Light-switchable gene expression system and the methods for controlling gene expression in prokaryotic bacterium

Abstract

Provided is an optically controlled gene expression system of prokaryotic bacterium, comprising: a) a photosensitive recombinant transcription factor encoding gene, the photosensitive recombinant transcription factor is one fusion protein comprising a first polypeptide as the DNA bonding domain and a second polypeptide as the photosensitive domain; b) a target transcription unit comprising promoter or promoter-reaction element or reaction element-promoter containing at least one reaction element recognized/bound by the first polypeptide and the nucleic acid sequence to be transcribed. Also provided is a prokaryotic expression vector comprising said optically controlled gene expression system, and a method for regulating gene expression in a prokaryotic host cell by using the optically controlled gene expression system. Also provided is a reagent kit containing different components of the optically controlled gene expression system. The optically controlled gene expression system of prokaryotic bacterium has a quick, effective and powerful induction, is safer than other inducers, is of little or no toxicity, and can control gene expression both spatially and temporally, and can regulate many life processes of prokaryotic bacterium.

Claims

1. A light-switchable gene expression system of prokaryotic bacterium comprising: a prokaryotic bacterium comprising a gene expression system, the gene expression system comprising: a) a gene encoding a photosensitive recombinant prokaryotic light-switchable transcription factor, said recombinant light-switchable transcription factor is one fusion protein including the first polypeptide as DNA-binding domain and the second polypeptide as light-switchable domain, wherein said first polypeptide is selected from helix-turn-helix DNA-binding domain, zinc finger motif or zinc cluster DNA-binding domain, leucine zipper DNA-binding domain, winged helix DNA-binding domain, winged helix-turn-helix DNA-binding domain, helix-loop-helix DNA-binding domain, high mobility family DNA-binding domain and B3 DNA-binding domain, wherein said second polypeptide is selected from LOV2 domain of Neurospora crassa VIVID, AsLOV2 domain of oat phytochrome gene 1, AuLOV domain in aureochrome1 of Stramenophile algae Vaucheria frigida, and LOV domain in PpSB1-LOV of Pseudomonas putida; and b) a target transcription unit, including promoter or promoter-reaction element or reaction element promoter containing at least one reaction element recognized/bound by the first polypeptide and a vacancy for the nucleic acid sequence to be transcribed.

2. The light-switchable gene expression system of prokaryotic bacterium according to claim 1, wherein said first polypeptide and second polypeptide are linked each other directly or operatively, and/or wherein said promoter or promoter-reaction element or reaction element-promoter and the nucleic acid sequence to be transcribed in the target transcription unit are linked each other directly or operatively.

3. The light-switchable gene expression system of prokaryotic bacterium according to claim 1, wherein said first polypeptide is selected from DNA binding domain of E. coli LexA protein, DNA binding domain of phage cI repression protein, DNA binding domain of LacI repression protein, DNA binding domain of yeast Gal4 protein and DNA binding domain of tetracycline repression protein TetR.

4. The light-switchable gene expression system of prokaryotic bacterium according to claim 1, the gene expression system further comprising a third peptide recruiting other components of RNA polymerase, said third polypeptide being linked with the first and the second polypeptides directly or via a linker peptide.

5. The light-switchable gene expression system of prokaryotic bacterium according to claim 4, wherein said third polypeptide is selected from w protein and a protein of E. coli.

6. The light-switchable gene expression system of prokaryotic bacterium according to claim 1, wherein said reaction element is a DNA motif which can be specifically recognized and bound by the first polypeptide, wherein said reaction element is selected from LexA binding element, cI binding element, LacI binding element, Gal4 binding element and TetR binding element.

7. A prokaryotic expression vector comprising the gene encoding said recombinant light-switchable transcription factor and/or the target transcription unit of said light-switchable gene expression system according to claim 1.

8. The prokaryotic expression vector according to claim 7, wherein said gene encoding said recombinant light-switchable transcription factor has a nucleotide sequence selected from SEQ. ID. NO: 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 109; said recombinant light-switchable transcription factor has an amino acid sequence selected from SEQ. ID. NO: 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 110.

9. A method for the regulation of gene expression in prokaryotic host cells with a light-switchable gene expression system of prokaryotic bacterium, comprising the steps of: a) constructing the light-switchable gene expression system according to claim 1 containing a gene to be regulated in prokaryotic expression vectors; b) introducing the prokaryotic expression vectors into the prokaryotic host cells; and c) inducing the prokaryotic host cells via illumination to express the gene being regulated.

10. The method for the regulation of gene expression according to claim 9, further comprising the selection of light source and the selection of illumination method, wherein said light source is selected from LED lamp, fluorescent lamp, laser and incandescent lamp; said illumination method is a continuous or discontinuous illumination.

11. The method for the regulation of gene expression according to claim 9, wherein said selection of light source and said selection of illumination method comprises the spatial control of the cellular gene expression level in the different locations by using scan, projection or optical molds.

12. The method for the regulation of gene expression according to claim 9, wherein the illumination method further comprises the spatial control of the cellular gene expression level in the different locations by using a printing projection film or a neutral gray film.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the principle of overlapping PCR.

(2) FIG. 2 is a schematic diagram of homologous recombination ligation.

(3) FIG. 3 shows the principle of reverse PCR.

(4) FIG. 4 is a schematic diagram of construction of T7 promoter driven expression of prokaryotic expression vectors containing the light-switchable transcription factors with different linkers. Top panel is schematic diagram of the light-switchable transcription factor fusion protein with different linkers. Bottom panel is schematic diagram of orbicular expression vectors, wherein the backbone of these vectors is pCDFDuet1.

(5) FIG. 5 is a schematic diagram of construction of Amp promoter driven expression of prokaryotic expression vectors containing the light-switchable transcription factors with different linkers. Top panel is schematic diagram of the light-switchable transcription factor fusion protein with different linkers. Bottom panel is schematic diagram of orbicular expression vectors, wherein the backbone of these vectors is pCDFDuet1.

(6) FIG. 6 is a schematic diagram of construction of prokaryotic expression vectors containing the light-switchable transcription factors with different VVD mutants, AsLOV2 or AuLOV as the second peptide. Top panel is schematic diagram of the light-switchable transcription factor fusion protein with different linkers. Bottom panel is schematic diagram of orbicular expression vectors, wherein the backbone of these vectors is pCDFDuet1.

(7) FIG. 7 is a schematic diagram of construction of prokaryotic expression vectors containing the light-switchable transcription factors with cI, Lad, Gal4 or TetR as the first peptide. Top panel is schematic diagram of the light-switchable transcription factor fusion protein with different linkers. Bottom panel is schematic diagram of orbicular expression vectors, wherein the backbone of these vectors is pCDFDuet1.

(8) FIG. 8 is a schematic diagram of construction of prokaryotic expression vectors containing the light-switchable transcription factors with co as the third peptide. Top panel is schematic diagram of the light-switchable transcription factor fusion protein with different linkers. Bottom panel is schematic diagram of orbicular expression vectors, wherein the backbone of these vectors is pCDFDuet1.

(9) FIG. 9 is a schematic diagram of construction of prokaryotic expression vectors containing the light-switchable transcription factors with a as the third peptide. Top panel is schematic diagram of the light-switchable transcription factor fusion protein with different linkers. Bottom panel is schematic diagram of orbicular expression vectors, wherein the backbone of these vectors is pCDFDuet1.

(10) FIG. 10 is a schematic diagram of construction of prokaryotic expression vectors containing the target transcription units corresponding to LexA, cI, Lad, Gal4 or TetR. Top panel is a schematic diagram of respective target transcription units, and the bottom panel is a schematic diagram of orbicular expression vectors. The backbone of these vectors is pRSETb.

(11) FIG. 11 is a schematic diagram of construction of prokaryotic expression vectors containing the target transcription units corresponding to LexA, cI, Lad, Gal4 or TetR regulated by the recombinant light-switchable transcription factors with co or a as the third peptide. Top panel is a schematic diagram of respective target transcription units, and the bottom panel is a schematic diagram of orbicular expression vectors. The backbone of these vectors is pRSETb.

(12) FIG. 12 is a schematic diagram of construction of prokaryotic expression vectors using cI repressor for the indirect regulation. Top panel is a schematic diagram of respective target transcription units, and the bottom panel is a schematic diagram of orbicular expression vectors. The backbone of the vector is pRSETb.

(13) FIG. 13 shows the mCherry expression levels regulated by illuminating JM109(DE3,sulA.sup.,LexA.sup.) cells transformed by the T7 promoter driven expression of light-switchable transcription factors with different linkers. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(14) FIG. 14 shows the mCherry expression levels regulated by illuminating JM109(DE3,sulA.sup.,LexA.sup.) cells transformed by the Amp promoter driven expression of light-switchable transcription factors with different linkers. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(15) FIG. 15 shows the LacZ expression levels regulated by light-switchable transcription factor LV-L0. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of LacZ.

(16) FIG. 16 shows the mCherry expression levels regulated by prokaryotic expression vectors containing light-switchable transcription factor LV-L0 with other three reaction element corresponding to LexA. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(17) FIG. 17 shows the mCherry expression levels regulated by illuminating JM109(DE3,sulA.sup.,LexA.sup.) cells transformed by the transcription factor expression vectors with several VVD mutants as the second peptide. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(18) FIG. 18 shows the mCherry expression levels regulated by illuminating JM109(DE3,sulA.sup.,LexA.sup.) cells transformed by the transcription factor expression vectors with cI, Lad, Gal4 or TetR as the first peptide. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(19) FIG. 19 shows the mCherry expression levels regulated by illuminating JM109(DE3,sulA.sup.,LexA.sup.) cells transformed by the transcription factor expression vectors with AsLOV2 or AuLOV as the second peptide. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(20) FIG. 20 shows the mCherry expression levels regulated by illuminating JM109(DE3,sulA.sup.,LexA) cells transformed by the transcription factor expression vectors with co as the third peptide. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(21) FIG. 21 shows the mCherry expression levels regulated by illuminating JM109(DE3,sulA.sup.,LexA.sup.) cells transformed by the transcription factor expression vectors with a as the third peptide. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(22) FIG. 22 shows the mCherry expression levels regulated by light-switchable transcription factor LV-L0 at different temperature. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(23) FIG. 23 shows the mCherry expression levels regulated by light-switchable transcription factor LV-L0 using cI repressor for the indirect regulation. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(24) FIG. 24 shows the mCherry expression levels regulated by single plasmid containing both the light-switchable transcription factor LV-L0 and target transcription unit. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(25) FIG. 25 shows the mCherry expression levels regulated by single plasmid containing both the light-switchable transcription factor LaV(wt) and target transcription unit in Bacillus subtilis cells. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(26) FIG. 26 shows the mCherry expression levels regulated by the light-switchable transcription factor LV-L0 expressed by JM109(DE3,sulA.sup., LexA::Amp-LV-L0) itself. The lateral axis is the names of co-transformed plasmids; the vertical axis is the relative expression of mCherry.

(27) FIG. 27 shows the time course the light-induced gene expression in cells expressing the recombinant light-switchable transcription factor LV-L0. The lateral axis is the time of illumination which represents the time point when the sample is transferred from light to dark; the vertical axis is the relative expression of mCherry.

(28) FIG. 28 shows the reversibility of the light-induced gene expression in cells expressing the recombinant light-switchable transcription factor LV-L0. The lateral axis is the time of illumination which represents the time point when the sample is transferred from light to dark; the vertical axis is the relative expression of mCherry.

(29) FIG. 29 shows the light dependent light-induced gene expression in cells expressing the recombinant light-switchable transcription factor LV-L0. The lateral axis is the light intensity; the vertical axis is the relative expression of mCherry.

(30) FIG. 30 is the Stop pattern obtained from taking photograph of the cells expressing the recombinant light-switchable transcription factor LV-L0 using a printed patterns projection film. The left panel is the photograph of culture dish affixed with projection film, and the right panel is the image of fluorescent cells.

(31) FIG. 31 is the white light imaging result of the regulation on bacteria mobility by the recombinant light-switchable transcription factor LV-L0, the right panel is dark condition, the right panel is light condition, 1, 2,3 represent different plasmids for co-transformation.

