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US 9,315,816 B2Granted
Method for rapidly developing gene switches and gene circuits
AssigneeNational University Corporation Chiba University
InventorsDaisuke Umeno, Hiroki Fukutomi, Kyoichi Saito
Life of this patent
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Abstract
Provided are: a selection method for a gene switch and a gene circuit, including using, as a selector, an expression vector containing at least a gene sequence whose expression is controlled by a transcription regulatory factor to be expressed when a genetic switch and a genetic circuit including the genetic switch operate, and a promoter sequence operably linked to the gene sequence upstream thereof; and an expression vector to be used in the selection method. This enables an effective selection method for a genetic switch and a genetic circuit, the selection method being able to be conducted within a short time period and with high selection efficiency and less leakiness.
Claims20
A selection method for a genetic switch and a genetic circuit, the selection method comprising:
- incubating cells transfected with
- a genetic circuit expression vector comprising a genetic circuit having a genetic switch expression sequence, and
- a transcription regulatory factor gene sequence encoding a transcription regulatory factor whose expression is induced by a genetic switch encoded by the genetic switch expression sequence, and
- a controllable expression vector having a controlled gene sequence whose expression is controlled by the transcription regulatory factor, the incubation being performed
- in a presence or absence of an activating compound that activates the genetic switch, with addition of a first death-determining compound that induces cell death under expression of the controlled gene sequence, and collecting viable cells, and/or
- in a presence or absence of the activating compound with addition of a second death-determining compound that induces cell death under non-expression of the controlled gene sequence;
- and
- collecting viable cells that survive the incubation,
- wherein the controllable expression vector comprises a first promoter sequence operably linked to and upstream of the controlled gene sequence, and
- the controlled gene sequence encodes an alkylated DNA repair enzyme;
- and
- the genetic circuit expression vector comprises a target sequence of the genetic switch operably linked to the transcription regulatory factor gene sequence and a second promoter sequence different from the first promoter sequence, the transcription regulatory factor gene sequence encoding a transcription factor being operable on the first promoter sequence, and the second promoter sequence being operably linked to the genetic switch expression sequence, incubation being performed
- (1) when the transcription regulatory factor is a transcription repression factor, in the absence of the activating compound with addition of an alkylating agent, or
- (2) when the transcription regulatory factor is a transcription activation factor, in the presence of the activating compound with addition of the alkylating agent.
11 more elements
A selection method for a genetic switch and a genetic circuit, the selection method comprising:
- incubating an alkylating agent-hypersensitive Escherichia coli strain transfected with
- a controllable expression vector comprising at least sequences according to the following items (a) and (b):
- (a) an enzyme gene sequence encoding alkyladenine DNA glycosidase (AAG);
- and
- (b) a first promoter sequence operably linked to the enzyme gene sequence according to the item (a) upstream of the enzyme gene sequence, and
- a genetic circuit expression vector comprising at least sequences according to the following items (c) to (f):
- (c) a second promoter sequence different from the first promoter sequence, the second promoter sequence being operably linked to a genetic switch expression sequence downstream thereof;
- (d) the genetic switch expression sequence;
- (e) a target sequence of a genetic switch encoded by the genetic switch expression sequence;
- and
- (f) a transcription regulatory factor gene sequence having the target sequence operably linked thereto, the transcription regulatory factor gene sequence encoding a CI protein that operates on the first promoter sequence according to the item (b), the incubation being performed
- in an absence of a compound that activates the genetic switch, with addition of methanesulfonic acid (MMS) for 15 minutes to 60 minutes;
- and
- collecting viable cells that survive the incubation.
