CN114150002A

CN114150002A - Light-operated gene switch and application thereof

Info

Publication number: CN114150002A
Application number: CN202010928794.3A
Authority: CN
Inventors: 胡晓清; 吴佳欣; 王小元; 薛岚; 刘世傲; 刘昭君; 罗双华; 余克洋
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2022-03-08

Abstract

The invention discloses a light-operated gene switch and application thereof, belonging to the technical field of biology. The invention provides a light-operated gene switch, which is a recombinant plasmid for expressing a gene for coding a regulatory protein BluR, a gene for coding a blue light receptor protein BluF, an ycgZ promoter and a target gene, wherein when no blue light is irradiated, the regulatory protein BluR coded by the BluR can be combined on the ycgZ promoter to inhibit ycgZ, and when the blue light is irradiated, the blue light receptor protein BluF coded by the BluF gene can relieve the inhibition of the regulatory protein BluR on the ycgZ to start the expression of the target gene; the light-operated gene switch can control the expression quantity of the target protein by controlling the irradiation dose of blue light so as to achieve the aim of accurately controlling the expression of the target protein in time and space.

Description

Light-operated gene switch and application thereof

Technical Field

The invention relates to a light-operated gene switch and application thereof, belonging to the technical field of biology.

Background

In the field of synthetic biology, artificial precise regulation of gene expression for controlling a target protein has become an indispensable means. Currently, there are many systems for inducing protein expression through artificial regulation in the world, and the systems for inducing and regulating the expression of target proteins under specific conditions through chemical inducers or physical methods.

In recent years, the induction and regulation of gene expression systems by chemical substances has been greatly developed. However, these chemical inducers have potential problems of toxicity to the bacteria themselves, high purification cost, and serious environmental pollution. Also, when these chemical inducers are induced to express in bacteria for fermentative production, there is a possibility that they may affect the normal metabolic processes of the bacteria. In addition, it is difficult to achieve precise control of chemical substances in terms of time and space when the expression of genes is induced and controlled.

In fermentation production, a temperature-controlled molecular switch can dynamically regulate metabolic pathways through a synthetic biosensor and redirect metabolic flows through a gene loop, improving yield and productivity. However, most biosensors require extensive debugging of genetic circuit elements to adapt to a target strain under a specific fermentation condition, and thus have a large load on the microbial cells. Furthermore, high temperatures can affect the activity of certain enzymes and proteins during the growth of the strain.

Light is an ideal inducer of gene expression and can be used as a very ideal inducer due to its low toxicity, easy acquisition, easy manipulation, high spatial and temporal resolution, etc. In addition, compared with a chemical substance regulation system, the regulation system induced by a physical method is more and more concerned by people due to the advantages of convenience, no need of adding other inducers and the like. Therefore, the light is used as an inducer to regulate the expression of target protein so as to control fermentation production or disease treatment, and the application value is very high.

At present, strong ultraviolet rays have been used to release a locked transcription factor or a chemical inducer to induce gene expression, but strong ultraviolet rays are likely to cause irreversible damage to cells, and there is a difficulty in introducing the locked transcription factor into living cells. The gene expression system mediated by the heat shock effect is controlled by far infrared light to generate heat, but the far infrared laser control of the heat shock effect can activate the expression of other endogenous genes of the thallus, and the equipment is complex and expensive.

Therefore, it is urgently needed to find a method which is harmless, has low cost and can precisely regulate the expression amount of the target protein in space and time.

Disclosure of Invention

[ problem ] to

The technical problem to be solved by the invention is to provide a method which is harmless and low in cost and can accurately regulate and control the expression quantity of a target protein in space and time.

[ solution ]

In order to solve the technical problems, the invention provides a light-operated gene switch, which is characterized in that:

(a) is a recombinant plasmid for expressing the ycgZ promoter and a target gene;

(b) is a recombinant plasmid for connecting a gene encoding a blue light receptor protein bluF on (a);

(c) is a recombinant plasmid for connecting a gene encoding a regulatory protein bluR and a gene encoding a blue light receptor protein bluF on (a).

In one embodiment of the present invention, when the light-operated gene switch is (a), the gene of interest is located downstream of the ycgZ promoter; when the light-operated gene switch is (b), the target gene is located downstream of the ycgZ promoter, and the gene encoding the blue light receptor protein, bluF, is located upstream of the ycgZ promoter; when the light-dependent gene switch is (c), the gene of interest is located downstream of the ycgZ promoter, the gene encoding blue light receptor protein blu f is located upstream of the ycgZ promoter, and the gene encoding regulatory protein blu r is located upstream of the gene encoding blue light receptor protein blu f.

In one embodiment of the present invention, the recombinant plasmid is pET-28a plasmid, pCL1920 plasmid, pFT24 plasmid or pBAD33 plasmid.

In one embodiment of the invention, the ycgZ promoter is derived from a strain having NCBI accession number CP046033.1, CP014768.1, CP024695.1, CP041513.1, CP026797.1, CP026866.1, CP026814.1, CP026811.1, CP026762.1, CP026731.1, CP024470.1, CP011511.1, CP001063.1, CP032513.1, CP026795.1, CP026872.1, CP026098.1, CP041620.1, CP026875.1, CP026845.1, CP026877.1, CP026836.1, CP026846.1, CP026824.1, CP027027.1 or CP 026867.1.