(32) FIG. 32 is the regulation on bacteria cell lysis by the recombinant light-switchable transcription factor LV-L0, the lateral axis is the names of co-transformed plasmids; the vertical axis is the lysis efficency.

(33) FIG. 33 is the elution result of ion-exchange chromatography using AKTA purifier. The lateral axis is the number of elution tubes; the vertical axis is the value of UV absorption.

(34) FIG. 34 is the result of 12% SDS PAGE of protein from some tubes after ion-exchange chromatography.

(35) FIG. 35 is the result of 12% SDS PAGE of the obtained protein after merging.

(36) FIG. 36 shows homology analysis of LOV domains derived from six photosensitive proteins, wherein the black color represents 100% homologous amino acid sequence, the dark grey represents the higher homologous sequences and french gray represents the medium homologous Sequences.

PREFERABLE EMBODIMENTS

(37) The invention will be described in detail by using following examples. These examples are only used for the illustration of the invention without any restriction on the scope of protection. It is not difficult for those skilled in the art to successfully implement the invention on the basis of the examples with some modifications or alternatives. All these modifications or alternatives are within the scope of the attached claims. The methods used in the samples were the routine methods of molecular biology cloning in genetic engineering and cell biology field, such as: Lab Ref: A handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench written by Roskams, J. et al, Molecular Cloning: A Laboratory Manual (the third edition, August in 2002, Science press, Beijing) written by Sambrook J and Russell D W, and translated by Peitang Huang et al.; Chapters in book Short protocols in Protein Science (Science press, background) written by Coligan J. E. et al, and translated by Shentao Li et al.

(38) pCDFDuet1 vector was purchased from Novagen company; pRSETb and pBAD/His A were purchased from Invitrogen company; pKD3, pKD, pCP20 and pKD46 were gifts from Prof. Jie Bao of East China University of Science and Technology. All the primers were synthesized, purified and identified via mass spectrometry by Shanghai Generay Biotech Co. Ltd. All the vectors obtained in the examples were verified via sequencing by BGI and JIE LI Biology Company. Taq DNA polymerase used in the examples was purchased from DongSheng Biotech Company; pfu DNA polymerase was purchased from TianGen Biotech (Beijing) Co. LtD., and PrimeStar DNA polymerase was purchased from TaKaRa. All the three polymerases contained corresponding buffer and dNTP when purchased. Restriction enzyme such as BamHI, BglII, HindIII, NdeI, XhoI, SacI, EcoRI, SpeI et al., T4 ligase and T4 phosphatase (T4 PNK) were purchased, together with 10 Tango buffer, from Fermentas. CloneEZ PCR clone kit was purchased from GenScript (Nanjing). Unless otherwise mentioned, inorganic salt chemical reagents were purchased from Sinopharm Chemical Reagent Co.; Kanamycin, Ampicillin and ONPG were purchased from Ameresco; 384 well white plates for luminescence detection and 384 well black plates for fluorescene detection were purchased from Grenier.

(39) The kit for DNA purification was purchased from BBI (Canada); common plasmid kit was purchased from TianGen Biotech (Beijing) Co. LtD.; E. coli strain Mach1 was purchased from Invitrogen; E. coli strain JM109 (DE3) was purchased from Promega; E. coli strain BL21 (DE3) was purchased from Novagen;

(40) Main equipments: Biotek Synergy 2 multi-mode microplate reader (BioTek, US), X-15R high speed refriger (Beckman, US), Microfuge22R high speed refriger (Beckman, US), PCR Amplifier (Biometra, Germany), In-Vivo Multispectral System FX (Kodak, US), Luminometer (Sanwa, Japan), electrophoresis apparatus (shanghai Biocolor BioScience &Technolgy Co.).

(41) The meaning of abbreviations: h=hour, min=minute, s=second, L=microliter, mL=milliliter, L=liter, bp=base pair, mM=millimole, M=Micromolar.

(42) 20 Amino Acids and Abbreviations

(43) TABLE-US-00001 Abbreviation Abbreviation Name by three letters by one letter Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Proline Pro P Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W Serine Ser S Threonine Thr T Cysteine Cys C Methionine Met M Asparagine Asn N Glutamine Gln Q Asparagic acid Asp D Glutamate Glu E Lysine Lys K Arginine Arg R Histidine His H
Methods Used in the Examples

(44) (1) Polymerase Chain Reaction (PCR):

(45) 1. Amplification of Gene Fragment by PCR:

(46) TABLE-US-00002 The reaction system of PCR amplification of target gene Template 0.5-1 L Forward primer (25 M) 0.5 L Reverse primer (25 M) 0.5 L 10 pfu buffer 5 L Pfu DNA polymerase 0.5 L dNTP (10 mM) 1 L ddH.sub.2O 41.5-42 L Total volume 50 L

(47) Amplification Process (Bp Represents the Number of Nucleotide being Amplified):

(48) TABLE-US-00003 Process of PCR amplification of target gene denaturation 95 C. 2-10 min 94-96 C. 30-45 s 30 cycles {open oversize brace} 50-65 C. 30-45 s 72 C. bp/(600 bp/min) extension 72 C. 10 min

(49) 2. PCR Amplification of Long Fragment (>2500 bp):

(50) TABLE-US-00004 Reaction system of PCR amplification of long fragment(>2500 bp) template (10 pg-1 ng) 1 L Forward primer (25 M) 0.5 L Reverse primer (25 M) 0.5 L 5 PrimerSTAR buffer 10 L PrimerSTAR DNA polymerase 0.5 L dNTP (2.5 mM) 4 L ddH.sub.2O 33.5 L Total volume 50 L
Amplification Process (bp Represents the Number of Nucleotide being Amplified):

(51) TABLE-US-00005 Process of PCR amplification of long fragment denaturation 95 C. 5 min 98 C. 10 s 30 cycles {open oversize brace} 50-68 C. 5-15 s 72 C. bp/(1000 bp/min) extension 72 C. 10 min

(52) Or

(53) TABLE-US-00006 Process of PCR amplification of long fragment denaturation 95 C. 5 min 30 cycels 98 C. 10 s {open oversize brace} 68 C. bp /(1000 bp/min) extension 72 C. 10 min

(54) (2) Reaction System of Restriction Enzyme

(55) 1. The System of Double Digestion of Plasmid (n Represents the Required ddH.sub.2O to Reach the Total Volume (L):

(56) TABLE-US-00007 The system of double digestion of plasmid plasmid 20 L (about 1.5 g) 10 buffer 5 L restriction enzyme 1 1-2 L restriction enzyme 2 1-2 L ddH.sub.2O n L Total volume 50 L Reaction condition 37 C., 1~7 h

(57) 2. The System of Double Digestion of PCR Fragment (n Represents the Same Meaning as Above):

(58) TABLE-US-00008 The system of double digestion of PCR fragment PCR fragment 15-25 L(about1 g) 10 buffer 5 L restriction enzyme 1 1-2 L restriction enzyme 2 1-2 L ddH.sub.2O n L Total volume 50 L Reaction condition 37 C., 1~7 h

(59) 3. The System of Ligating the PCR Fragment into Plasmid by Double Digestion:

(60) TABLE-US-00009 Ligation system DNA of PCR fragment after double 1-7 L digestion Digested plasmids 0.5-7 L 10 T4 ligase buffer 1 L T4 DNA ligase 1 L ddH.sub.2O N L Total volume 10 L Reaction condition 16 C., 4~8 h Note: The ratio of PCR fragment to digested plasmid is about 2:1-6:1.

(61) (3) Cyclization Reaction of DNA Fragment after Phosphorylation at the 5 End:

(62) The terminal of plasmid or genome from microorganism has phosphate group, but PCR product has no, so addition reaction of phosphate group at the 5 end of PCR product is necessary for the ligation of DNA molecular. Cyclization reaction refers to ligation of the 3 and 5 ends of linearized fragment.

(63) TABLE-US-00010 Reaction system of phosphorylation DNA of PCR product 5-8 L 10 T4 ligase buffer 1 L T4 PNK 1 L ddH.sub.2O 0-3 L Total volume 10 L Reaction condition 37 C., 30 min~2 h

(64) T4 PNK is the abbreviation of polynueleotide kinase which is used for addition reaction of phosphate group at the 5 end of DNA molecular. Cyclization reaction system of DNA fragment with 5 end phosphorylation:

(65) TABLE-US-00011 Cyclization reaction system Phosphorylation product 10 L T4 ligase (5 U/L) 0.5 L Total volume 10.5 L Reaction condition 16 C., 4~16 h

(66) (4) Overlapping PCR

(67) Overlapping PCR is commonly used in ligating two different genes. Such as FIG. 1, to ligate gene AD with gene BC, two pairs of primers A and D, C and B are used to amplify gene AD and gene BC, the 5 end of primer D and primer C contains certain length of complementary sequences. The amplified products AD and BC of the first round are used as the template of the second round after recovery. 10 cycles of conventional PCR progress is carried out from the second round, the PCR system is:

(68) TABLE-US-00012 Reaction system of PCR amplification of target gene AD 1 L BC 1 L 10 pfu buffer 5 L Pfu DNA polymerase 0.5 L dNTP (10 mM) 1 L ddH2O 39.5 L Total volume 48 L
Addition of primer A and primer B after the second round, additional 30 cycles of amplification is carried out to obtain the ligation product of AD and BC.

(69) (5) Reverse PCR

(70) Reverse PCR technology is used in the sample for site mutagenesis, truncation mutagenesis and insertion mutagenesis. The basic principle is based on the experiment progress of MutaBEST kit from Takara Company. As is shown in FIG. 3, reverse PCR primers are located at the mutation site; 5 end of one of the primers contains the mutation sequence. The amplification product undergoes purification, phosphorylation at the 5 end, cyclization, and then is transformed into competent cells.

(71) (6) Preparation of Competent Cells and Transformation

(72) Preparation of Competent Cells:

(73) 1. Pick single clone (such as Mach1) into 5 ml LB medium, culture at 37 C. overnight.

(74) 2. Transfer 0.5-1 ml of the overnight cultures to 50 ml LB medium, culture at 220 rpm/min for 3-5 h to reach OD6000.5;

(75) 3. Incubate the cells on ice for 2 h;

(76) 4. Centrifuge the cells at 4000 rpm/min for 10 min at 4 C.

(77) 5. Discard the supernatant, resuspend cells with 5 mL of ice cold suspension buffer, mix completely and then add 45 ml of the suspension buffer after;

(78) 6. Keep the cells on ice for 45 min;

(79) 7. Centrifuge the cells at 4000 rpm/min for 10 min at 4 C., resuspend cells using 5 mL of ice stock buffer;

(80) 8. Dispense 100 uL to sterile Eppendorf vials. Snap-freeze in dry ice or 80 C.

(81) Suspension buffer: CaCl.sub.2) (100 mM), MgCl.sub.2(70 mM), NaAc (40 mM)

(82) Stock buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10CaCl.sub.2) (1M), 1 mL 10MgCl.sub.2 (700 mM), 1 mL 10NaAc (400 mM), 4.6 mL ddH.sub.2O

(83) Transformation:

(84) 1. take 100 ul competent cells to thaw on ice;

(85) 2. Add the ligation product, mix and incubate on ice for 30 min. Usually, the volume of ligation product should be less than 1/10 of competent cells;

(86) 3. Heat shock at 42 C. for 90 s, rapidly transfer the cells to ice for 5 min;

(87) 4. Add 500 l LB and grow in 37 C. shaking incubator for 1 h.;

(88) 5. Centrifuge the cells at 4000 rpm/min for 3 min, resuspend cells using the remained 200 l supernatant, and plate the cells onto a plate containing the appropriate antibiotic. Incubate plates at 37 C. overnight.

