was recovered from the culture solution. Further, E. coli MG1655 was cotransformed with the recovered P.sub.ars-GFP and ArsR (Plasmid 4: see Table 7). Next, colonies on solid medium were isolated, and confirmed to show weak fluorescence as compared to the one showing strong fluorescence obtained from the above-mentioned colonies.
(265) TABLE-US-00006 TABLE 6 No. Name Sequence Note 1 SpeI-lux-Fwd TTTT ACTAGT GAAAACATAAATGCCGACGACACA Fwd primer for PCR of vector for ArsR-LuxR 2 SpeI-tetR-Rev TTTT ACTAGT Rev primer for PCR of vector GGACCCACTTTCACATTTAAGTTGTTTTT for ArsR-LuxR 3 pET23d-XhoI-Fwd TTTT CTCGAG Fwd primer for PR of ArsR ATGTCATTTCTGTTACCCATCCAATTGTTC domain and ArsR insert 4 SpeI-eArsR-Rev TTTT ACTAGT Rev Primer for PCR of ArsR ACTGCAAATGTTCTTACTGTCCCCG domain 5 BamHI-eArsR-Rev TTTT GGATCC Rev primer for PCR of ArsR ACTGCAAATGTTCTTACTGTCCCCG insert 6 pACmod3 TTTT GGTCTC a Rev primer for PCR of P.sub.ars-GFP PT5-arsO-sfgfp AAACATATATGACTTAACGAATGTGTATT Rev ATACAGAAAAATTTTCCTGAAAGCAAATAAATTT TTCATG 7 pACmod3 TTTT GGTCTC a Fwd primer for PCR of P.sub.ars-GFP PT5-arsO-sfgfp GTTTTTGACTTTAGCACGAGCTSAA Fwd KAATACARGRAGACKMTCAATGGGGTCTAAAGGC GAAGAAC 8 BamHI-His-Fwd TTTT GGATCC CATCATCACCATCAC 9 luxR-seq-Rev-1 AGCATTCCGAAGCCATTGTTAGC 10 ROP-seq-Fwd GATAAAGCGGGCCATGTTAAGG 11 Y-T7tn-gfp-sq-R TCAGCAAAAAACCCCTCAAGACCCGTTTA 12 pUc vector Rev TTTT GGTACCCCTGGGGTGCCTAATGAGTG (KpnI) 13 pUC vector Fwd TTTT (HindIII) AAGCTTCATATGGTGCACTCTCAGTACAATC 14 Pet23d-Fwd (KpnI) TTTT GGTACCGTGAGGGTAAACAACTGGCG 15 prpC-luxR-Fwd GTTATATCTCGGAGGTTTAC Fwd primer for PCR of PrpC ATGAGCGACACAACGATCCT domain 16 prpC-luxR-Fwd TCGGCATTTATGTTTTCACTAGT Fwd primer for PCR of PrpC CTGGCGCTTATCCAGCG domain 17 prpD-luxR-Fwd GTTATATCTCGGAGGTTTAC Fwd primer for PCR of PrpD ATGTCAGCTCAAATCAACAACATC domain 18 prpD-luxR-Fwd Fwd primer for PCR of PrpD domain 19 mTagBFP Fwd CCATTTGATCGCAAATATCAAGACG 20 pMCvec-Plux-ApaI TTTATTCGACTATAACAAACCATTTTCTTGCGTA PCR1 Rev AACCTGTACGATCCTACAGGTGGTACC TATAAACGCAGAAAG 21 pMCvec-Plux-ApaI TTTT GGGCCC PCR2 Rev TTTATTCGACTATAACAAACCATTTTCTTGCG 22 sfGFP standard PBS TTTT GGGCCC RMSCYYTAWGGAGGY CTCGAG library Fwd ATGGGGTCTAAAGGCGAAGAAC (ApaI-XhoI) 23 aph-HindIII Rev TTTT AAGCTT A TCAGAAGAACTCGTCAAGAAGGC 24 Fusion-ml-inv-Fwd CATCATCACCATCACCACTAATGAA Fwd primer for amplifying vector for library plasmid 25 Fusion-ml-inv-Rev GTAAACCTCCGAGATATAACTAGAG Fwd primer for amplifying vector for library plasmid 26 Fusion-mutlibrary- CTCTAGTTATATCTCGGAGGTTTAc Fwd primer for generating Fwd mutant libraries of PrpC-LuxR and PrpD-LuxR 27 Fusion-mutlibrary- TTCATTAGTGGTGATGGTGATGATG Rev primer for generating Rev mutant libraries of PrpC-LuxR and PrpD-LuxR