In one embodiment of the invention, the nucleotide sequence of the ycgZ promoter is shown in SEQ ID No. 1.

In one embodiment of the invention, the gene encoding the regulatory protein blu is derived from a strain having NCBI accession number CP, HE, CP, LR, CP, HE, CP, LR, CP or CP.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the blue light receptor protein blu is shown in SEQ ID No. 2.

In one embodiment of the present invention, the gene encoding the blue light receptor protein bluF is derived from strains having NCBI accession No. CP051430.1, CP046033.1, CP041513.1, CP026795.1, CP026797.1, CP026814.1, CP026731.1, CP011511.1, CP001063.1, CP047094.1, CP024695.1, CP026867.1, CP026866.1, CP026762.1, CP026872.1, CP026839.1, CP024470.1, CP045522.1, CP026803.1, CP032513.1, CP026098.1, CP026875.1, CP026845.1, CP026877.1, CP044191.1, CP044151.1, CP041511.1, CP032523.1, CP029794.1, CP027027.1, CP026802.1, CP026836.1, HE616528.1, CP045526.1, CP045524.1, CP046286.1, CP045932.1, CP041322.1, CP LR 041322.1, CP041322.1, or a strain.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the blue light receptor protein bluF is shown in SEQ ID No. 3.

In one embodiment of the invention, the gene of interest is a gene encoding green fluorescent protein.

In one embodiment of the invention, the nucleotide sequence of the gene encoding green fluorescent protein is shown as SEQ ID No. 4.

The invention also provides a host cell, which carries the light-operated gene switch; alternatively, the genome of the host cell has integrated therein a gene segment comprising the ycgZ promoter and the gene of interest; alternatively, the genome of the host cell has integrated a gene segment comprising a gene encoding the blue light receptor protein, bluF, the ycgZ promoter, and the gene of interest; alternatively, the genome of the host cell has integrated a gene segment comprising a gene encoding the regulatory protein blu, a gene encoding the blue light receptor protein blu f, the ycgZ promoter and the gene of interest.

In one embodiment of the invention, when the genome of the host cell incorporates a gene segment comprising the ycgZ promoter and a gene of interest, the gene of interest is located downstream of the ycgZ promoter; when the genome of the host cell is integrated with a gene fragment containing a gene encoding blue light receptor protein bluF, the ycgZ promoter, and a gene of interest located downstream of the ycgZ promoter, the gene encoding blue light receptor protein bluF is located upstream of the ycgZ promoter; when the genome of the host cell is integrated with a gene fragment containing a gene encoding a regulatory protein blu, a gene encoding a blue light receptor protein blu, a ycgZ promoter, and a gene of interest, the gene of interest is located downstream of the ycgZ promoter, the gene encoding the blue light receptor protein blu is located upstream of the ycgZ promoter, and the gene encoding the regulatory protein blu is located upstream of the gene encoding the blue light receptor protein blu.

In one embodiment of the invention, the host cell is a bacterium or a fungus.

In one embodiment of the invention, the host cell is a gram-negative bacterium.

In one embodiment of the invention, the host cell is E.coli.

The invention also provides a method for controlling the expression quantity of the target protein, which comprises the steps of inoculating the host cell into a culture medium for fermentation to obtain fermentation liquor, and then separating the fermentation liquor to obtain the target protein; in the fermentation process, the expression amount of the target protein is controlled by controlling the irradiation dose of the blue light.

In one embodiment of the present invention, the target protein is green fluorescent protein.

The invention also provides the application of the light-operated gene switch or the host cell or the method in producing target protein.

[ advantageous effects ]

(1) The invention provides a light-operated gene switch, which is a recombinant plasmid for expressing a gene for coding a regulatory protein BluR, a gene for coding a blue light receptor protein BluF, an ycgZ promoter and a target gene, wherein when no blue light is irradiated, the regulatory protein BluR coded by the BluR can be combined on the ycgZ promoter to inhibit ycgZ, and when the blue light is irradiated, the blue light receptor protein BluF coded by the BluF gene can relieve the inhibition of the regulatory protein BluR on the ycgZ to start the expression of the target gene; the light-operated gene switch can control the expression quantity of the target protein by controlling the irradiation dose of blue light so as to achieve the aim of accurately controlling the expression of the target protein in time and space.

(2) The invention provides a method for controlling the expression quantity of a target protein, by using the method, the expression quantity of the target protein can be controlled by controlling the irradiation dose of blue light, so as to achieve the aim of accurately controlling the expression of the target protein in space and time, and the blue light irradiation has the advantages of no harm to host cells and low cost.

Drawings

FIG. 1: light-operated gene switch pET28a-P_ycgZPlasmid map of gfp.

FIG. 2: fluorescence intensity of fermentation liquor obtained by fermenting different recombinant escherichia coli.

FIG. 3: recombinant Escherichia coli BL21/pET28a-P_ycgZFluorescence intensity of the fermentation broth obtained at different times of gfp fermentation.

FIG. 4: light-operated gene switch pET28a-blu R-P_ycgZPlasmid map of gfp.