(89) (7) Determination of mCherry Fluorescent Protein Expressed by E. coli

(90) Single clones on the transformation plate is picked into 48-well plate, each well contains 700 l LB, each sample has six replicates, the cells grow overnight at 30 C. The cells are diluted 200 folds into two 48-well plates containing fresh LB. Unless otherwise mentioned, the culture condition is 30 C., the speed of shaking incubator is 280 rpm/min, light intensity is 0.125 mW/cm.sup.2. Cells are harvested by 4000 rpm for 20 min after 18 h. The supernatant is discarded and 200 l PBS is added into each well, the cells are resuspended using the agitator. 5 l of the cells is added into 96-well white plate, then 115 l PBS is added into each well and mix completely, determine the OD600 using Biotek Synergy 2 multi-mode microplate reader. The OD600 of each well is adjusted to the same according to the OD600 value from the reader. After adjustment, 100 l of the cells is added into 96-well plate for fluorescence determinant, the fluorescence of the cells is determined using Biotek Synergy 2 multi-mode microplate reader using the filters Ex590/20 and Em645/40. The dark samples are wrapped by aluminum foil; other manipulation methods are the same.

(91) (8) Determination of -Galactosidase Activity

(92) Single clones on the transformation plate is picked into 48-well plate, each well contains 700 l LB, each sample has six replicates, the cells grow overnight at 30 C. The cells are diluted 200 folds into two 48-well plates containing fresh LB, the cells are cultured in the light or in the dark at 30 C., the light intensity of blue light is 0.125 mW/cm.sup.2. 5 l of the cells is added to the 96-well white plate after 18 h, 20 l of the membrane permeable solution is added into each well, shake the 96-well plate using agitator for 1 min and incubate the cells at 37 C. for 10 min. 150 l of the substrate solution is added and the kinetics of OD420 is determined after 30 s shaking. The obtained slope of the curve is proportional to LacZ activity.

(93) Preparation of Solutions for Determination of LacZ Activity:

(94) Substrate solution: Na.sub.2HPO.sub.4 60 mM, NaH.sub.2PO.sub.4 40 mM, ONPG 1 mg/ml, -mercaptoethanol 2.7 l/ml.

(95) Membrane permeable solution: Na.sub.2HPO.sub.4 100 mM, KCl 20 mM, MgSO.sub.4 2 mM, CTAB 0.8 mg/ml, sodium deoxycholate 0.4 mg/ml, -mercaptoethanol 5.4 l/ml.

(96) (9) Gene Knock-Out on E. coli Genome

(97) 1. transform pKD46 vector into the target strain, culture on the plate containing Ampicillin at 30 C. overnight.

(98) 2. Pick the single clone into 5 mL LB medium containing Ampicillin and culture overnight;

(99) 3. The overnight cells are diluted 100 folds into 50 ml 2YT medium and culture at 30 C., add L-arabinose (final concentration is 30 mM) when the OD600 reaches about 0.2-0.3, culture at 30 C. for induction for 90 min;

(100) 4. The cells after induction are placed on ice for 1 h, centrifuge the cells at 4000 rpm for 10 min at 4 C. and discard the supernatant, resuspend the cells with 20 ml ice-cold ddH.sub.2O, centrifuge the cells at 4000 rpm for 10 min at 4 C., repeat this process for 4 times. Resuspend the cells using 1.5 ml ddH.sub.2O for the last time, dispense 80-100 uL to each sterile Eppendorf vials.

(101) 5. Adding 10 l linearized fragment used for knock-out into the competent cells, rapidly mix and add into the electroporation cuvette, put the electroporation cuvette in the electropolator for electrotransformation, add 500 l fresh LB immediately after electrotransformation, recover the cells at 37 C. for 1-2 h.

(102) 6. Centrifuge the cells at 4000 rpm and plate the cells onto a plate containing the appropriate antibiotic. Incubate plates at 37 C. overnight; identify the positive clone the next day.

(103) (10) Elimination of the Antibiotics Gene from the Genome of E coli

(104) 1. Transform pCP20 into the strain that is ready to eliminate the antibiotics gene, culture at 30 C. overnight.

(105) 2. Pick the single clone from the transformation plate into Eppendorf tube containing fresh LB (no antibiotics), culture the cells at 37 C. for 8 h for heat induction;

(106) 3. Transfer the Eppendorf tube to 42 C. and grow overnight to remove pCP20 plasmid, plate little cells onto a plate containing no antibiotics and grow at 37 C. overnight.

(107) 4. Identify the positive clone using corresponding primers.

(108) (11) Transformation of Bacillus subtilis WB800

(109) 1. Grow Bacillus subtilis WB800 in a 3 mL LB broth overnight.

(110) 2. Transfer 2.6 ml of the overnight culture to 40 ml medium (LB+0.5 M sorbic alcohol) and culture at 37 C. with 200 rpm shaking to OD600=0.850.95.

(111) 3. Incubate the cells on ice for 10 min; centrifuge the cells at 5000 g for 5 min to harvest the cells.

(112) 4. Resuspend the cells with 50 ml ice-cold transformation medium (0.5 Msorbic alcohol, 0.5 Mmannitol, 10% glucose), 4 C. centrifuge the cells at 5000 g for 5 min, discard the supernatant. Repeat this process for 4 times.

(113) 5. Resuspend the cells with 1 ml transformation medium; dispense 120 L to each sterile Eppendorf vials.

(114) 6. Add 50 ng DNA (18 l) into 60 L of the competent cells, incubate on ice for 2 min, add the cells into pre-cooled transformation medium, pulse once.

(115) 7. remove the cuvette and immediately add 1 ml RM (LB+0.5 Msorbic alcohol+0.38 M mannitol) to the cuvette, incubate the cells at 37 C. with 200 rpm shaking for 3 h, plate aliquots of the cells on plate. Incubate the plate at 37 C. overnight.

(116) Preparation of the Solutions:

(117) 40 ml (LB+0.5 M sorbic alcohol): typtone 10 g/l, yeast extract 5 g/l, NaCl 10 g/1, 3.6 g sorbic alcohol pH=7.2

(118) 10 ml RM (0.5 M M sorbic alcohol, 0.38 M mannitol): 0.9 g sorbic alcohol, 0.7 g mannitol.

(119) Two 50 ml centrifuge tubes, 0.22 M filter.

Example 1: Construction of E. coli Strains JM109(DE3,sulA,LexA), JM109(DE3, sulA,LexA,CheZ), JM109(DE3,sulA,LexA,), and JM109(DE3,sulA,LexA::Amp-LV-L0)

(120) The linearized fragment containing the homologous arm of sulA gene at the ends used in sulA gene knock-out was amplified from pKD3 plasmid by PCR using primer P1, P2, P3 and P4 (SEQ ID NO: 112). The knock-out of sulA gene was carried out on the basis of JM109(DE3) strain, the resulted transitional strain was JM109(DE3,sulA::Cam). The linearized fragment containing the homologous arm of LexA gene at the ends used in LexA gene knock-out was amplified from pKD4 plasmid by PCR using primer P5, P6, P7 and P8 (SEQ ID NO: 113). The knock-out of LexA gene was carried out on the basis of JM109 (DE3, sulA::Cam) transitional strain, the resulted transitional strain was JM109(DE3, sulA::Cam,LexA::kan). The Cam and Kan resistance genes of JM109 (DE3,sulA::Cam,LexA::kan) strain were removed, resulting in JM109(DE3,sulA.sup.,LexA.sup.) strain.

(121) The linearized fragment containing the homologous arm of CheZ gene at the ends used in CheZ gene knock-out (SEQ ID NO: 114) was amplified from pKD4 plasmid by PCR using primer P9, P10, P11 and P12. The linearized fragment containing the homologous arm of w gene at the ends used in w gene knock-out (SEQ ID NO: 115) was amplified from pKD4 plasmid by PCR using primer P9, P10, P11 and P12. The knock-out of CheZ and w genes were carried out on the basis of JM109(DE3,sulA.sup.,LexA.sup.) strain, resulting in JM109(DE3,sulA.sup.,LexA.sup.,CheZ::kan) and JM109(DE3,sulA.sup.,LexA.sup.,::kan) strains, respectively. The kan resistance gene in the genome of these two strains was removed, resulting JM109 (DE3,sulA.sup.,LexA.sup.,.sup.) strain.

(122) TABLE-US-00013 Primers for the amplification of the linearized fragment used in sulA gene-knock: Forward primer 1 (P1): 5-TAACTCACAGGGGCTGGATTGATTGTGTAGGCTGGAGCTGCTT-3 Forward primer 2 (P2): 5-GATGTACTGTACATCCATACAGTAACTCACAGGGGCTGGATT-3 Reverse primer 1 (P3): 5-TTCCAGGATTAATCCTAAATTTACATGGGAATTAGCCATGGTC-3 Reverse primer 2 (P4): 5-CATTGGCTGGGCGACAAAAAAAGTTCCAGGATTAATCCTAAATT-3 Primers for the amplification of the linearized fragment used in LexA gene-knock: Forward primer 1 (P5): 5-CAACAAGAGGTGTTTGATCTCATCCTGAGCGATTGTGTAGGCTG-3 Forward primer 2 (P6): 5-GAAAGCGTTAACGGCCAGGCAACAAGAGGTGTTTGAT-3 Reverse primer 1 (P7): 5-ACGACAATTGGTTTAAACTCGCCATATGAATATCCTCCTTAG-3 Reverse primer 2 (P8): 5-GAAGCTCTGCTGACGAAGGTCAACGACAATTGGTTTAAACTC-3 Primers for the amplification of the linearized fragment used in CheZ gene-knock: Forward primer 1 (P9): 5-GGTCACGCCACATCAGGCAATACAAATGAGCGATTGTGTAGGCTG- 3 Forward primer 2 (P10): 5-CTTATCAGACCGCCTGATATGACGTGGTCACGCCACATCAGGCAA- 3 Reverse primer 1 (P11): 5-AACTGGGCATGTGAGGATGCGACTCATATGAATATCCTCCTTAG-3 Reverse primer 2 (P12): 5-AGGAAAAACTCAACAAAATCTTTGAGAAACTGGGCATGTGAGGATG- 3 Primers for the amplification of the linearized fragment used in gene-knock: Forward primer 1 (P13): 5-GTAACCGTTTTGACCTGGTACTGTGAGCGATTGTGTAGGCTG-3 Forward primer 2 (P14): 5-AGGACGCTGTAGAGAAAATTGGTAACCGTTTTGACCTGGT-3 Reverse primer 1 (P15): 5-AATTCAGCGGCTTCCTGCTCTTGCATATGAATATCCTCCTTAG-3 Reverse primer 2 (P16): 5-GCAATAGCGGTAACGGCTTGTAATTCAGCGGCTTCCTGCTC-3

(123) To obtain the JM109(DE3,sulA.sup.,LexA::Amp-LV-L0) containing said recombinant light-switchable transcription factor LV-L0 encoding cassette, pALV-L0 was amplified by PCR using primers P17 and P18, kan gene fragment containing kan resistance gene encoding cassette was amplified from pKD4 by PCR using primers P19, P20, P21 and P22, and inserted into the linearized pALV-L0 by the same double digestion, the resulting vector was named as pALV-L0-kan. Amp-LV-L0-kan fragment amplified from pALV-L0-kan by PCR using primers P23, P24, P25 and P26, the nucleotide sequence is SEQ. ID. No:116. The knock-out was carried out on the basis of JM109(DE3, sulA.sup.) strain, resulting in JM109(DE3,sulA.sup.,LexA::AmpLV-L0-kan) strain whose kan resistance was removed from its genome to obtain JM109(DE3,sulA.sup.,LexA::Amp-LV-L0) strain.

