Multi-Input/Multi-Output Gene Switch, And Method For Producing Same

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 B1 or a transcription factor T1, which responds to a ligand L1, and a binder B2 or a transcription factor T2, which responds to a ligand L2, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the binder B1 or the transcription factor T1 and a gene sequence encoding the binder B2 or the transcription factor T2, and a reporter expression vector carrying a gene sequence encoding a promoter P1 to be controlled by the transcription factor T1 and/or a gene sequence encoding a promoter P2 to be controlled by the transcription factor T2, and a gene sequence encoding a reporter Rx functionally linked to the promoter sequence P1 and/or the promoter sequence P2, where X represents an integer of 1 or more;
• (2) a step of adding the ligand L1 and/or the ligand L2 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 B1 or the transcription factor T1 and the binder B2 or the transcription factor T2 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 B1 or a transcription factor T1, which responds to a ligand L1, a binder B2 or a transcription factor T2, which responds to a ligand L2, and a binder BN or a transcription factor TN, which responds to a ligand LN, the library being obtained by introducing mutations into a genetic construct carrying a gene sequence encoding the binder B1 or the transcription factor T1, a gene sequence encoding the binder B2 or the transcription factor T2, and a gene sequence encoding the binder BN or the transcription factor TN, and a reporter expression vector carrying a gene sequence encoding a promoter P1 to be controlled by the transcription factor T1, a gene sequence encoding a promoter P2 to be controlled by the transcription factor T2, and/or a gene sequence encoding a promoter PN to be controlled by the transcription factor TN, and a gene sequence encoding a reporter Rx functionally linked to the promoter sequence P1, the promoter sequence P2, and/or the promoter sequence PN, where N represents an integer of 3 or more;
• (2) a step of introducing the ligand L1, the ligand L2, and/or the ligand LN 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 B1 or the transcription factor T1, the binder B2 or the transcription factor T2, and the binder BN or the transcription factor TN as a genetic switch or a transcription factor for forming the switch through use of an expression amount of the reporter as an indicator.