FIG. 5: fluorescence intensity of fermentation liquor obtained by fermenting different recombinant escherichia coli.

FIG. 6: light-operated gene switch pET28a-bluF-P_ycgZPlasmid map of gfp.

FIG. 7: recombinant Escherichia coli BL21/pET28a-bluF-P_ycgZFluorescence intensity of the fermentation broth obtained at different times of gfp fermentation.

Detailed Description

Coli (Escherichia coli) BL21 and E.coli (Escherichia coli) W3110 referred to in the examples below were purchased from Youbao; the pET-28a plasmid and the pWSK129 plasmid referred to in the following examples were purchased from Biovector plasmid vector cell gene collection center; the One-Step Cloning Kit referred to in the following examples was purchased from Biotech Inc. of Najing Nodezam, Inc., having a commercial number of C113-01.

The media and reagents involved in the following examples are as follows:

LB liquid medium: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract.

LB solid medium: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract and 15g/L of agar.

Example 1: light-operated gene switch pET28a-P_ycgZConstruction of-gfp

The method comprises the following specific steps:

(1) light-operated gene switch pET28a-P_ycgZConstruction of-gfp

Synthesizing a gene gfp (SEQ ID No.4) for coding green fluorescent protein; carrying out double enzyme digestion on gfp gene encoding green fluorescent protein and pET-28a plasmid through BamH I and EcoRI, and then connecting by using a One-Step Cloning Kit Clon express Multi One Step Cloning Kit to obtain a connected product; transforming the ligation product into escherichia coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 30. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 200rpm, extracting plasmids, and performing PCR verification to obtain recombinant escherichia coli BL21/pET28a-gfp and recombinant plasmid pET28a-gfp after verification is correct;

the primers used for PCR verification are as follows:

pET28a-gfp+(SEQ ID No.5)：

CAGCAAATGGGTCGCGGATCCATGAGTAAAGGAGAAGAACTTTTCACTG；

pET28a-gfp-(SEQ ID No.6)：

TTGTCGACGGAGCTCGAATTCCTATTTGTATAGTTCATCCATGCCATG。

synthesizing the ycgZ promoter (SEQ ID No.1) derived from Escherichia coli (Escherichia coli) MG 1655; the ycgZ promoter and the recombinant plasmid pET28a-gfp were digested by Bgl I and Xba I, and then ligated by using the One-Step Cloning Kit Clon express Multi S One Step Cloning Kit to obtainTo the ligation product; transforming the ligation product into escherichia coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 12.5. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 200rpm, extracting plasmids for PCR verification, and obtaining recombinant escherichia coli BL21/pET28a-P after verification is correct_ycgZGfp and the recombinant plasmid pET28a-P_ycgZGfp (plasmid map see fig. 1, nucleotide sequence of plasmid SEQ ID No. 7);

the primers used for the one-step cloning are as follows:

pET28a-P_ycgZ-gfp promoter+(SEQ ID No.8)

CGGCGTAGAGGATCGAGATCTTCTTTTAATACCAAAACATAACC；

pET28a-P_ycgZ-gfp promoter-(SEQ ID No.9)

AACAAAATTATTTCTAGAGCTACGCCTCTGTTAAA；

the primers used for PCR validation were as follows:

pET28a-P_ycgZ-gfp promoter test+(SEQ ID No.10)：

CTCTCCCTTATGCGACTCCT；

pET28a-P_ycgZ-gfp promoter test-(SEQ ID No.11)：

GCCAACTAGTGTTCAACTTTTTCCA。

(2) light-operated gene switch pET28a-P_ycgZFirst verification of gfp

Recombinant Escherichia coli BL21/pET28a-gfp was used as a control, and recombinant Escherichia coli BL21/pET28a-P was used_ycgZInoculating a gfp single colony into an LB liquid culture medium, performing shake culture for 3h under the conditions of 37 ℃, 200rpm and natural illumination, and fermenting for 15h under the conditions of 37 ℃, 200rpm and blue light illumination or 37 ℃, 200rpm and darkness respectively to obtain a fermentation liquid; wherein, the blue light is emitted by an 415nm LED lamp, and the illumination frequency is 15min off/15 min on.

Detecting the recombinant Escherichia coli BL21/pET28a-gfp and the recombinant Escherichia coli BL21/pET28a-P by an enzyme-labeling instrument_ycgZFluorescence intensity (excitation wavelength) of fermentation broth obtained by gfp fermentation488nm, emission wavelength 516nm), the detection result is shown in FIG. 2.

As shown in FIG. 2, when the recombinant Escherichia coli BL21/pET28a-gfp was fermented without IPTG induction, the fluorescence intensity of the fermentation broth obtained by fermentation was 2000AU/OD₆₀₀Recombinant Escherichia coli BL21/pET28a-P_ycgZFluorescence intensity of fermentation broth from gfp fermentation 80000AU/OD₆₀₀. Thus, the light-operated gene switch pET28a-P_ycgZThe gfp can regulate the expression of the target gene gfp, and the expression level of the target gene gfp gradually increases with the increase of the illumination time.