(124) TABLE-US-00014 Primers for the amplification of pALV-L0 vector: Forward primer (P17): 5-CCCCTCGAGCTGCCACCGCTGAGCAATAACT-3 Reverse primer (P18): 5-CCCGAATTCTCATTCCGTTTCGCACTGGAA-3 Primers for the amplification of kan resistance gene: Forward primer (P19): 5-CCCGAATTCGCGATTGTGTAGGCTGGAGCTGC-3 Reverse primer 1 (P20): 5-CTTTTGCTGTATATACTCATGAATATCCTCCTTAGTTC-3 Reverse primer 2 (P21): 5-GTTTATGGTTCCAAAATCGCCTTTTGCTGTATATACTCAT-3 Reverse primer 3 (P22): 5-GGGCTCGAGGTTTATTGTGCAGTTTATGGTTCCAAAATCG-3 Primers for the amplification of pALV-L0-kan vector: Forward primer 1 (P23): 5-ATTGGTTTAAACTCGCTATTTTCGTGCGCGGAACCCCTATTTG-3 Forward primer 2 (P24): 5-CTGCTGACGAAGGTCAACGACAATTGGTTTAAACTCGCTA-3 Forward primer 3 (P25): 5-GAAGCTCTGCTGACGAAGGTCAACG-3 Reverse primer (P26): 5-GTTTATTGTGCAGTTTATGGTTCCAAAATC-3

Example 2 Construction of Prokaryotic Bacterium Expression Vectors Containing T7 Promoter Driven Expression of Different Linkers of LexA (1-87)-VVD36(C71V)

(125) Gene fragment encoding 1-87 amino acid of LexA was amplified from the genome of JM109(DE3) using primers P27 and P28. VVD36 (C71V) gene fragment was amplified from pGAVP(C71V) plasmid (preserved by our lab, the corresponding paper: Wang, X. et al, Nat Methods, 2012.) using primers P29 and P30, LexA(1-87)-VVD36 (C71V) gene fragment was obtained by fusing LexA (1-87) to VVD36 (C71V) using overlaping PCR. pCDFDuet1 was amplified using P31 and P32, the obtained linearized vector was ligated with LexA(1-87)-VVD36(C71V) gene fragment by XhoI and EcoRI double digestion, the resulting vector was named as pLV-L0 containing the gene of recombinant light-switchable transcription factor LexA(1-87)-VVD36(C71V) (abbreviated to LV-L0, SEQ. ID. No:48 (polynucleotide) and 49 (polypeptide)). On the basis of pLV-L0, the linker between LexA(1-87) and VVD36(C71V) was replaced using primers P33, P34, P35, P36, P37, P38, P39 and P40, the resulting plasmids containing seven different linkers (L1, L2, L3, L4, L5, L6 and L7) of the fusion protein LexA(1-87)-VVD36(C71V) were named as pLV-L1. pLV-L2, pLV-L3, pLV-L4, pLV-L5, pLV-L6 and pLV-L7, respectively (FIG. 4). The fusion protein polynucleotide encoding sequences are SEQ. ID. No: 50, 52, 54, 56, 58, 60 and 62; the amino acid sequences are SEQ. ID. No: 51, 53, 55, 57, 59, 61 and 63.

(126) TABLE-US-00015 Primers for the amplification of LexA (1-87) gene fragment: Forward primer (P27): 5-CCCCTCGAGCATGAAAGCGTTAAC-3 Reverse primer (P28): 5-CGGTTCACCGGCAGCCACACGACCTACCAG-3 Primers for the amplification of VVD36 (C71V) gene fragment: Forward primer (P29): 5-CTGCCGGTGAACCGCATACGCTCTACGCTCCCGGCG-3 Reverse primer (P30): 5-CCCGAATTCTCATTCCGTTTCGCACTGGAA-3 Primers for the amplification of pCDFDuet1 vector: Forward primer (P31): 5-CCCGAATTCCTGCCACCGCTGAGCAATAACT-3 Reverse primer (P32): 5-GGGCTCGAGCCCTGGCTGTGGTGATGATGGTG-3 Primers for changing the linkers of LexA (1-87)-VVD36 (C71V): The common reverse primer (P33): 5-CGGTTCACCGGCAGCCACACGACCTACCAG-3 Forward primer for linker 1 (P34): 5-TGTCGTGGGCATACGCTCTACGCTCCCGGC-3 Forward primer for linker 2 (P35): 5-GTGTTTCATACGCTCTACGCTCCCGGC-3 Forward primer for linker 3 (P36): 5-TATAAGCATACGCTCTACGCTCCCGGC-3 Forward primer for linker 4 (P37): 5-GGATCCCATACGCTCTACGCTCCCGGC-3 Forward primer for linker 5 (P38): 5-GAACCTCATACGCTCTACGCTCCCGGC-3 Forward primer for linker 6 (P39): 5-CTGGCCGAGGCCGCTGCCCATACGCTCTACGCTCCCGGC-3 Forward primer for linker 7 (P40): 5-ACCGAGTTCCCCGGCGTGGACCAGCATACGCTCTACGCTCCCGGC- 3

Example 3 Construction of Prokaryotic Bacterium Expression Vectors Containing Amp Promoter Driven Expression of Different Linkers of LexA(1-87)-VVD36(C71V)

(127) Amp promoter fragment was amplified from pRSETb vector by PCR using primers P41 and P42 (SEQ. ID. No:136 (polynucleotide)), pCDFDuet1 vector was amplified by PCR using P43 and P44, the obtained linearized fragment was ligated with Amp promoter fragment by NdeI and XhoI double digestion, the resulting plasmids was named as pAmp. LexA(1-87)-VVD36 (C71V) fragments containing different linkers constructed in example 2 were amplified using primers P45 and P46, the resulting LexA(1-87)-VVD36 (C71V) fragments containing different linkers were ligated into pAmp by NdeI and XhoI double digestion, the resulting plasmids were named as pALV-L0, pALV-L1, pALV-L2, pALV-L3, pALV-L4, pALV-L5, pALV-L6 and pALV-L7 containing the genes of recombinant protein LexA(1-87)-VVD36 (C71V) with eight different linkers (L0, L1, L2, L3, L4, L5, L6, L7) (FIG. 5, SEQ. ID. No: 48, 50, 52, 54, 56, 58, 60, 62 (polynucleotide) and 49, 51, 53, 55, 57, 59, 61, 63 (polypeptide)).

(128) TABLE-US-00016 Primers for amplification of Amp promoter: Forward primer (P41): 5-GGCTGCAGGTGCGCGGAACCCCTATTTG-3 Reverse primer (P42): 5-GGCTCGAGTACTCATATGCTTCCTTTTTCAA-3 Primers for amplification of pCDFDuet1 vector: Forward primer (P43): 5-GGCTGCAGGTGCGCGGAACCCCTATTTG-3 Reverse primer (P44): 5-GGCTCGAGTACTCATATGCTTCCTTTTTCAA-3 Primers for amplification of LexA (87)-VVD36(C71V) containing different linkers: Forward primer (P45): 5-GATTCCATATGAAAGCGTTAACGGCC-3 Reverse primer (P46): 5-CCCCTCGAGTCATTCCGTTTCGCACTGGAA-3

Example 4 Construction of Prokaryotic Bacterium Expression Vectors Containing the Recombinant Light-Switchable Transcription Factor with Different VVD Mutants, Phot1-LOV2, and Aurochrome as the Second Polypeptide

(129) pALV-L0 vector was amplified by reverse PCR using primers P47 and P48, the resulting transitional vector was named as pALV-L0(wt). Prokaryotic bacterium expression vectors containing genes of the recombinant protein LexA(1-87)-VVD36 with VVD mutants N56K or Y50W or N56K C71V or I52A C71V or I52S C71V or N56R C71V were constructed by reverse PCR using primers P49 and P50, P51 and P52 for pALV-L0(wt) vector, P53 and P54, P55 and P56, P57 and P58, P59 and P60 for pALV-L0, the resulting vectors were named as pALV-L0 (N56K), pALV-L0 (Y50W), pALV-L0 (N56K C71V), pALV-L0 (I52A C71V), pALV-L0 (I52S C71V), and pALV-L0 (N56R C71V), respectively (FIG. 6, SEQ. ID. No: 64, 66, 68, 70, 72, 74, (polynucleotide) and 65, 67, 69, 71, 73, 75 (polypeptide)).

(130) TABLE-US-00017 Primers for amplification of pALV-L0 (wt): Forward primer (P47): 5-GCTCTGATTCTGTGCGACCTGAAGC-3 Reverse primer (P48): 5-GCATGACGTGTCAACAGGTCCCAGTTC-3 Primers for amplification of VVD36 (N56K) mutant: Forward primer (P49): 5-GAGGCCAAACCCCCAAGTAGAACTG-3 Reverse primer (P50): 5-TTCATAATCTGAATCAGATAGCCCAT-3 Primers for amplification of VVD36 (Y50W) mutant: Forward primer (P51): 5-GCTGATTCAGATTATGAACAGGCC-3 Reverse primer (P52): 5-CAGCCCATAATGTCATAACCGCCGGG-3 Primers for amplification of VVD36 (N56K C71V) mutant: Forward primer (P53): 5-GCTGATTCAGATTATGAAGAGGCCAAACC-3 Reverse primer (P54): 5-CAGCCCATAATGTCATAACCGCCGGGAG-3 Primers for amplification of VVD36 (I52S C71V) mutant: Forward primer (P55): 5-TCCCAGATTATGAACAGGCCAAACCC-3 Reverse primer (P56): 5-CAGATAGCCCATAATGTCATAACCG-3 Primers for amplification of VVD36 (I52A C71V) mutant: Forward primer (P57): 5-GCGCAGATTATGAACAGGCCAAACCC-3 Reverse primer (P58): 5-CAGATAGCCCATAATGTCATAACCG-3 Primers for amplification of VVD36 (N56R C71V) mutant: Forward primer (P59): 5-GCCAAACCCCCAAGTAGAACTGGGAC-3 Reverse primer (P60): 5-CTGCGCATAATCTGAATCAGATAGC-3

(131) For constructing the expression vector containing light-switchable transcription factor with LOV2 domain of phototropin1 (abbreviated to AsLOV2, a kind gift from Gardner lab, The University of Texas at Dallas) as the second peptide, AsLOV2 gene fragment was amplified from cDNA using primers P61 and P62, LexA(1-87) gene fragment was amplified from pALV-L0 vector constructed in example 3 using primers P63 and P64, then AsLOV2 gene fragment and LexA(1-87) gene fragment were fused using overlapping PCR, the obtained recombinant gene fragment LexA(1-87)-AsLOV2 was ligated with pALV-L0 vector constructed in example 3 by NdeI and XhoI double digestion, the resulting vector was named as pALA containing recombinant protein LexA(1-87)-AsLOV2 (abbreviated to LA, SEQ. ID. No: 76 (polynucleotide) and 77 (polypeptide)).

(132) TABLE-US-00018 Primers for amplification of AsLOV2 gene fragment: Forward primer (P61): 5-GCTGCCGGTGAACCGTCCTTCTTGGCTACTACACTTGAAC-3 Reverse primer (P62): 5-ACGGGCTCGAGAATAAGTTCTTTTGCCGCCTC-3 Primers for amplification of LexA (1-87) gene fragment: Forward primer (P63): 5-GATTCCATATGAAAGCGTTAACGGCC-3 Reverse primer (P64): 5-ATCGGTTCACCGGCAGCCACACGACCTAC-3

(133) For constructing the expression vector containing light-switchable transcription factor with LOV2 domain of aurochrome (abbreviated to AuLOV, a kind gift from Hironao Kataoka lab, Ritsumeikan University) as the second peptide, AuLOVgene fragment was amplified from cDNA using primers P65 and P66, and then was ligated with LexA(1-87) in this example using overlapping PCR, the obtained recombinant gene fragment LexA(1-87)-AsLOV2 was ligated with pALV-L0 vector constructed in example 3 by NdeI and XhoI double digestion, the resulting vector was named as pALAu containing recombinant protein LexA(1-87)-AuLOV (abbreviated to LAu, SEQ. ID. No: 78 (polynucleotide) and 79 (polypeptide)).

(134) TABLE-US-00019 Forward primer (P65): 5-GCTGCCGGTGAACCGTCCTTCTTGGCTACTACACTTGAAC-3 Reverse primer (P66): 5-CTACTACACACACGAAGTTCTTTTGCCGCCTC-3

Example 5 Construction of Prokaryotic Bacterium Expression Vectors Containing the Light-Switchable Transcription Factors with cI, LacI, Gal4, and TetR as the First Polypeptide

(135) For constructing the expression vector containing light-switchable transcription factor with cI(1-102) as the first polypeptide, cI(1-102) gene fragment was amplified from the genome of phage by PCR using primers P67 and P68, the obtained fragment was ligated with pALV-L4 by NdeI and BamHI double digestion, the resulting vector was named as pACV containing recombinant light-switchable transcription factor with cI as the first peptide (FIG. 7), the corresponding recombinant light-switchable transcription factor was abbreviated to CV, whose polynucleotide and polypeptide sequence are SEQ. ID. No: 80 and SEQ. ID. No: 81, respectively.