(266) TABLE-US-00007 TABLE 7 No. Name Construct 1 TL (Tet R-LuxR.sub.N86K and C245W) pET23d-P.sub.J23116-tetR::luxR.sub.N86K-C245W::his 2 GFP pACmod3-P.sub.T5-sfgp-HSVtk:aph 3 ArsR::LuxR (FIG. 11) pET23d-P.sub.J23116-arsR::luxR.sub.N86K-C245W::his 4 ArsR (FIG. 12) pET23d-P.sub.J23116-arsR::his 5 P.sub.lux-GFP (FIG. 13) pACmod3-P.sub.lux-sfgfp-HSVtk:aph 6 P.sub.ars-GFP (FIG. 14) pACmod3-P.sub.TS/arsO-.sup.highsfgfp-HSVtk:aph 7 ArsR::LuxR pET23d-P.sub.J23116-(arsR::luxR)::his 8 pUC-TAL pUC19-P.sub.J23116-tal::his 9 pUC-ArsR pUC19-P.sub.J23116-arsR::his 10 pUC-ArsR::LuxR pUC19-P.sub.J23116-(arsR::luxR)::his 11 pUC-phi pUC19-P.sub.J23116-phi 12 AL (AraC-LuxR.sub.N86K and C245W) pET23d-P.sub.J23116-araC::luxR.sub.N86K-C245W 13 pMC-P.sub.lux-GFP pMC-P.sub.lux-sfgfp-HSVtk::aph 14 pET23d-PrpC::LuxR pET23d-P.sub.J23116-(prpC::luxR.sub.N56K-C245W)::his 15 pET23d-PrpD::LuxR pET23d-P.sub.J23116-(prpD::luxR.sub.N86K-C245W)::his 16 pMC-BFP pMC-P.sub.N25-mTagBFP 17 LuxR pTrcHis2-P.sub.tra-luxR 18 pET23d-PrpC::LuxRmut pET23d-P.sub.J23116-(prpC.sub.K318R::luxR.sub.N86K-C245W)::his 19 pET23d-PrpD::LuxRmut pET23d-P.sub.J23116-(prpD.sub.S163T-A225G-I285V::luxR.sub.N86K-C245W)::his
(267) (2-1) Generation of ArsR::LuxR Library
(268) (2-1-1) Preparation of Vector Fragment
(269) Plasmid 3 (see Table 7) was used as a template and subjected to PCR (KOD plus) treatment with Primers 8 and 9 (see Table 6: SEQ ID NOS: 8 and 9) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction. The extract was subjected to digestion with Xhol, BamHI-HF, and rSAP, and further subjected to gel extraction.
(270) (2-1-2) Preparation of ArsR::LuxR Fragment Having Random Mutations Introduced Throughout Entire Gene
(271) Plasmid 3 (see Table 7) was used as a template and subjected to PCR (KOD plus) treatment with Primers 10 and 11 (see Table 6: SEQ ID NOS: 10 and 11) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction. The gel extraction product was used as a template and subjected to PCR (Taq, 10 μM or 50 μM MnCl.sub.2) treatment with Primers 10 and 11 (see Table 6) to give a PCR product. Next, the PCR product was subjected to gel extraction. The extract was subjected to digestion with XhoI and BamHI-HF, and further subjected to gel extraction.