(3) Light-operated gene switch pET28a-P_ycgZSecond verification of gfp

To prove the light-operated gene switch pET28a-P of the invention_ycgZThe gfp can control the expression quantity of the target protein, and the recombinant Escherichia coli BL21/pET28a-P_ycgZInoculating a gfp single colony into an LB liquid culture medium, performing shake culture for 3h under the conditions of 37 ℃, 200rpm and natural illumination, and fermenting for 9h under the conditions of 37 ℃, 200rpm and blue light illumination or 37 ℃, 200rpm and darkness respectively to obtain a fermentation liquid; wherein, the blue light is emitted by an 415nm LED lamp, and the illumination frequency is 15min off/15 min on.

Detection of recombinant Escherichia coli BL21/pET28a-P by enzyme labeling instrument_ycgZFluorescence intensity (excitation wavelength 488nm, emission wavelength 516nm) of fermentation broth obtained at different time of gfp fermentation, and the detection results are shown in fig. 3.

As shown in FIG. 3, fluorescence intensity/OD before blue light irradiation₆₀₀Almost the same, and at 15h on blue light irradiation, E.coli BL21/pET28a-P_ycgZFluorescence intensity of fermentation broth from gfp fermentation 80000AU/OD₆₀₀The expression level of the same strain is only 45000AU/OD under dark conditions₆₀₀。

Example 2: light-operated gene switch pET28a-blu R-blu F-P_ycgZConstruction of-gfp

The method comprises the following specific steps:

(1) light-operated gene switch pET28a-blu R-blu F-P_ycgZConstruction of-gfp

Synthesis of a Gene encoding the regulatory protein BluR derived from Escherichia coli (Escherichia coli) MG1655Gene (SEQ ID No.2) and a gene encoding the blue light receptor protein bluF (SEQ ID No.3) derived from Escherichia coli (Escherichia coli) MG 1655; carrying out double enzyme digestion on a gene for coding a regulatory protein bluR, a gene for coding a blue light receptor protein bluF and a recombinant plasmid pET28a-gfp by Bgl I and BamH I, and then connecting by using a One-Step Cloning Kit Clon express Multi One Step Cloning Kit to obtain a connection product; transforming the ligation product into escherichia coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 30. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 200rpm, extracting plasmids for PCR verification, and obtaining recombinant escherichia coli BL21/pET28a-bluR-bluF-P after verification is correct_ycgZGfp and the recombinant plasmid pET28a-blu R-blu F-P_ycgZGfp (plasmid map see fig. 4, nucleotide sequence of plasmid SEQ ID No. 12);

the primers used for the one-step cloning are as follows:

pET28a-bluR-bluF-P_ycgZ-gfp+(SEQ ID No.13)

CAGCAAATGGGTCGCGGATCCATGAGTAAAGGAGAAGAACTTTTCACTG；

pET28a-bluR-bluF-P_ycgZ-gfp-(SEQ ID No.14)

TTGTCGACGGAGCTCGAATTCCTATTTGTATAGTTCATCCATGCCATG；

the primers used for PCR validation were as follows:

pET28a-bluR-bluF-P_ycgZ-gfp test+(SEQ ID No.15)：

GTAGGTGTTCCACAGGGTAGC；

pET28a-bluR-bluF-P_ycgZ-gfp test-(SEQ ID No.16)：

CTCCCGCATTATGAGCAGTA。

(2) light-operated gene switch pET28a-blu R-blu F-P_ycgZVerification of gfp

Inoculating a single colony of recombinant escherichia coli BL21/pET28a-gfp serving as a control into an LB liquid culture medium, performing shake-flask culture for 3h at 37 ℃ under 200rpm under natural illumination, adding IPTG (isopropyl-beta-thiogalactoside) into the LB liquid culture medium until the final concentration is 1mmol/L, and continuing to perform shake-flask culture for 15h at 37 ℃ under 200rpm under natural illumination to obtain a fermentation liquid.

Recombinant Escherichia coli BL21/pET28a-P_ycgZGfp and BL21/pET28a-blu R-blu F-P_ycgZInoculating a gfp single colony into an LB liquid culture medium, performing shake culture for 3h under the conditions of 37 ℃, 200rpm and natural illumination, and fermenting for 15h under the conditions of 37 ℃, 200rpm and blue light illumination or 37 ℃, 200rpm and darkness respectively to obtain a fermentation liquid; wherein, the blue light is emitted by an 415nm LED lamp, and the illumination frequency is 15min off/15 min on.

Detecting recombinant escherichia coli BL21/pET28a-gfp and recombinant escherichia coli BL21/pET28a-P by an enzyme-labeling instrument_ycgZGfp and recombinant E.coli BL21/pET28a-blu R-blu F-P_ycgZFluorescence intensity of fermentation broth from gfp fermentation (excitation wavelength 488nm, emission wavelength 516nm), the results are shown in fig. 4.

As shown in fig. 4, both photoswitches have a regulating effect, and the protein expression level gradually increases with the increase of the illumination dose. And the two switches are compared to find that the recombinant plasmid pET28a-bluR-bluF-P_ycgZGfp effect after blue light irradiation compared to recombinant plasmid pET28a-P with only ycgZ promoter added_ycgZThe effect of gfp is more pronounced after blue light irradiation. Recombinant plasmid BL21/pET28a-blu R-blu F-P_ycgZThe fluorescence intensity of gfp can reach 100000AU/OD after 15 hours of blue light irradiation₆₀₀The above.