(136) TABLE-US-00020 Primers for amplification of cI (1-102): Forward primer (P67): 5-GGCGCATATGTCTACCAAGAAGAAACC-3 Reverse primer (P68): 5-CCCGGATCCATATTCTGACCTCAAAGACG-3

(137) For constructing the expression vector containing light-switchable transcription factor with lad (1-62) as the first polypeptide, the gene fragment of DNA binding domain (1-62 amino acid) was amplified from pCDFDuet1 vector (Novagen company) using primers P69 and P70 and ligated with pALV-L4 by NdeI and BamHI double digestion, the resulting transitional vector was named as pALaV(wt). pALaV (wt) was then amplified from pALaV (wt) vector by PCR using primers P71 and P72, the linearized vector fragment was phosphorylated and ligated to obtain the expression vector named as pALaV containing the recombinant light-switchable transcription factor with Lad (1-62) as the first peptide (FIG. 7), the corresponding recombinant light-switchable transcription factor was abbreviated to LaV, whose polynucleotide and polypeptide sequence are SEQ. ID. No: 82 and SEQ. ID. No: 83, respectively.

(138) TABLE-US-00021 Primers for amplification of LacI (1-62): Forward primer (P69): 5-GGCGCATATGAAACCAGTAACGTTATAC-3 Reverse primer (P70): 5-CCCGGATCCCAACGACTGTTTGCCCGCC-3 Primers for amplification of pALaV vector: Forward primer (P71): 5-CGTTTCCAACGTGGTGAACCAGGCC-3 Reverse primer (P72): 5-CGTTTCCAACGTGGTGAACCAGGCC-3

(139) For constructing the expression vector containing light-switchable transcription factor with Gal4(1-65) as the first polypeptide, gene fragment of Gal4(1-65) DNA binding domain was amplified from pBIND vector (Promega company) using primers P73 and P74, the obtained fragment was ligated with pALV-L4 by NdeI and BamHI double digestion, the resulting vector was named as pAGV containing recombinant light-switchable transcription factor with Gal4(1-65) as the first peptide (FIG. 7), the corresponding recombinant light-switchable transcription factor was abbreviated to GV, whose polynucleotide and polypeptide sequence are SEQ. ID. No: 84 and SEQ. ID. No: 85, respectively.

(140) TABLE-US-00022 Primers for amplification of Gal4 (1-65): Forward primer (P73): 5-GGCGCATATGAAGCTACTGTCTTCTATC-3 Reverse primer (P74): 5-CCCGGATCCTTCCAGTCTTTCTAGCCTTG-3

(141) For constructing the E. coli expression vector containing light-switchable transcription factor with TetR(1-63) as the first polypeptide, gene fragment of TetR DNA binding domain (1-63 amino acid) synthesized by Shanghai Generay Biotech Co. Ltd. was amplified by PCR using primers P75 and P76, and then was ligated with pALV-L4 by NdeI and BamHI double digestion, the resulting vector was named as pATV containing recombinant light-switchable transcription factor with TetR(1-63) as the first peptide (FIG. 7), the corresponding recombinant light-switchable transcription factor was abbreviated to TV, whose polynucleotide and polypeptide sequence are SEQ. ID. No: 86 and SEQ. ID. No: 87, respectively.

(142) TABLE-US-00023 Primers for amplification of TetR (1-63): Forward primer (P75): 5-GGCGCATATGTCTAGGCTAGATAAGAG-3 Reverse primer (P76): 5-CCCGGATCCGTGTCTATCCAGCATCTCG-3

Example 6 Construction of Prokaryotic Bacterium Expression Vectors Containing the Light-Switchable Transcription Factors with and as the Third Polypeptide

(143) gene fragment was amplified from the genome of BL21(DE3) using primers P77 and P78, w-linker gene fragment was amplified by overlapping PCR using primers P77, P79 and P80, LV-L0 gene fragment was amplified from pALV-L0 constructed in example 3 using primers P81 and P82, the gene fragment of recombinant protein -linker-LV-L0 (abbreviated to AX, SEQ. ID. No:90 (polynucleotide) and SEQ. ID. No:91 (polypeptide)) was obtained by fusing -linker to LV-L0 gene fragment by overlapping PCR, and then was ligated with pALV-L0 vector constructed in example 3 by NdeI and XhoI double digestion, the resulting vector was named as pALV. CV, LaV, GV and TV gene fragments were amplified from pACV, pALaV, pAGV and pATV vectors constructed in example 5 using primers P82 and P83, P82 and P84, P82 and P85, P82 and P86, and then were ligated with pALV vector by SpeI and XhoI double digestion, the resulting plasmids were named as pACV, pALaV, pAGV and pATV, respectively (FIG. 8). These recombinant transcription factors were abbreviated to CV, LaV, GV and TV respectively, the polynucleotide sequences are SEQ. ID. No: 94, 98, 102, 106 and polypeptide sequences are SEQ. ID. No: 95, 99, 103 and 107.

(144) TABLE-US-00024 Primers for amplification of gene fragment: Forward primer 1 (P77): 5-GCGGCATATGGCACGCGTAACTGTTC-3 Reverse primer 2 (P78): 5-TCCTTGTAGTCCGCGGCCGCACGACCTTCAGCAATAG-3 Primers for amplification of -linker gene fragment: Forward primer (P79): 5-CATGGGGGGGTGTCTTGGAACCGGTCCGGAACTTGTCGTCGTC ATCCTTGTAGTCCGCG-3 Reverse primer 2 (P80): 5-CGTTAACGCTTTCATACTAGTGTGGGGGGGTGTCTTGG-3 Primers for amplification of LV-L0 gene fragment: Forward primer (P81): 5-ATGAAAGCGTTAACGGCCAGGCAAC-3 Reverse primer (P82): 5-CCCGAATTCTCATTCCGTTTCGCACTGGAA-3 Primers for amplification of CV, LaV, GV and TV gene fragments: Forward primer (P83): 5-GGGACTAGTATGAGCACAAAAAAGAAACC-3 Forward primer (P84): 5-GGGACTAGTATGAAACCAGTAACGTTA-3 Forward primer (P85): 5-CCCACTAGTATGAAGCTACTGTCTTCTATC-3 Forward primer (P86): 5-CCCACTAGTATGTCTAGGCTAGATAAGAGC-3

(145) For construction of prokaryotic bacterium expression vectors containing the light-switchable transcription factors with a as the third polypeptide, LV-L0 gene fragment was amplified from pALV-L0 vector constructed in example 3 using primers P87 and P88, LV-L0-linker was obtained by overlapping PCR using primers P87, P79 and P89, gene fragment was amplified from the genome of BL21(DE3) using primers P90 and P91, LV-L0-linker gene fragment and a gene fragment were fused by overlapping PCR to obtain LV-L0-linker- gene fragment (abbreviated to LV, SEQ. ID. No:88 (polynucleotide) and 89 (polypeptide)) which was ligated with pALV-L0 vector by NdeI and XhoI double digestion, the resulting vector was named as pALV. CV, LaV, GV and TV gene fragments were amplified from pACV, pALaV, pAGV and pATV vectors constructed in example 5 using primers P88 and P92, P88 and P93, P88 and P94, P88 and P95, and then were ligated with pALV vector by NdeI and SpeI double digestion, the resulting plasmids were named as pACV, pALaV, pAGV and pATV, respectively (FIG. 9). These recombinant transcription factors were abbreviated to CV, LaV, GV and TV respectively, the polynucleotide sequences are SEQ. ID. No: 92, 96, 100, 104 and polypeptide sequences are SEQ. ID. No: 93, 97, 101 and 105.

(146) TABLE-US-00025 Primers for amplification of LV-L0 gene fragment: Forward primer 1 (P87): 5-GATTCCATATGAAAGCGTTAACGGCC-3 Reverse primer 2 (P88): 5-CCTTGTAGTCCGCGGCCGCACTAGTTTCCGTTTCGCACTGGAA-3 Primers for amplification of LV-L0-linker gene fragment: Reverse primer (P89): 5-CCTGCATGGTACCGTGGGGGGGTGTCTTGGA-3 Primers for amplification of gene fragment: Forward primer 1 (P90): 5-CATGGTACCATGCAGGGTTCTGTGACAGAG-3 Reverse primer 2 (P91): 5-GCCCTCGAGTTACTCTGGTTTCTCTTCTTTC-3 Primers for amplification of CV, LaV, GV and TV gene fragments: Forward primer (P92): 5-GGGCATATGAGCACAAAAAAGAAACC-3 Forward primer (P93): 5-GGGCATATGAAACCAGTAACGTTA-3 Forward primer (P94): 5-CCCCATATGAAGCTACTGTCTTCTATC-3 Forward primer (P95): 5-CCCCATATGTCTAGGCTAGATAAGAGC-3

Example 7 Construction of Prokaryotic Bacterium Expression Vectors Containing Target Transcription Units with the Reaction Elements of LexA, cI, LacI, Gal4 and TetR

(147) To detect the effect of recombinant light-switchable transcription factor with LexA as the first peptide on the transcriptional regulation of mCherry fluorescent protein and LacZ galactosidase genes, prokaryotic bacterium expression vector containing the target transcription unit with LexA reaction element and fluorescent protein reporter gene was constructed. colE promoter fragment was amplified by overlapping PCR using primers P96, P97, P98, P99 and P100; mCherry gene fragment was amplified from pU5-mCherry vector (preserved by our lab, the corresponding paper: Wang, X. et al, Nat Methods, 2012.) using primers P101 and P102. rrnB transcription termination fragment was amplified from pBAD/His A vector by PCR using primiers P103 and P104; there three fragments were amplified by overlapping PCR to obtain colE-mCherry-rrnB gene fragment. pRSETb vector was amplified by primers P105 and P106, the obtained linearized fragment was ligated with colE-mCherry-rrnB gene fragment by BamHI and XhoI double digestion, the resulting transitional plasmid was amplified by primers P107 and P108, the obtained linearized fragment was digested by KpnI and NheI and then ligated with rrnB fragment amplified by primers P109 and P110, the resulting prokaryotic bacterium expression vector pB-colE-mCherry contains the target transcription unit with LexA reaction element and mCherry fluorescent protein reporter gene, the polynucleotide sequence is SEQ. ID. No:117. LacZ gene was amplified from the genome of BL21(DE3) using primers P111 and P112, pB-colE-mCherry vector was amplified using primers P113 and P114, the obtained LacZ gene fragment and linearized pB-colE-mCherry fragment were ligated after HindIII and BglII double digestion, the resulting prokaryotic bacterium expression vector pB-colE-LacZ contains the target transcription unit with LexA reaction element and LacZ 13 galactosidase gene, the polynucleotide sequence is SEQ. ID. No:118. sulA, RecA and umuDC fragments were amplified from the genome of JM109(DE3) strain using primers P115 and P116, P117 and P118, P119 and P120, and then were fused with mCherry gene fragment obtained in this sample, the resulting fragments sulA-mCherry, RecA-mCherry and umuDC-mCherry were ligated with pB-colE-mCherry by BamHI and EcoRI double digestion to generate other three prokaryotic bacterium expression vectors pB-sulA-mCherry, pB-umuDC-mCherry and pB-RecA-mCherry containing the target transcription unit with LexA reaction element and mCherry fluorescent protein gene, respectively (FIG. 10), the corresponding polynucleotide sequences are SEQ. ID. No:119, 120 and 121.