(272) (2-1-3) Recovery of ArsR::LuxR Library
(273) The vector fragment of the section “2-1-1” and the insert fragment of the section “2-1-2” were subjected to ligation. Next, the ligation product was subjected to column purification. Next, an E. coli strain BW25113 was trans formed with the purified ligation product and subjected to liquid culture, and a library was recovered from the resultant culture solution.
(274) (2-2) Evaluation of Reporter (GFP) Expression Amount
(275) MG1655 was transformed with an arbitrary reporter plasmid. Next, one transformant was selected, and competent cells (E. coli) were generated. The E. coli was transformed with the library (plasmids). An arbitrary number of isolated colonies were selected, and subjected to liquid culture overnight. A1/100 amount of the preculture solution was inoculated into liquid medium containing As(III) or AHL at an arbitrary concentration, and was cultured for 12 hours. Next, 200 μL of a measurement sample obtained by diluting the culture solution 10-fold with physiological saline was prepared. Cell density (OD.sub.595) and fluorescence at 535 nm at the time of excitation at 485 nm were measured using FilterMax F5 (commercially available absorbance detection apparatus). As a blank sample (control), 200 μL of a blank sample obtained by diluting liquid medium 10-fold with physiological saline was also subjected to measurement simultaneously. Data analysis was performed with a value obtained by subtracting the measured value of the blank sample from the measured value of the sample.
(276) (2-3) Selection of ArsR::LuxR Library
(277) MG1655 was transformed with a reporter P.sub.lux-GFP (Plasmid 5: see Table 7). Then, one transformant was selected, and competent cells (E. coli) were generated. The E. coli was transformed with the ArsR::LuxR library of the section “2-1”, inoculated into 10 mL of liquid medium, and cultured overnight. Next, the preculture solution in an amount corresponding to 10.sup.7 cells was inoculated into 10 mL of liquid medium containing 1 μM AHL and 5,000 ppb (67 μM) As(III), and was cultured for 1 hour. Next, 100 μg/mL kanamycin was added, followed by culture for 3 hours. Next, the culture solution was centrifuged and the supernatant was removed. Further, the resultant was resuspended in 10 mL of liquid medium containing 1 μM AHL and 5,000 ppb (67 μM) As(III). The culture solution was again centrifuged and the supernatant was removed (an operation of removing kanamycin was performed). The operation of removing kanamycin was performed again. Next, the resultant was resuspended in 10 mL of liquid medium containing 1 μM AHL and 5,000 ppb (67 μM) As(III), and was cultured overnight. Finally, cells were collected from the culture solution to recover concentrated library plasmids.
(278) (3-1) Generation of High-Sensitivity 2-Input AND-Type Arsenic Switch (ArsR-LuxR)
(279) MG1655 was transformed with a reporter plasmid P.sub.lux-GFP (Plasmid 5: see Table 7). Next, one transformant was selected, and competent cells (E. coli) were generated. The competent cells (E. coli) were transformed with the ArsR-LuxR library of the section “2-1”. 89 of colonies formed on solid medium were randomly selected.
(280) Fluorescence intensities per cell density in the case of culture under separate conditions of “medium containing 1 μM AHL” and “medium containing 1 μM AHL and 5,000 ppb As(III)” were evaluated by the method of the section “2-2”. A mutant showing weak fluorescence under the condition of adding AHL alone, and showing strong fluorescence under the condition of adding AHL and As(III) was recovered.
(281) The recovered mutant was used as a template to generate a library by the method of the section “2-1”. The competent cells (E. coli) were transformed with the generated library. 59 of colonies formed on solid medium were randomly selected. Fluorescence intensities per cell density in the case of culture under separate conditions of “medium containing 1 μM AHL” and “medium containing 1 μM AHL and 5 ppb As(III)” were evaluated by the method of the section “2-2”. A mutant showing weak fluorescence under the condition of adding AHL alone, and showing strong fluorescence under the condition of adding AHL and As(III) was recovered.