Example 3: light-operated gene switch pET28a-bluF-P_ycgZConstruction of-gfp

The method comprises the following specific steps:

(1) light-operated gene switch pET28a-bluF-P_ycgZConstruction of-gfp

Synthesizing a gene encoding blue light receptor protein bluF (SEQ ID No.3) derived from Escherichia coli (Escherichia coli) MG 1655; carrying out double enzyme digestion on a gene for coding a blue light receptor protein bluF and a recombinant plasmid pET28a-gfp by Bgl I and BamH I, and then connecting by using a One-Step Cloning Kit Clon express Multi One Step Cloning Kit to obtain a ligation product; transforming the ligation product into escherichia coli BL21 to obtain a transformation product; the transformation product was plated on LB solid medium (containing30μg·mL^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 200rpm, extracting plasmids for PCR verification, and obtaining recombinant escherichia coli BL21/pET28a-bluF-P after verification is correct_ycgZGfp and the recombinant plasmid pET28a-bluF-P_ycgZGfp (plasmid map see fig. 6, nucleotide sequence of plasmid SEQ ID No. 17);

the primers used for the one-step cloning are as follows:

pET28a-bluF-P_ycgZ-gfp+(SEQ ID No.18)

TGGGGCCGCCATGCCGGCTTATTTTTTCTCTGGCCACGCT；

pET28a-bluF-P_ycgZ-gfp-(SEQ ID No.19)

TTAAACAAAATTATTTCTAGAGCTACGCCTCTGTTAAAAATGTTAA；

the primers used for PCR validation were as follows:

pET28a-bluF-P_ycgZ-gfp test+(SEQ ID No.20)：

GTAGGTGTTCCACAGGGTAG；

pET28a-bluF-P_ycgZ-gfp test-(SEQ ID No.21)：

CTATTTGTATAGTTCATCCATGCCATG。

(2) light-operated gene switch pET28a-bluF-P_ycgZVerification of gfp

Recombinant Escherichia coli BL21/pET28a-bluF-P_ycgZInoculating a single colony of gfp into an LB liquid culture medium, performing shake culture for 3h under the conditions of 37 ℃, 200rpm and natural illumination, and fermenting for 6.5h under the conditions of 37 ℃, 200rpm and blue light illumination or 37 ℃, 200rpm and darkness respectively to obtain a fermentation liquid; wherein, the blue light is emitted by an 415nm LED lamp, and the illumination frequency is 15min off/15 min on.

Detection of recombinant Escherichia coli BL21/pET28a-bluF-P by enzyme-labeling instrument_ycgZFluorescence intensity of fermentation broth from gfp fermentation (excitation wavelength 488nm, emission wavelength 525nm), the results are shown in fig. 7.

As shown in FIG. 7, the fluorescence intensity of the fermentation broth obtained by fermentation of recombinant E.coli was different from the first two due to the different detection wavelengthsThe photoswitches differ. The fluorescence intensity of the fermentation liquor is 2300AU/OD when being irradiated by blue light for 6.5h₆₀₀300AU/OD only in dark conditions₆₀₀. Thus, the light-operated gene switch BL21/pET28a-bluF-P_ycgZGfp can regulate the expression of the gene of interest gfp. Furthermore, the expression level of the target gene gfp gradually increases with the increase of the illumination time, so that the light-operated gene switch BL21/pET28a-bluF-P_ycgZSensitivity to gfp is better.

Example 4: construction of other light-operated Gene switches

The method comprises the following specific steps:

based on example 1, the ycgZ promoter from Escherichia coli (Escherichia coli) MG1655 was replaced with the ycgZ promoters from different sources in table 1, and effect detection shows that the ycgZ promoters in table 1 all have similar effects when used in a light-operated gene switch expression system.

TABLE 1 sources of the ycgZ promoter and its NCBI numbering

Promoter source	NCBI number
		Salmonella sp.HNK130	CP046033.1
Shigella sp.PAMC 28760	CP014768.1
		Shigella boydii strain 192	CP024695.1
Shigella boydii strain 83-578	CP026814.1

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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ggcctttatt tcattaagta aaaaagagac tgcgtcaggt tcgttaacca gggtcatagg 480