(148) TABLE-US-00026 Primers for amplification of colE promoter fragment: Forward primer 1(P96): 5-GATCGTTTTCACAAAAATGGAAGTCCACAGTCTTGACAGGGAAAA TGCAGCGGCGTAG-3 Forward primer 2 (P97): 5-GGGGATCCTGTTTTTTTGATCGTTTTCACAAAAAT-3 Reverse primer 1 (P98): 5-TATAAAATCCTCTTTGACTTTTAAAACAATAAGTTAAAAATAAATA CTGTACATATAAC-3 Reverse primer 2(P99): 5-CGCCCTTGCTCACCATTATAAAATCCTCTTTGAC-3 Reverse primer 3(P100): 5-CGCCCTTGCTCACCATTATAAAATCCTCTTTGAC-3 Primers for amplification of mCherry gene fragment: Forward primer (P101): 5-ATGGTGAGCAAGGGCGAGGAGCTGTTC-3 Reverse primer (P102): 5-GGGGAATTCTTACTTGTACAGCTCGTCCAT-3 Primers for amplification of rrnB transcription terminator: Forward primer (P103): 5-CAAGTAAGAATTCCCCCTGTTTTGGCGGATGAGAG-3 Reverse primer (P104): 5-CAAGTAAGAATTCCCCCTGTTTTGGCGGATGAGAG-3 Primers for amplification of pRSET vector: Forward primer (P105): 5-GACCTCGAGCGCAGCCTGAATGGCGAATG-3 Reverse primer (P106): 5-CGGGATCCATTTCGCGGGATCGAGATC-3 Primers for amplification of the transitional vector: Forward primer (P107): 5-CCCGCTAGCGGATCCATAGGGTTGATCTT-3 Reverse primer (P108): 5-GGGGGTACCATTTCGCGGGATCGAGA-3 Primers for amplification of rrnB transcription terminator: Forward primer (P109): 5-CCCGGTACCCCCCTGTTTTGGCGGATGAGAG-3 Reverse primer (P110): 5-CCCGCTAGCGCAAACAACAGATAAAACGAAA-3 Primers for amplification of LacZ gene: Forward primer (P111): 5-CCCAAGCTTATGGTCGTTTTACAACGTCGTG-3 Reverse primer (P112): 5-CCTAGATCTTTATTTTTGACACCAGACCAAC-3 Primers for amplification of pB-colE-mCherry vector: Forward primer (P113): 5-CCCAGATCTCCCCTGTTTTGGCGGATGAGAGAAG-3 Reverse primer (P114): 5-CCCAAGCTTATCCTCTTTGACTTTTAAAACAAT-3 Primers for amplification of sulA promoter: Forward primer (P115): 5-CCCGGATCCATAGGGTTGATCTTTGTTG-3 Reverse primer (P116): 5-GCCCTTGCTCACCATAATCAATCCAGCCCCTGTG-3 Primers for amplification of RecA promoter: Forward primer (P117): 5-CCCGGATCCCAATTTCTACAAAACACTTGATACT-3 Reverse primer (P118): 5-CGCCCTTGCTCACCATTTTTACTCCTGTCATGCCGGG-3 Primers for amplification of umuDC promoter: Forward primer (P119): 5-CCCGGATCCGCCTATGCAGCGACAAATATT-3 Reverse primer (P120): 5-CGCCCTTGCTCACCATAATAATCTGCCTGAAGTTATA-3

(149) To detect the effect of recombinant light-switchable transcription factor with cI as the first peptide on the transcriptional regulation of mCherry fluorescent protein gene, prokaryotic bacterium expression vector containing the target transcription unit with cI reaction element and fluorescent protein reporter gene was constructed. P.sub.O12 promoter fragment was amplified by overlapping PCR using primers P121, P122, P123, P124 and P125, mCherry fragment was amplified from pB-colE-mCherry vector using primers P126 and P127, P.sub.O12-mCherry gene fragment was obtained by fusing P.sub.O12 promoter fragment to mCherry gene fragment using overlapping PCR, and then was ligated with pB-colE-mCherry vector by BamHI and EcoRI double digestion, the resulting prokaryotic bacterium expression vector pB-P.sub.O12-mCherry contains the target transcription unit with cI reaction element and mCherry fluorescent protein gene (FIG. 10), the polynucleotide sequence is SEQ. ID. No:122.

(150) TABLE-US-00027 Primers for amplification of P.sub.O12 promoter: Forward primer 1 (P121): 5-TATCTAACACCGTGCGTGTTGACTATTTTACCTCTG-3 Forward primer 2 (P122): 5-CCCGGATCCTATCTAACACCGTGCGTG-3 Reverse primer 1 (P123): 5-GCAACCATTATCACCGCCAGAGGTAAAATAGT-3 Reverse primer 2 (P124): 5-AGTACCTCCTTAGTACATGCAACCATTATCACCG3 Reverse primer 3 (P125): 5-GCCCTTGCTCACCATACTAGTACCTCCTTAGTAC-3 Primers for amplification of mCherry gene fragment: Forward primer (P126): 5-ATGGTGAGCAAGGGCGAGGAGCTGTTC-3 Reverse primer (P127): 5-GGGGAATTCTTACTTGTACAGCTCGTCCAT-3

(151) To detect the effect of recombinant light-switchable transcription factor with Lad, Gal4 or TetR as the first peptide on the transcriptional regulation of mCherry fluorescent protein gene, prokaryotic bacterium expression vector containing the target transcription unit with Lad, Gal4 or TetR reaction element and fluorescent protein reporter gene was constructed. T7 promoter-lac operator fragment was amplified from pCDFDuet1 vector using primers P128 and P129, the obtained fragment was ligated with pB-P.sub.O12-mCherry vector by BamHI and SpeI double digestion, the resulting transitional vector was named as pB-T7lacop-mCherry (wt). pB-T7lacop-mCherry(wt) vector was amplified by PCR using primers P130 and P131, P132 and P133, P134 and P135, the obtained linearized vector fragments were phosphorylated by T4 PNK and ligated to obtain the prokaryotic expression vectors pB-T7lacop-mCherry, pB-T7galop-mCherry, pB-T7tetop-mCherry containing the target transcription unit with Lad, Gal4 or TetR reaction element, respectively (FIG. 10). The polynucleotide sequences are SEQ. ID. No:123, 124, 125.

(152) TABLE-US-00028 Primers for amplification of T7 promoter-lac operator: Forward primer (P128): 5-CCCGGATCCGGAAATTAATACGACTCACTA-3 Reverse primer (P129): 5-GGGACTAGTTCTCCTTATTAAAGTTAAAC-3 Primers for amplification of the vector containing the target transcription unit with LacI reaction element: Forward primer (P130): 5-CTAAAAATTCCCCTGTAGAAATAATTTTGTT-3 Reverse primer (P131): 5-CGCTAAAAATTCCCCTATAGTGAGTCGTATTA-3 Primers for amplification of the vector containing the target transcription unit with Gal4 reaction element: Forward primer (P132): 5-GTCCTCCGCCCCTGTAGAAATAATTTTGT-3 Reverse primer (P133): 5-AGTACTCCGCCCCTATAGTGAGTCGTATTAA-3 Primers for amplification of the vector containing the target transcription unit with TetR reaction element: Forward primer (P134): 5-TGATAGAGACCCCTGTAGAAATAATTTTG-3 Reverse primer (P135): 5-CTGATAGGGACCCCTATAGTGAGTCGTATTAA-3

Example 8 Construction of Prokaryotic Bacterium Expression Vectors Containing the Target Transcription Units with the Reaction Elements of LexA, cI, LacI, Gal4 or TetR and mCherry Fluorescent Protein Gene Corresponding to Recombinant Light-Switchable Transcription Factor with LexA, cI, LacI, Gal4 and TetR as the First Peptide, with and as the Third Peptide

(153) For construction of prokaryotic bacterium expression vectors containing the target transcription unit of the light-switchable transcription factors with and as the third polypeptide, lac minimal promoter was amplified using primers P136, P137 and P138, pB-colE-mCherry constructed in example 7 was amplified using primers P139 and P140, the linearized fragment was ligated with lac minimal promoter by BamHI and HindIII double digestion, the resulting transitional vector was named as pB-lac-mCherry. pB-lac-mCherry vector was amplified using primers P141 and P142, P143 and P144, P145 and P146, P147 and P148, P149 and P150, the obtained linearized fragment was phosphorylated and ligated to generate prokaryotic bacterium expression vectors containing the target transcription units with the reaction elements and mCherry fluorescent protein gene corresponding to recombinant light-switchable transcription factor with LexA, cI, lacI, Gal4 and TetR as the first peptide, with and as the third peptide, these vectors were named as pB-lexAop-lac-mCherry, pB-o12-lac-mCherry, pB-lacop-lac-mCherry, pB-galop-lac-mCherry and pB-tetop-lac-mCherry (FIG. 11), the target transcription unit sequences are SEQ. ID. No:126, 127, 128, 129 and 130.

(154) TABLE-US-00029 Primers for amplification of lac minimal promoter: Forward primer 1 (P136): 5-AGGCACCCCGGGCTTTACACTTTATGCTTCCGGCTCGTATGTTG TGTCGACCGAGCGGAT-3 Forward primer 2 (P137): 5-CCGGATCCCATTAGGCACCCCGGGCTTTACA-3 Reverse primer (P138): 5-CCAAGCTTTTCCTGTGTGAAAGTCTTATCCGCTCGGTCGAC-3 Primers for amplification of pB-colE-mCherry vector: Forward primer (P139): 5-CCCAAGCTTATGGTGAGCAAGGGCGAGGAG-3 Reverse primer (P140): 5-ACAGGATCCGCTAGCGCAAACAACAGATAAAAC-3 Primers for PCR amplification of pB-lac-mCherry vector to generate the prokaryotic bacterium plasmids containing LexA target transcription unit: Forward primer (P141): 5-GTTATATGTACAGTACCATTAGGCACCCCGGGCTTT-3 Reverse primer (P142): 5-CACTGGTTTTATATACAGGGATCCGCTAGCGCAAACAA-3 Primers for PCR amplification of pB-lac-mCherry vector to generate the prokaryotic bacterium plasmids containing cI target transcription unit: Forward primer (P143): 5-TATTTTACCTCTGGCGGTGATAATGCATTAGGCACCCCGGGCTTT- 3 Reverse primer (P144): 5-GTCAACACGCACGGTGTTAGATAGGATCCGCTAGCGCAAACAA-3 Primers for PCR amplification of pB-lac-mCherry vector to generate the prokaryotic bacterium plasmids containing lacI target transcription unit: Forward primer (P145): 5-GCTCACAATTCATTAGGCACCCCGGGCTTT-3 Reverse primer (P146): 5-GCTCACAATTGGATCCGCTAGCGCAAACAA-3 Primers for PCR amplification of pB-lac-mCherry vector to generate the prokaryotic bacterium plasmids containing Gal4 target transcription unit: Forward primer (P147): 5-CGGAGTACTGTCCTCCGCATTAGGCACCCCGGGCTTT-3 Reverse primer (P148): 5-CTCGGAGGACAGTACTCCGGGATCCGCTAGCGCAAACAA-3 Primers for PCR amplification of pB-lac-mCherry vector to generate the prokaryotic bacterium plasmids containing TetR target transcription unit: Forward primer (P149): 5-CTCCCTATCAGTGATAGAGACATTAGGCACCCCGGGCTTT-3 Reverse primer (P150): 5-CTCTCTATCACTGATAGGGAGGATCCGCTAGCGCAAACAA-3

Example 9 Construction of Prokaryotic Bacterium Expression Vectors Containing cI Repressor for the Indirect Regulation

(155) For construction of prokaryotic bacterium expression vectors containing cI repressor for the indirect regulation, rrnB-colE-mCherry gene fragment obtained in example 7 was amplified using primers P151 and P152, pRSETb vector was amplified using primers P153 and P154, The obtained linearized fragment was ligated with rrnB-colE-mCherry gene fragment by KpnI and BglII double digestion to generate the transitional vector pB-rrnB-colE-mCherry. ColE promoter was amplified using primers P155 and P156, cI gene fragment (SEQ. ID. No:131 (polynucleotide) and 132 (polypeptide)) was amplified from the genome of phage using primers P157 and P158, colE-cI fragment was obtained by overlapping PCR and ligated with pB-rrnB-colE-mCherry vector by BamHI and BglII double digestion to generate the transitional vector pB-rrnB-colE-cI. P.sub.O12 promoter fragment was amplified by overlapping PCR using primers P159, P160, P161, P162 and P163, mCherry fragment was amplified from pB-colE-mCherry vector using primers P164 and P165, rrnB transcription terminator fragment was amplified using primers P166 and P167, P.sub.O12-mCherry-rrnB gene fragment was obtained by overlapping PCR. pB-rrnB-colE-mCherry constructed in this sample was amplified using primers P168 and P169, the obtained linearized fragment was ligated with P.sub.O12-mCherry-rrnB by SacI and XhoI double digestion to generate the indirect regulation vector pB-colE-cI-P.sub.O12-mCherry (FIG. 12), the polynucleotide sequence of the target transcription unit is SEQ. ID. No:133.