(282) It was confirmed by the method of the section “2-2” that a high-sensitivity AND-type arsenic switch had been generated (see
(283) (3-2) Generation of 2-Input AND-Type Arsenic Switch Having High Stringency (ArsR-LuxR)
(284) MG1655 was transformed with a reporter-plasmid P.sub.lux-GFP (Plasmid 5: see Table 7). Next, one transformant was selected, and competent cells (E. coli) were generated. The competent cells (E. coli) were transformed with the ArsR-LuxR library of the section “2-1”.
(285) Mutants showing fluorescence when 1 μM AHL and 5,000 ppb As(III) were added were concentrated by the method of the section “2-3”. Next, the competent cells (E. coli) were transformed with the concentrated library. Next, 80 colonies showing weak fluorescence on solid medium containing 1 μM AHL were selected.
(286) Fluorescence intensities per cell density in the case of culture under separate conditions of “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)” were evaluated by the method of the section “2-2”. A mutant showing weak fluorescence under the condition of adding AHL alone, and showing strong fluorescence under the condition of adding AHL and 5 ppb As(III) and under the condition of adding AHL and 500 ppb As(III) was recovered.
(287) It was confirmed by the method of the section “2-2” that an AND-type arsenic switch having high stringency had been generated (see
(288) (Evaluation of Arsenic Responsiveness of ArsR/P.sub.ars)
(289) In this Example, the performance of a 2-input AND-type arsenic switch in the case of not using P.sub.lux in a reporter plasmid was evaluated.
(290) (4-1) Change to pUC Vector
(291) (4-1-1) Preparation of Vector
(292) pUC-TAL (Plasmid 8: see Table 7, TetR-AraC-LuxR.sub.N86K and C245W) was used as a template and subjected to PCR (KOD plus) treatment with Primers 12 and 13 (see Table 6: SEQ ID NOS: 12 and 13) to give a PCR product. Next, the PCR product was subjected to gel extraction. The extract was subjected to digestion with KpnI-HF (known restriction enzyme), HindIII-HF, and rSAP, and further subjected to gel extraction.
(293) (4-1-2) Preparation of Insert
(294) ArsR and ArsR::LuxR (Plasmids 4 and 7: see Table 7) were each used as a template and subjected to PCR (KOD plus) treatment with Primers 11 and 14 (see Table 6: SEQ ID NOS: 11 and 14) to give a PCR product. Next, the PCR product was subjected to gel extraction. The extract was subjected to digestion with KpnI-HF and HindIII-HF, and further subjected to gel extraction.
(295) (4-1-3) Recovery of pUC Product
(296) The vector fragment of the section “1-2-1” and the insert fragment of the section “1-2-2” were subjected to ligation. Next, XL10-Gold was transformed with the ligation product, and clones were recovered.
(297) (4-2) Arsenic Responsiveness Evaluation with P.sub.ars-GFP (See
(298) MG1655 was transformed with a reporter plasmid P.sub.ars-GFP (Plasmid 6: see Table 7). One transformant was selected, and competent cells (E. coli) were generated.
(299) The competent cells (E. coli) were transformed with pUC-ArsR, pUC-ArsR::LuxR, or pUC-phi (Plasmids 9 to 11: see Table 7). Three of colonies formed on solid medium were randomly selected.
(300) Fluorescence intensities per cell density in the case of culture in the presence of 100 μM AHL and at arbitrary different As(III) concentrations were evaluated by the method of the section “2-2” (see
Example 5
(301) (Selection Method for Sensitivity-Variable Genetic Switch)
(302) The inventors of the present invention confirmed from the results of
(303) Specifically, when the EC50 value of the sensor including the 2-input AND-type arsenic switch generated in Example 4 was plotted against the concentration of AHL present in the system, it was confirmed that the EC50 value reduced as the AHL concentration increased (see
(304) This phenomenon is a unique effect of the sensor including the genetic switch obtained by the method of the present invention. The AND-type genetic switch retains a functional structure in a manner dependent both on stabilization by binding to AHL and on stabilization by binding to arsenic. In the case of a genetic switch having such properties, as the concentration of AHL in the system increases, the effective concentration of ArsR serving as an arsenic sensor increases, and hence apparent sensitivity of “arsenic+ArsR.Math.arsenic⋅ArsR” increases.