taatagattg attgaaatca ttttatcacc gagctcaagt gcgtgtgcca tcgtgaatgc 540

aagcgcctta cttttgagat ccgctgtgta gatttccccg tctttacgct gcccaaccgc 600

tatggctgat gggctatctt catttttttg cacaatggct tcaaaagcga ttatccgccg 660

cgataaggga tcgacaatag gatgaaaggc aaagtggtcg tttatagttg gtgaaagggc 720

acaggaatca agttctttat cagatccgtc agcgataaaa agccaggagt cttcggcagg 780

gatctcgaaa taggttgatt gttcggttgc aaggacaaaa gtacgaaaaa attgtagcgc 840

tctgtcatca taagttagct gaaattttga tgtgcctttg tcgaatacgg cctgtaaaac 900

gtcatctcgc tcgtgcaggc gcaaatcaaa taattccatt cccgctttgc caaaacggcg 960

agcaggcgcg taatcgcaca gcagctcaac aatattatag tgccgtggat cctggcatat 1020

agcccgatat atcattttaa cctgttcttc cggaccttcc agaagctgga aaaaatgaga 1080

accattaaac agtaagatcc ctgttacgtc agactgcatg ttcctgcgat ttgctatcga 1140

aaccatttct tcgatttttt tgacaggttc gtcgtcacgt atatggctac gataaataag 1200

ggtggtaagc at 1212

<210> 4

<211> 717

<212> DNA

<213> Artificial sequence

<400> 4

atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60

gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120

aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180

gtcactactt tgacttatgg tgttcaatgc ttttcaagat acccagatca tatgaaacgg 240

catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc 300

aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360

aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420

ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480

atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540

cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600

ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660

cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaatag 717

<210> 5

<211> 49

<212> DNA

<213> Artificial sequence

<400> 5

cagcaaatgg gtcgcggatc catgagtaaa ggagaagaac ttttcactg 49

<210> 6

<211> 48

<212> DNA

<213> Artificial sequence

<400> 6

ttgtcgacgg agctcgaatt cctatttgta tagttcatcc atgccatg 48

<210> 7

<211> 6275

<212> DNA

<213> Artificial sequence

<400> 7

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctt cttttaatac 4980

caaaacataa ccgtttcttt acattgataa aaaatggaaa aagttgaaca ctagttggcg 5040

aaaaatcttg tatagattgt cagttaaatg atgcaatatg ttttatcata acacattgtt 5100

ttatatgcat tagcactaat tgcaaaaaat taatttatca ttctgtacac atatttcgta 5160

caagtttgct attgttactt cacttaacat tgattaacat ttttaacaga ggcgtagctc 5220

tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat 5280

catcatcaca gcagcggcct ggtgccgcgc ggcagccata tggctagcat gactggtgga 5340

cagcaaatgg gtcgcggatc catgagtaaa ggagaagaac ttttcactgg agttgtccca 5400

attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt 5460

gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta 5520

cctgttccat ggccaacact tgtcactact ttgacttatg gtgttcaatg cttttcaaga 5580

tacccagatc atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta 5640

caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag 5700

tttgaaggtg atacccttgt taatagaatc gagttaaaag gtattgattt taaagaagat 5760

ggaaacattc ttggacacaa attggaatac aactataact cacacaatgt atacatcatg 5820

gcagacaaac aaaagaatgg aatcaaagtt aacttcaaaa ttagacacaa cattgaagat 5880

ggaagcgttc aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc 5940

cttttaccag acaaccatta cctgtccaca caatctgccc tttcgaaaga tcccaacgaa 6000

aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac acatggcatg 6060

gatgaactat acaaatagga attcgagctc cgtcgacaag cttgcggccg cactcgagca 6120

ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc 6180

tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 6240

gggttttttg ctgaaaggag gaactatatc cggat 6275

<210> 8

<211> 44

<212> DNA

<213> Artificial sequence

<400> 8

cggcgtagag gatcgagatc ttcttttaat accaaaacat aacc 44

<210> 9

<211> 35

<212> DNA

<213> Artificial sequence

<400> 9

aacaaaatta tttctagagc tacgcctctg ttaaa 35

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<400> 10

ctctccctta tgcgactcct 20

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence

<400> 11

gccaactagt gttcaacttt ttcca 25

<210> 12

<211> 5495

<212> DNA

<213> Artificial sequence

<400> 12

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggct tagggggcat gaaagatgac tttataacct tgctcacccc agtgttgtaa 3240