(156) TABLE-US-00030 Primers for amplification of rrnB-colE-mCherry gene fragment: Forward primer (P151): 5-CCCGGTACCCCCCTGTTTTGGCGGATGAGAG-3 Reverse primer (P152): 5-GGGAGATCTTTACTTGTACAGCTCGTCCAT-3 Primers for amplification of pRSETb vector: Forward primer (P153): 5-CGAAGCTTGAAGATCTGCTTGATCCGGCTGCAAAC-3 Reverse primer (P154): 5-GGGGGTACCATTTCGCGGGATCGAGA-3 Primers for amplification of colE promoter: Forward primer (P155): 5-GGGGATCCTGTTTTTTTGATCGTTTTCACAAAAAT-3 Reverse primer (P156): 5-TATAAAATCCTCTTTGACTTTTAAA-3 Primers for amplification of cI gene fragment: Forward primer (P157): 5-AAAGAGGATTTTATAATGAGCACAAAAAAGAAACC-3 Reverse primer (P158): 5-GGGAGATCTTTAGCCAAACGTCTCTTCAGG-3 Primers for amplification of P.sub.O12 promoter: Forward primer 1 (P159): 5-TATCTAACACCGTGCGTGTTGACTATTTTACCTCTG-3 Forward primer 2 (P160): 5-CCCGAGCTCTATCTAACACCGTGCGTG-3 Reverse primer 1 (P161): 5-GCAACCATTATCACCGCCAGAGGTAAAATAGT-3 Reverse primer 2 (P162): 5-AGTACCTCCTTAGTACATGCAACCATTATCACCG-3 Reverse primer 3 (P163): 5-GCCCTTGCTCACCATACTAGTACCTCCTTAGTAC-3 Primers for amplification of mCherry gene fragment: Forward primer (P164): 5-ATGGTGAGCAAGGGCGAGGAGCTGTTC-3 Reverse primer (P165): 5-GGGGAATTCTTACTTGTACAGCTCGTCCAT-3 Primers for amplification of rrnB transcription terminator: Forward primer (P166): 5-CAAGTAAGAATTCCCCCTGTTTTGGCGGATGAGAG-3 Reverse primer (P167): 5-GGGCTCGAGCAAACAACAGATAAAACGAAAGG-3 Primers for amplification of pB-rrnB-colE-mCherry vector: Forward primer (P168): 5-GACCTCGAGCGCAGCCTGAATGGCGAATG-3 Reverse primer (P169): 5-CAGGAGCTCCAACTGTTGGGAAGGGCGATC-3

(157) For construction of prokaryotic bacterium expression vectors containing cI repressor for the indirect regulation and CheZ or SRRz gene cassette as the reporter gene, CheZ gene and SRRz gene cassette were amplified from the genome of JM109(DE3) and phage using primers P170 and P171, P172 and P173, respectively, the obtained gene fragments were ligated with pB-colE-cI-P.sub.O12-mCherry constructed in this sample by SpeI and EcoRI double digestion to generate the indirect regulation vectors pB-colE-cI-P.sub.O12-CheZ and pB-colE-cI-P.sub.O12-SRRz (FIG. 12), the polynucleotide sequences of the target transcription unit are SEQ. ID. No:134 and 135.

(158) TABLE-US-00031 Primers for amplification of CheZ gene fragment: Forward primer (P170): 5-CCCACTAGTATGATGCAACCATCAATCAAACCTG-3 Reverse primer (P171): 5-GGGGAATTCTCAAAATCCAAGACTATCCAA-3 Primers for amplification of SRRz gene cassette: Forward primer (P172): 5-CCCACTAGTATGAAGATGCCAGAAAAACATGACC-3 Reverse primer (P173): 5-CCCGAATTCTAGGCATTTATACTCCGCTGGA-3

(159) For large scale production of sulfhydryl oxidase Ero1 using the light-switchable gene expression system of prokaryotic bacterium, the vector containing the indirect regulation of cI and Ero1 as the reporter gene, Ero1(56-424) gene fragment was amplified from the genome of Saccharomyces cerevisiae BY4741 using primers P174 and P175, and was then ligated with pB-colE-cI-P.sub.O12-mCherry constructed in this sample by SpeI and EcoRI double digestion to generate the indirect vector pB-colE-cI-P.sub.O12-Ero1, the polynucleotide and polypeptide sequences of Ero1(56-424) are SEQ. ID. No:46 and 47.

(160) TABLE-US-00032 Primers for amplification of Ero1 (56-424) gene fragment: Forward primer (P174): 5-CCCACTAGTATGTTCAATGAATTAAATGC-3 Reverse primer (P175): 5-CCCGAATTCTTATAACCTTTTCCCGTAC-3

Example 10 Construction of Single Expression Vector Containing Light-Switchable Transcription Factor and Target Transcription Unit

(161) For construction of single expression vector containing both light-switchable transcription factor and target transcription unit. Amp-LV-L0 fragment was amplified from pALV-L0 vector constructed in example 3 using primers P176 and P177 and ligated with pB-colE-mCherry-P.sub.O12-mCherry constructed in example 9 by SacI and EcoRI double digestion, the resulting transitional vector was named as pB-colE-mCherry-Amp-LV-L0. pB-colE-mCherry-Amp-LV-L0 was double digested by KpnI and XhoI and ter-colE-mCherry-Amp-LV-L0-ter fragment was recovered, pCDFDuet1 vector was amplified by PCR using primers P178 and P179, the obtained linearized fragment was ligated with colE-mCherry-Amp-LV-L0 fragment by KpnI and XhoI double digestion, the resulting single vector containing both light-switchable transcription factor and target transcription unit was named as pD-colE-mCherry-Amp-LV, the polynucleotide sequence of ter-colE-mCherry-Amp-LV-ter is SEQ. ID. No: 108.

(162) TABLE-US-00033 Primers for amplification of Amp-LV-L0 fragment: Forward primer (P176): 5-CCCGAGCTCGTGCGCGGAACCCCTATTTG-3 Reverse primer (P177): 5-CCCGAATTCTCATTCCGTTTCGCACTGGAA-3 Primers for amplification of pCDFDuet1 vector: Forward primer (P178): 5-CCCCTCGAGCTGCCACCGCTGAGCAATAACT-3 Reverse primer (P179): 5-GCCGGTACCGAGCGTCGAGATCCCGGACAC-3

(163) For construction of another single expression vector containing both light-switchable transcription factor and target transcription unit, LacI(1-62,wt)-VVD36(C71V) fragment was amplified from pALaV(wt) vector constructed in example 5 using primers P180 and P181, Bacillus subtilis vector pHT01 was amplified using primers P182 and P183, the obtained linearized fragment was ligated with LacI(1-62,wt)-VVD(C71V) fragment by infusion technology, the resulting transitional vector was named as pHT01-LaV(wt). mCherry gene fragment was amplified using primers P184 and P185 and ligated with pHT01-LaV(wt) vector by BamHI and XbaI double digestion, the resulting single vector containing both light-switchable transcription factor and target transcription unit was named as pHT01-LaV(wt)-P.sub.grac-mCherry, the polynucleotide sequence of LaV(wt)-P.sub.grac-mCherry is SEQ. ID. No:111.

(164) TABLE-US-00034 Primers for amplification of LacI-VVD36 (C71V) fragment: Forward primer (P180): 5-AGGGAGACGATTTTGATGAAACCAGTAACGTTA-3 Reverse primer (P181): 5-TTAATTGCGTTGCGCTCATTCCGTTTCGCACTGGAA-3 Primers for amplification of pHT01 vector: Forward primer (P182): 5-CAAAATCGTCTCCCTCCGTTTGAATATTTG-3 Reverse primer (P183): 5-GCGCAACGCAATTAATGTGAGTTAAGGCC-3 Primers for amplification of mCherry gene fragment: Forward primer (P184): 5-CCCGGATCCATGGTGAGCAAGGGCGAGGA-3 Reverse primer (P185): 5-GGGTCTAGATTACTTGTACAGCTCGTCCAT-3

Example 11 Regulation of Gene Expression by Recombinant Light-Switchable Transcription Factor in E. coli Cells

(165) The vectors constructed in the samples containing different light-switchable transcription factors and the reporter vector using mCherry as the reporter gene were Co-transformed into the corresponding strains to test light-regulated gene expression by recombinant light-switchable transcription factor.

(166) To firstly detect the regulation of gene expression by light-switchable transcription factor containing LexA(1-87)-VVD36 (C71V) with different linkers, pB-colE-mCherry constructed in sample 7 and pLV-Ln (n=0, 1, 2, 3, 4, 5, 6, 7) constructed in sample 2 were co-transformed into JM109 (DE3,sulA.sup.,LexA.sup.) strains respectively, the difference of mCherry expression levels before and after blue light illumination was determined. Co-transformation of pB-colE-mCherry and pCDFDuet1 containing no light-switchable transcription factor was used as the control to detect the effect of blue light illumination on the bacteria growth and protein expression. High expression levels was observed in the cells containing no light-switchable transcription factor both in the dark and light, indicating that blue light illumination had no effect on bacteria protein expression. The mCherry fluorescence of cells containing light-switchable transcription factor with different linkers significantly decreased after blue light illumination and was much lower than cells in the dark, indicating that these light-switchable transcription factors could be used in controlling gene expression in E. coli. In detailed, the gene expression of cells containing pLV-L0 in the light was 13 folds lower than in the dark, the gene expression of cells containing pLV-L4 in the light was 32 folds lower than in the dark, other recombinant light-switchable transcription factors with different linkers also had marked inhibition on mCherry expression upon blue light illumination (FIG. 13).

(167) To detect the effect of light-switchable transcription factors when its expression driven by T7 background expression was replaced with Amp promoter, pALV-Ln (n=0, 1, 2, 3, 4, 5, 6, 7) constructed in example 3 and pB-colE-mCherry reporter plasmids were co-transformed into JM109 (DE3,sulA.sup.,LexA.sup.) strains, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. Co-transformation of pB-colE-mCherry and pCDFDuet1 containing no light-switchable transcription factor was used as the control to detect the effect of blue light illumination on the bacteria growth and protein expression. The results of the applicants showed that replacement of T7 background expression with Amp promoter had more marked inhibition after blue light illumination. In detailed, the gene expression of cells containing pALV-L0 in the light was 150 folds lower than in the dark, the gene expression of cells containing pALV-L4 in the light was more than 50 folds lower than in the dark (FIG. 14).

(168) To detect the effect of recombinant light-switchable transcription factors on the regulation of LacZ expression, pB-colE-LacZ constructed in example 7 and pALV-L0 constructed in example 3 were con-transformed into constructed in example 7 strains, the difference of LacZ expression before and after blue light illumination was detected according to the methods described in embodiment 8. The results of the applicants showed that the expression level of LacZ in the light was 126 folds lower than in the dark (FIG. 15).

(169) To detect other three prokaryotic bacteria expression vectors containing LexA reaction unit and mCherry fluorescent protein gene, pB-sulA-mCherry, pB-umuDC-mCherry and pB-RecA-mCherry constructed in example 7 were co-transformed with pALV-L0 into JM109(DE3,sulA.sup.,LexA.sup.) strains respectively, pCDFDuet1 empty vector was used as the control, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. The results of the applicants showed that the response of target transcription unit of sulA, umuDC and RecA promoter to light switchable transcription factor was significantly lower than colE promoter, In detailed, the inhibition effect of sulA and umuDC on mCherry expression was less than 3 folds while the RecA had only 5-fold (FIG. 16).

(170) To detect the regulation of gene expression by light-switchable transcription factor with VVD mutants as the second polypeptide relative to LV-L0, pALV-L0(N56K), pALV-L0(Y50W), pALV-L0(N56K C71V), pALV-L0(I52A C71V), pALV-L0(I52S C71V), pALV-L0(N56R C71V) constructed in example 4 and pALV-L0 were co-transformed with pB-colE-mCherry reporter vector into JM109(DE3,sulA.sup.,LexA.sup.) strain, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. The results of the applicants showed that VVD(C71V) and VVD(N56K C71V) had higher inhibition ratios relative to other VVD mutants, due to the lower mCherry expression of VVD(C71V) in the light, VVD(C71V) was chosen as the second peptide of light-switchable transcription factor in following experiments (FIG. 17).