11 more elements
A selection method for a genetic switch and a genetic circuit, the selection method comprising:
- incubating an alkylating agent-hypersensitive Escherichia coli strain transfected with
- a controllable expression vector comprising at least sequences according to the following items (a) and (b):
- (a) a first enzyme gene sequence encoding alkyladenine DNA glycosidase (AAG) and a second enzyme gene sequence encoding AP endonuclease (APE1);
- and
- (b) a first promoter sequence operably linked to the two enzyme gene sequences according to the item (a) upstream of the first and second enzyme gene sequences, and
- a genetic circuit expression vector comprising at least sequences according to the following items (c) to (f):
- (c) a second promoter sequence different from the first promoter sequence, the second promoter sequence being operably linked to a genetic switch expression sequence downstream thereof;
- (d) the genetic switch expression sequence;
- (e) a target sequence of a genetic switch encoded by the genetic switch expression sequence;
- and
- (f) a CI protein gene sequence having the target sequence operably linked thereto, the CI protein gene sequence encoding a CI protein that operates on the first promoter sequence according to the item (b), the incubation being performed
- in an absence of a compound that activates the genetic switch with addition of methanesulfonic acid (MMS) for 15 minutes to 60 minutes;
- and
- collecting viable cells that survive the incubation.
11 more elements
A selection method for a genetic switch and a genetic circuit, the selection method comprising:
- incubating an alkylating agent-hypersensitive Escherichia coli strain transfected with
- a controllable expression vector comprising at least sequences according to the following items (a) and (b):
- (a) a first enzyme gene sequence encoding alkyladenine DNA glycosidase (AAG) and a second enzyme gene sequence encoding a human herpes virus derived thymidine kinase;
- and
- (b) a first promoter sequence operably linked to the first and second enzyme gene sequences according to the item (a) upstream of the first and second enzyme gene sequences, and
- a genetic circuit expression vector comprising at least sequences according to the following items (c) to (f):
- (c) a second promoter sequence different from the first promoter sequence, the second promoter sequence being operably linked to a genetic switch expression sequence downstream thereof;
- (d) the genetic switch expression sequence;
- (e) a target sequence of a genetic switch encoded by the genetic switch expression sequence;
- and
- (f) a CI protein gene sequence having the target sequence operably linked thereto, the CI protein gene sequence encoding a CI protein that operates on the first promoter sequence according to the item (b)
- in a presence of an activating compound that activates the genetic switch for 5 minutes to 60 minutes with addition of 6-(β-D-2-deoxyribo-furanosyl)-3,4-dihydro-8H-pyrimido[4,5c-][1,2]oxazin-7-one (dP) in a first incubation stage;
- collecting viable cells that survive the first incubation stage;
- incubating the collected cells for 15 minutes to 60 minutes with addition of methanesulfonic acid (MMS) in the absence of the activating compound in a second incubation stage;
- and
- collecting viable cells that survive the second incubation stage.
13 more elements
A selection method for a genetic switch and a genetic circuit, the selection method comprising:
- incubating an alkylating agent-hypersensitive Escherichia coli strain transfected with
- a controllable expression vector comprising at least sequences according to the following items (a) and (b):
- (a) a first enzyme gene sequence encoding alkyladenine DNA glycosidase (AAG), a second enzyme gene sequence encoding a human herpes virus derived thymidine kinase, and a third enzyme gene sequence encoding AP endonuclease (APE1);
- and
- (b) a first promoter sequence operably linked to the first, second, and third enzyme gene sequences according to the item (a) upstream of the first, second, and third enzyme gene sequences, and
- a genetic circuit expression vector comprising at least sequences according to the following items (c) to (f):
- (c) a second promoter sequence different from the first promoter sequence, the second promoter sequence being operably linked to a genetic switch expression sequence downstream thereof;
- (d) the genetic switch expression sequence;
- (e) a target sequence of a genetic switch encoded by the genetic switch expression sequence;
- and
- (f) a CI protein gene sequence having the target sequence operably linked thereto, the CI protein gene sequence encoding a CI protein that operates on the first promoter sequence according to the item (b)
- in a presence of an activating compound that activates the genetic switch for 5 minutes to 60 minutes with addition of 6-(β-D-2-deoxyribo-furanosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]oxazin-7-one (dP) in a first incubation stage;
- collecting viable cells that survive the first incubation stage;
- incubating the collected cells for 15 minutes to 60 minutes with addition of methanesulfonic acid (MMS) in the absence of the activating compound in a second incubation stage;
- and
- collecting viable cells that survive the second incubation stage.
13 more elements
Dependencies are inferred from the claim text.