aagttcgttt tgccttctcg ttggtgccat gcctgtccag acaatcaatg tttgcgtcgg 3300

gaacagttcc gggcgcggcg attcaatggg atcagcaaga acagaaatgt gccatcctga 3360

caatgataaa cgccatgctt ccagccacaa tcgtgctctc tcttcaacat tccacgccat 3420

cagaatggct tctttaccgg gcttacggcg catttcgaaa agcgaggttg ctgcgtattc 3480

aattaatgcg ccgtcaaaca tactgctcat aatgcgggag gtgttgtgat caagcacgag 3540

acgctggcga acaggaaggt aaacatgatt aatcaattga tctactgggt actctcgacc 3600

cagtgaaata attctcgcgc gtagtttggc aggattagcc atgcgaagaa ttgacatcat 3660

ctcttcttgc aggcggctcc agtcatcttc cgtatcctgg ctggtggttt ccagtaatgc 3720

tttaactttg cctacaggga cgccattact tatccaacgc ttgatctctt cgatgcgttg 3780

tatgtcttct tcatcaaaga gtcggtgtcc gccttcactg cgctgtggtt ttaacaaacc 3840

gtagcggcgt tgccaggccc ggagagtgac aggattaatc ccgcaacgtt cagcaacatc 3900

accaatgctg taataagcca caattcctcc ttgcggtcac aaatctccgt cgcctgtaca 3960

cgacccaata atactttgta caatatacgc taaaattgta caaagtataa ataagatttc 4020

agctaaattg gatgaaacat tatttttaat gtggattaaa tttaaacgta acgtattcat 4080

tttcacgatg atttactgaa atcatgtgaa agaatgtgct gaaaattatt ttttctctgg 4140

ccacgctatg gaagggatac cattcaattt agctttagca aacagatctt cttttaatac 4200

caaaacataa ccgtttcttt acattgataa aaaatggaaa aagttgaaca ctagttggcg 4260

aaaaatcttg tatagattgt cagttaaatg atgcaatatg ttttatcata acacattgtt 4320

ttatatgcat tagcactaat tgcaaaaaat taatttatca ttctgtacac atatttcgta 4380

caagtttgct attgttactt cacttaacat tgattaacat ttttaacaga ggcgtagctc 4440

tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat 4500

catcatcaca gcagcggcct ggtgccgcgc ggcagccata tggctagcat gactggtgga 4560

cagcaaatgg gtcgcggatc catgagtaaa ggagaagaac ttttcactgg agttgtccca 4620

attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt 4680

gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta 4740

cctgttccat ggccaacact tgtcactact ttgacttatg gtgttcaatg cttttcaaga 4800

tacccagatc atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta 4860

caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag 4920

tttgaaggtg atacccttgt taatagaatc gagttaaaag gtattgattt taaagaagat 4980

ggaaacattc ttggacacaa attggaatac aactataact cacacaatgt atacatcatg 5040

gcagacaaac aaaagaatgg aatcaaagtt aacttcaaaa ttagacacaa cattgaagat 5100

ggaagcgttc aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc 5160

cttttaccag acaaccatta cctgtccaca caatctgccc tttcgaaaga tcccaacgaa 5220

aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac acatggcatg 5280

gatgaactat acaaatagga attcgagctc cgtcgacaag cttgcggccg cactcgagca 5340

ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc 5400

tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 5460

gggttttttg ctgaaaggag gaactatatc cggat 5495

<210> 13

<211> 49

<212> DNA

<213> Artificial sequence

<400> 13

cagcaaatgg gtcgcggatc catgagtaaa ggagaagaac ttttcactg 49

<210> 14

<211> 48

<212> DNA

<213> Artificial sequence

<400> 14

ttgtcgacgg agctcgaatt cctatttgta tagttcatcc atgccatg 48

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence

<400> 15

gtaggtgttc cacagggtag c 21

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> 16

ctcccgcatt atgagcagta 20

<210> 17

<211> 5771

<212> DNA

<213> Artificial sequence

<400> 17

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggct tattttttct ctggccacgc tatggaaggg ataccattca atttagcttt 3240