(305) That is, in the sensor including the genetic switch of the present invention, the sensitivity of the genetic switch to a ligand can be easily adjusted by the following method.
(306) When the sensitivity of the ligand L.sub.1 is to be increased, the addition concentration of the ligand L.sub.2 is increased.
(307) When the sensitivity of the ligand L.sub.1 is to be decreased, the addition concentration of the ligand L.sub.2 is decreased.
(308) When the sensitivity of the ligand L.sub.2 is to be increased, the addition concentration of the ligand L.sub.1 is increased.
(309) When the sensitivity of the ligand L.sub.2 is to be decreased, the addition concentration of the ligand L.sub.1 is decreased.
Example 6
(310) (Selection Method for High-Sensitivity Genetic Switch)
(311) The inventors of the present invention confirmed from the results of the foregoing Examples that the production method of the present invention was able to improve, for example, the sensitivity, output intensity, and stringency of the sensor.
(312) For example, when the transcription factor LuxR is used, intentional detection with an arsenic detection switch based on the expression of a reporter gene downstream of the P.sub.1 promoter has the following advantages.
(313) (1) Output intensity and stringency: LuxR has the highest stringency (least expression leakage in a non-induced state) among transcription factors, and has a high maximum output when induced (transcription enhancing efficiency). Meanwhile, an ArsR/ArsP (P.sub.ars) system has a low output intensity, and also has low stringency. Accordingly, the following result was obtained: the signal-to-noise ratio at the time of its response to arsenic was extremely low (see “ArsR.fwdarw.P.sub.ars” of
(314) (2) High-sensitivity detection of ligand: For example, in the case of the combination of the ArsR-LuxR transcription factor and the promoter ArsP, the expression profile of the reporter gene downstream of ArsP had an inflection point around 50 ppb. This detection sensitivity is nearly the same detection sensitivity as that in the case where ArsR alone is allowed to act on ArsP. However, in the case of the combination of the ArsR-LuxR transcription factor and the promoter P.sub.lux, the expression profile of the reporter gene downstream of P.sub.lux had an inflection point around 5 ppb. This sensitivity is the world's highest sensitivity for a biosensor. An arsenic concentration serving as a WHO standard is 10 ppb. In the case of using an ArsR/ArsP system, the inflection point is present at a concentration that is one digit higher than the foregoing, and hence the ArsR/ArsP system cannot be used. However, in the case of the combination of the ArsR-LuxR transcription factor and the promoter P.sub.lux, the combination can be used as it is for environmental monitoring.
(315) This increase in sensitivity results from liberation from the operation mechanism of As-ArsR/ArsP as a “depressor”. ArsR can originally bind to As with high affinity, but tightly binds to ArsP under an As-free state. That is, the ArsR structure in a repressed state is significantly stabilized by the binding to ArsP. Arsenic cancels the stabilized structure to release ArsR from ArsP.
(316) That is, the equilibrium of “As+ArsR.Math.As-ArsR” is significantly shifted to the left due to the presence of ArsP (that is, the sensitivity is decreased). It is considered that, in the absence of ArsP, As-ArsR binding to be read by LuxP is liberated from the sensitivity-decreasing effect, and hence the nearly one-digit increase in sensitivity is achieved.
Example 7
(317) In this Example, in order to ascertain that it was possible to produce the 2-input-type sensor developed in the foregoing even by using an enzyme as a material, an enzyme PrpC or PrpD was used in place of the arabinose sensor (AraC) or the arsenic sensor (ArsR). The details are as described below. PrpC and PrpD are E. coli-derived enzymes called 2-methylcitrate synthase (EC:2.3.3.5) and 2-methylcitrate dehydratase (EC:4.2.1.3), respectively.
(318) (1-1) Generation of PrpC::LuxR.sub.N86K-C245W and PrpD::LuxR.sub.N86K-C245W
(319) (1-1-1) Preparation of Vector for PrpC::LuxR.sub.N86K-C245W and PrpD::LuxR.sub.N86K-C245W
(320) Plasmid-12 (see Table 7) was subjected to digestion with NcoI-HF (commercially available restriction enzyme) and SpeI-HF (commercially available restriction enzyme), and subjected to gel extraction.
(321) (1-1-2) Preparation of PrpC and PrpD Domains
(322) The genome of the E. coli strain MG1655 was used as a template and subjected to PCR (Q5 ploymerase) treatment with Primer-15 and Primer-16 or Primer-17 and Primer-18 (see Table 6) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction.
(323) (1-1-3) Recovery of PrpC::LuxR.sub.N86K-C245W and PrpC::LuxR.sub.N86K-C245W
(324) The vector fragment of the section “1-1-1” and the insert fragment of the section “1-1-2” were subjected to the Gibson assembly method using a commercially available enzyme to assemble the DNA fragments. Next, XL10-Gold (commercially available competent cells) was transformed with the Gibson assembly product, and clones were recovered (Plasmid-14 and Plasmid-15).
(325) (1-2) Generation of pMC-P.sub.lux-GFP
(326) (1-2-1) Preparation of Vector for pMC-P.sub.lux-GFP
(327) Plasmid-16 (see Table 7) was subjected to PCR (KOD plus) treatment with Primer-19 and Primer-20 (see Table 6) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction. The extract was subjected to PCR (KOD plus) treatment with Primer-19 and Primer-21 (see Table 6) to give a PCR product. Next, the PCR product was subjected to gel extraction. The extract was subjected to digestion with ApaI (commercially available restriction enzyme), HindIII-HF (commercially available restriction enzyme), and rSAP (commercially available phosphatase), and further subjected to gel extraction.
(328) (1-2-2) Preparation of GFP-hsvTK::APH Domain with Randomized RBS
(329) Plasmid-5 (see Table 7) was used as a template and subjected to PCR (KOD plus) treatment with Primer-22 and Primer-23 (see Table 6) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction. The extract was subjected to digestion with ApaI and HindIII-HF, and further subjected to gel extraction.
(330) (1-2-3) Recovery of RBS Library of pMC-P.sub.lux-GFP
(331) The vector fragment of the section “1-2-1” and the insert fragment of the section “1-2-2” were subjected to ligation. Next, XL10-Gold (commercially available competent cells) was transformed with the ligation product, and clones were recovered.
(332) (1-2-4) Recovery of pMC-P.sub.lux-GFP
(333) The E. coli strain BW25113 was transformed with the library plasmids of the section “1-2-3” and Plasmid-17. 30 of isolated colonies were randomly selected, and subjected to liquid culture overnight. A 1/100 amount of the preculture solution was inoculated into medium containing or not containing 1 μM AHL, and was cultured for 12 hours. Next, 200 μL of a measurement sample obtained by diluting the culture solution 10-fold with physiological saline was prepared. Cell density (0D595) and fluorescence at 535 nm at the time of excitation at 482 nm were measured using FilterMax F5 (commercially available absorbance detection system). As a blank sample (control), 200 μL of a blank sample obtained by diluting liquid medium 10-fold with physiological saline was also subjected to measurement simultaneously. Data analysis was performed with a value obtained by subtracting the measured value of the blank sample from the measured value of the sample. On the basis of the obtained data, the RBS mutant having the largest change between the fluorescence intensity in the case of containing AHL and that in the case of not containing AHL was isolated and recovered.