agcaaacaga tctccctgaa acatctcaat tcctgcggat tcaagccaca tccactcttc 3300

tggtgttgcc acgcccatag cactgacttg aatttcaagt gatgtacagc attttatgat 3360

cgcctgaata attgcctgcc gtggcccact tttatgaaca ttggtaatca attcctgact 3420

gattttaatt ctgtcaggct ggaagcgtga caggagtaac aaaccagcaa aacctgcgcc 3480

aaaatgatca attgctacac tgataccagc agcctttagc gatttaatcg cttcggcaaa 3540

ctcatcaaac cgagatatga cttcactttc agtaaattca acgatgattt gttcaggcac 3600

cagagcattg gcctttattt cattaagtaa aaaagagact gcgtcaggtt cgttaaccag 3660

ggtcataggt aatagattga ttgaaatcat tttatcaccg agctcaagtg cgtgtgccat 3720

cgtgaatgca agcgccttac ttttgagatc cgctgtgtag atttccccgt ctttacgctg 3780

cccaaccgct atggctgatg ggctatcttc atttttttgc acaatggctt caaaagcgat 3840

tatccgccgc gataagggat cgacaatagg atgaaaggca aagtggtcgt ttatagttgg 3900

tgaaagggca caggaatcaa gttctttatc agatccgtca gcgataaaaa gccaggagtc 3960

ttcggcaggg atctcgaaat aggttgattg ttcggttgca aggacaaaag tacgaaaaaa 4020

ttgtagcgct ctgtcatcat aagttagctg aaattttgat gtgcctttgt cgaatacggc 4080

ctgtaaaacg tcatctcgct cgtgcaggcg caaatcaaat aattccattc ccgctttgcc 4140

aaaacggcga gcaggcgcgt aatcgcacag cagctcaaca atattatagt gccgtggatc 4200

ctggcatata gcccgatata tcattttaac ctgttcttcc ggaccttcca gaagctggaa 4260

aaaatgagaa ccattaaaca gtaagatccc tgttacgtca gactgcatgt tcctgcgatt 4320

tgctatcgaa accatttctt cgattttttt gacaggttcg tcgtcacgta tatggctacg 4380

ataaataagg gtggtaagca ttaacaatcc agggtaatgg gtgaggcgag agtaagacgg 4440

taacagacat atcttcttgt gtctttcttt taataccaaa acataaccgt ttctttacat 4500

tgataaaaaa tggaaaaagt tgaacactag ttggcgaaaa atcttgtata gattgtcagt 4560

taaatgatgc aatatgtttt atcataacac attgttttat atgcattagc actaattgca 4620

aaaaattaat ttatcattct gtacacatat ttcgtacaag tttgctattg ttacttcact 4680

taacattgat taacattttt aacagaggcg tagctctaga aataattttg tttaacttta 4740

agaaggagat ataccatggg cagcagccat catcatcatc atcacagcag cggcctggtg 4800

ccgcgcggca gccatatggc tagcatgact ggtggacagc aaatgggtcg cggatccatg 4860

agtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat 4920

gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa 4980

cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc aacacttgtc 5040

actactttga cttatggtgt tcaatgcttt tcaagatacc cagatcatat gaaacggcat 5100

gactttttca agagtgccat gcccgaaggt tatgtacagg aaagaactat atttttcaaa 5160

gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat 5220

agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg 5280

gaatacaact ataactcaca caatgtatac atcatggcag acaaacaaaa gaatggaatc 5340

aaagttaact tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat 5400

tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg 5460

tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt 5520

gagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa ataggaattc 5580

gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc 5640

ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact 5700

agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac 5760

tatatccgga t 5771

<210> 18

<211> 40

<212> DNA

<213> Artificial sequence

<400> 18

tggggccgcc atgccggctt attttttctc tggccacgct 40

<210> 19

<211> 46

<212> DNA

<213> Artificial sequence

<400> 19

ttaaacaaaa ttatttctag agctacgcct ctgttaaaaa tgttaa 46

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<400> 20

gtaggtgttc cacagggtag 20

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence

<400> 21

ctatttgtat agttcatcca tgccatg 27

Claims

1. A light-operated gene switch, wherein the light-operated gene switch is (a), (b), or (c):

2. The light-operated gene switch of claim 1, wherein when the light-operated gene switch is (a), the gene of interest is located downstream of the ycgZ promoter; when the light-operated gene switch is (b), the target gene is located downstream of the ycgZ promoter, and the gene encoding the blue light receptor protein, bluF, is located upstream of the ycgZ promoter; when the light-dependent gene switch is (c), the gene of interest is located downstream of the ycgZ promoter, the gene encoding blue light receptor protein blu f is located upstream of the ycgZ promoter, and the gene encoding regulatory protein blu r is located upstream of the gene encoding blue light receptor protein blu f.

3. The light-operated gene switch of claim 1 or 2, wherein the recombinant plasmid is a pET-28a plasmid, a pCL1920 plasmid, an pFT24 plasmid or a pBAD33 plasmid.

4. A light-operated gene switch as claimed in any one of claims 1 to 3, wherein the ycgZ promoter is derived from a strain having NCBI accession No. CP046033.1, CP014768.1, CP024695.1, CP041513.1, CP026797.1, CP026866.1, CP026814.1, CP026811.1, CP026762.1, CP026731.1, CP024470.1, CP011511.1, CP001063.1, CP032513.1, CP026795.1, CP026872.1, CP026098.1, CP041620.1, CP026875.1, CP026845.1, CP026877.1, CP026836.1, CP026846.1, CP026824.1, CP027027.1 or CP 026867.1.

5. A light-operated gene switch as claimed in any one of claims 1 to 4, characterized in that the gene coding for the regulatory protein blu is derived from a strain with NCBI accession number CP, HE, CP, LR, CP, or CP.

6. A light-operated gene switch as claimed in any one of claims 1 to 5, wherein the gene encoding the blue light receptor protein bluF is derived from a strain having NCBI accession number CP, HE, CP, LR, CP, or CP.

7. A host cell carrying the light-operated gene switch of any one of claims 1-6; alternatively, the genome of the host cell has integrated therein a gene segment comprising the ycgZ promoter and the gene of interest; alternatively, the genome of the host cell has integrated a gene segment comprising a gene encoding the blue light receptor protein, bluF, the ycgZ promoter, and the gene of interest; alternatively, the genome of the host cell has integrated a gene segment comprising a gene encoding the regulatory protein blu, a gene encoding the blue light receptor protein blu f, the ycgZ promoter and the gene of interest.

8. The host cell of claim 7, wherein when the genome of the host cell incorporates a gene segment comprising the ycgZ promoter and the gene of interest, the gene of interest is located downstream of the ycgZ promoter; when the genome of the host cell is integrated with a gene fragment containing a gene encoding blue light receptor protein bluF, the ycgZ promoter, and a gene of interest located downstream of the ycgZ promoter, the gene encoding blue light receptor protein bluF is located upstream of the ycgZ promoter; when the genome of the host cell is integrated with a gene fragment containing a gene encoding a regulatory protein blu, a gene encoding a blue light receptor protein blu, a ycgZ promoter, and a gene of interest, the gene of interest is located downstream of the ycgZ promoter, the gene encoding the blue light receptor protein blu is located upstream of the ycgZ promoter, and the gene encoding the regulatory protein blu is located upstream of the gene encoding the blue light receptor protein blu.

9. A method for controlling the expression level of a target protein, which comprises inoculating the host cell of claim 7 or 8 into a culture medium to ferment to obtain a fermentation broth, and then separating the fermentation broth to obtain the target protein; in the fermentation process, the expression amount of the target protein is controlled by controlling the irradiation dose of the blue light.

10. Use of the light-operated gene switch of any one of claims 1-6 or the host cell of claim 7 or 8 or the method of claim 9 for the production of a protein of interest.