(334) (1-3) Generation of PrpC.sub.K318R::LuxR.sub.N86K-C245W and PrpD.sub.S163T-A225G-I285V::LuxR.sub.N86K-C245W
(335) (1-3-1) Preparation of Vectors for PrpC::LuxR.sub.N86K-C245W and PrpD::LuxR.sub.N86K-C245W Libraries
(336) Plasmid-14 or Plasmid-15 (see Table 7) was subjected to inverse PCR (Q5 ploymerase) treatment with Primer-24 and Primer-25 (see Table 6) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction.
(337) (1-3-2) Preparation of PrpC-LuxR.sub.N86K-C245W and PrpD-LuxR.sub.N86K-C245W Domain Libraries
(338) Plasmid-14 or Plasmid-15 (see Table 7) was used as a template and subjected to error prone PCR (Taq ploymerase) treatment with Primer-26 and Primer-27 (see Table 6) to give a PCR product. Next, the PCR product was subjected to DpnI treatment and then subjected to gel extraction.
(339) (1-3-3) Recovery of PrpC::LuxR.sub.N86K-C245W and PrpD::LuxR.sub.N86K-C245W Library Plasmids
(340) The vector fragment of the section “1-3-1” and the insert fragment of the section “1-3-2” were subjected to the Gibson assembly method using a commercially available enzyme to assemble the DNA fragments. Next, BW25113 (commercially available competent cells) was transformed with the Gibson assembly product, and library plasmids were recovered.
(341) (1-3-4) Recovery of pET23d-PrpC.sub.K318R::LuxR.sub.N86K-C245W and pET23d-PrpD.sub.S163T-A225G-I285V::LuxR.sub.N86K-C245W
(342) The E. coli strain BW25113 was transformed with the library plasmids of the section “1-3-3” and Plasmid-23, and applied to a screening plate containing 10 μM AHL and 50 mM propionic acid. From isolated colonies, 48 colonies emitting GFP fluorescence were selected and subjected to liquid culture overnight. A 1/100 amount of the preculture solution was inoculated into liquid medium containing or not containing 50 mM propionic acid, and was cultured for 12 hours. Next, 200 μL of a measurement sample obtained by diluting the culture solution 10-fold with physiological saline was prepared. Cell density (0D595) and fluorescence at 535 nm at the time of excitation at 482 nm were measured using FilterMax F5 (commercially available absorbance detection system). As a blank sample (control), 200 μL of a blank sample obtained by diluting liquid medium 10-fold with physiological saline was also subjected to measurement simultaneously. Data analysis was performed with a value obtained by subtracting the measured value of the blank sample from the measured value of the sample. On the basis of the obtained data, the mutant having the largest change between the fluorescence intensity in the case of containing propionic acid and that in the case of not containing propionic acid was isolated and recovered, and its sequence was analyzed to identify mutation sites (full-length amino acid sequence of PrpC.sub.K318R-LuxR.sub.N86K and C245W: SEQ ID NO: 31, full-length amino acid sequence of PrpD.sub.S163T-A225G-I285V-LuxR.sub.N86K and C245W: SEQ ID NO: 32).
(343) (2) Propionic Acid Responsiveness Evaluation with pMC-P.sub.lux-GFP
(344) BW25113 was transformed with a reporter plasmid pMC-P.sub.lux-GFP (Plasmid 13: see Table 7). One transformant was selected, and competent cells (E. coli) were generated.
(345) The competent cells (E. coli) were transformed with pET23d-PrpC::LuxR.sub.mut (see
(346) The results are shown in
(347) (General Remark)
(348) As apparent from Examples described above, the production method for a genetic switch of the present invention is a method according to which not only a variable-type sensor having an integrated and advanced function is produced, but also an output can be made as the function of a more excellent transcription factor by compensating for the disadvantage of a sensor function of low basic performance.
INDUSTRIAL APPLICABILITY
(349) The production method for a multi-input/multi-output-type genetic switch or a transcription factor, and the multi-input/multi-output-type genetic switch or the transcription factor can be provided.