(171) To detect the regulation of gene expression by light-switchable transcription factor with cI, LacI, Gal4 or TetR as the first polypeptide, pACV, pALaV, pAGV and pATV constructed in example 7 were co-transformed with prokaryotic bacterium expression vectors containing target transcription unit and mCherry fluorescent protein into JM109 (DE3,sulA.sup.,LexA.sup.) strain, respectively, pCDFDuet1 empty vector was used as the control, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. The results indicated that the recombinant light-switchable transcription factor CV could result in reduction of mCherry expression in the light to half of that in the dark, mCherry expression in the light was 60%-70% of that in the dark for other three recombinant light-switchable transcription factors LaV, GV and TV, indicating light switchable characteristics of these recombinant light-switchable transcription factors (FIG. 18).

(172) To detect the regulation of gene expression by light-switchable transcription factor with phot1-LOV2 or LOV domain of aurochrome as the second polypeptide, pALA and pALAu constructed in example 4 were co-transformed with pB-colE-mCherry reporter vector constructed in example 7 into JM109(DE3,sulA.sup.,LexA.sup.) strain, respectively, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. The results indicated that the recombinant light-switchable transcription factor with AsLOV2 as the second peptide had greater inhibition in the dark, that the recombinant light-switchable transcription factor with AulOV as the second peptide had higher inhibition ratio of mCherry expression in the light (FIG. 19).

(173) To detect the regulation of gene expression by light-switchable transcription factor with as the third polypeptide, pALV, pACV, pALaV, pAGV and pATV constructed in example 8 were co-transformed with prokaryotic bacterium expression vectors containing target transcription unit and mCherry fluorescent protein gene into JM109(DE3,sulA.sup.,LexA.sup.,.sup.) strain, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. 5-fold activation of mCherry expression was observed for the recombinant light-switchable transcription factor LV in the light while CV and GV had 2-fold activation, LaV and TV also exhibited light-activated characteristics (FIG. 20).

(174) To detect the regulation of gene expression by light-switchable transcription factor with as the third polypeptide, pALV, pACV, pALaV, pAGV and pATV constructed in example 6 were co-transformed with prokaryotic bacterium expression vectors containing target transcription unit and mCherry fluorescent protein gene into BL21 (sulA.sup.,LexA.sup.,.sup.) strain, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. 4-fold activation of mCherry expression was observed for the recombinant light-switchable transcription factor LV in the light while CV, LaV and GV had 2-fold activation, TV also exhibited light-activated characteristics (FIG. 21).

(175) To detect the effect of temperature on the regulation of gene expression by light-switchable transcription factor, pALV-L0 constructed in example 3 was co-transformed with pB-colE-mCherry reporter vector constructed in example 7 into JM109(DE3,sulA.sup.,LexA.sup.) strain, the cells were cultured at 18 C., 25 C., 30 C. and 37 C., the difference of mCherry expression before and after blue light illumination was detected. The results showed that the recombinant light-switchable transcription factor LV-L0 had marked inhibition on mCherry expression at 18 C. both in the dark and light, in contrast to 18 C., the recombinant light-switchable transcription factor LV-L0 had little effect on mCherry expression whatever in the dark and light. Therefore, LV-L0 had no light-induced regulation on mCherry expression at these two temperatures. The recombinant light-switchable transcription factor LV-L0 had marked light-induced inhibition on mCherry expression at 25 C. and 30 C., the following measurements were carried out at 30 C. due to the higher growth rate at this temperature (FIG. 22).

(176) To detect the difference of mCherry expression before and after light illumination by light-switchable transcription factor for the prokaryotic bacterium expression vector using cI repressor as the indirect regulation, pB-colE-cI-P.sub.O12-mCherry constructed in example 9 was co-transformed with pALV-L0 constructed in example 3 into JM109(DE3,sulA.sup.,LexA.sup.) strain, pCDFDuet1 empty vector was used as the control, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. More than 50-fold activation of mCherry expression was observed for the recombinant light-switchable transcription factor LV-L0 in the light, demonstrating that such a regulation method using cI as the indirect regulation can be well used to control mCherry expression by light (FIG. 23).

(177) To detect the difference of mCherry expression before and after light illumination by single expression vector containing both light-switchable transcription factor and target transcription factor, pD-colE-mCherry-Amp-LV constructed in example 10 was transformed into JM109(DE3,sulA.sup.,LexA.sup.) strain, pCDFDuet1 empty vector was used as the control, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. The result showed that such single expression vector containing both light-switchable transcription factor and target transcription factor could well regulate mCherry expression of the target transcription unit by the recombinant light-switchable transcription factor LV-L0 expressed by itself, which avoids transformation of two vectors and has more important application prospect (FIG. 24).

(178) To detect the difference of mCherry expression before and after light illumination by Bacillus subtilis expression vector containing both light-switchable transcription factor and target transcription factor, pHT01-LaV(wt)-P.sub.grac-mCherry constructed in example 10 was transformed into Bacillus subtilis WB800, pHT01 vector was used as the control. mCherry expression in the dark and light was determined. The result showed that the recombinant light-switchable transcription factor LaV(wt) could well repress mCherry expression in Bacillus subtilis cells, the ratio could reach 3 folds (FIG. 25).

(179) To detect the effect of light-switchable transcription factor expressed by JM109(DE3,sulA.sup., LexA::Amp-LV-L0) strain on mCherry expression, pB-colE-mCherry constructed in example 7 was transformed into JM109(DE3,sulA.sup., LexA::Amp-LV-L0) strain, pRSETb vector was used as the control, the difference of mCherry expression before and after blue light illumination was detected according to the methods described in embodiment 7. The result showed that mCherry expression could be repressed by the light-switchable transcription factor expressed by the strain itself without introducing exogenous plasmid to express the light-switchable transcription factor, the repression ratio could reach 5 folds (FIG. 26).

Example 12 Characteristics of Gene Expression Regulation Upon Light Illumination

(180) Time course and reversibility of light-switchable transcription factor regulated gene expression were tested by co-transformation of pALV-L0 constructed in example 3 and pB-colE-mCherry reporter vector constructed in example 7 into JM109(DE3,sulA.sup.,LexA.sup.) strain, clones were picked into 48-well plate and divided into 6 groups, each group had four wells, all the cells were cultured at 30 C. upon light illumination. The cells were diluted 100 folds into two 48-well plates containing fresh LB, the two plates were labeled as A plate and B plate, respectively. A plate was cultured upon blue light exposure, mCherry expression was determined at 3 h, 5 h, and 7 h time points, after each measurement, one group was transferred from light to dark (residual wells in plate B) until the last time point. B plate was cultured in the dark, mCherry expression was determined at 3 h, 5 h, and 7 h time points, after each measurement, one group was transferred from dark to light (residual wells in plate A) until the last time point. The average of mCherry expression of each time point was plotted. The results showed that mCherry expression was greatly repressed when cells were illuminated by light from the beginning. The repression efficiency gradually weakened and mCherry expression increased when cells were transferred from light to dark, the curve rose slowly shown in the figure (FIG. 27). mCherry expression was not repressed and had high expression level when cells were kept in the dark, mCherry expression was gradually repressed when the cells were transferred to light conditions, the rising rate of the rising curve gradually decreased and the curve tended to horizontal at the last (FIG. 28). These results indicated that the regulation of gene expression by recombinant light-switchable transcription factor is reversible.

(181) To evaluate the gene expression regulated by light-switchable transcription factor in different light irradiance, mCherry was used as the reporter gene, pALV-L0 constructed in example 3 and pB-colE-mCherry reporter vector constructed in example 7 were co-transformed into JM109(DE3,sulA.sup.,LexA.sup.) strains. Clones were picked into 48-well plate and divided into 6 groups, each group had four wells, all the cells were cultured at 30 C. upon light illumination. The cells were diluted 100 folds into six 48-well plates containing fresh LB, 5 of the plates were cultured upon blue light illumination with the light irradiance 0.125 mW/cm.sup.2, 0.063 mW/cm.sup.2, 0.031 mW/cm.sup.2, 0.016 mW/cm.sup.2 and 0.009 mW/cm.sup.2 (light intensity determine by a laminator (Sanwa)), the last plate was cultured in the dark. mCherry expression was measured after 18 h. The result indicated that the gene expression regulated by light-switchable transcription factor is light irradiance dependent; more marked repression of mCherry expression was observed along with increase of light intensity. We also found that the recombinant light-switchable transcription factor LV-L0 functioned well even at extremely weak light (FIG. 29).

(182) To spatially control gene expression by light-switchable transcription factor, mCherry was used as the reporter gene, pALV-L0 constructed in example 3 and pB-colE-mCherry reporter vector constructed in example 7 were co-transformed into JM109(DE3,sulA.sup.,LexA.sup.) strains. Clones were picked into test tubes containing 5 ml fresh LB and cultured upon blue light illumination, the cells were harvested with 4000 rpm centrifugation and resuspended using 200 ul fresh medium the next day, then the OD600 was determined. The solid medium containing 1% agar, 0.5% tryptone, 0.25% yeast extract and 0.5% NaCl was prepared and cooled to 45 C. after autoclave sterilization, the above resuspended cells and antibiotics were added to make sure OD600-0.03, then the mixture was poured onto 90 mm dish. After coagulation, a Stop pattern was printed and a gradient slider on laser transparency film using a laser printer was used as photomask, the light intensity of transparent space detected by a luminator was 30 times more than the black space. The printed photomask was pasted on the bottom of the dish. 24 h after illumination, the imaging was conducted using the In-Vivo Multispectral System FX (Kodak) with 600 nm excitation and 670 nm emission filters, image was collected in 44 binning for 30 s exposure. Due to LV-L0 induced mCherry expression upon blue light illumination, the result showed that the left panel was the pattern we designed, the circle region of right panel was the fluorescence image of cells, so it could be concluded that the fluorescence image of the cells had the same pattern of the original image used as the mask, we could take photos for cells (FIG. 30), indicating that the system could spatially regulate gene expression.

Example 13 Regulation of Mobility of E. coli Cells by Light-Switchable Transcription Factor

(183) Detection of regulation of E. coli Swimming by recombinant light-switchable transcription factor was carried out on a special semisolid medium. The semisolid medium containing 1% tryptone, 0.5% NaCl and 0.25 agar was prepared and cooled to 50 C. after autoclave sterilization before addition of antibiotics, then 10 ml of the mixture was poured onto one 90 mm dish and harden at room temperature for 1 h. pALV-L0 constructed in example 3 and pB-colE-cI-P.sub.O12-CheZ constructed in example 9 were co-transformed into JM109 (DE3,sulA.sup.,LexA.sup.,CheZ.sup.) strain, clones were picked from the plate into test tubes and cultured overnight in the dark, the cells were diluted 200 folds into fresh medium and cultured in the dark. When the OD600 reached 0.1-0.2, 2 l of the cultured cells was spotted onto the semisolid medium and cultured at 30 C. in the light. The plates wrapped with foil and kept at the same conditions were used as the control in the dark. The imaging was conducted using the In-Vivo Multispectral System FX (Kodak) after 49 h. The result showed that light-switchable transcription factor LV-L0 could not repress cI expression in dark conditions, resulting in tight repression of CheZ expression by cI, so cells could not spread to form the bacteria ring due to that the bacteria containing no CheZ could not move. In contract to dark conditions, the light-switchable transcription factor LV-L0 could repress cI expression in light conditions, resulting in no effect on P.sub.O12 promoter activity, the expression of CheZ protein enabled the mobility of bacteria, so cells could spread around to form the bacteria ring (FIG. 31).

Example 14 Regulation of Lysis of E. coli Cells by Light-Switchable Transcription Factor

(184) To detect the regulation of E. coli lysis by light-switchable transcription factor, pALV-L0 constructed in example 3 and pB-colE-cI-P.sub.O12-SRRz constructed in example 9 were co-transformed into JM109(DE3,sulA.sup.,LexA.sup.,CheZ.sup.) strain, clones were picked from the plate into test tubes and cultured at 30 C. overnight in the dark, the cells were diluted 100 folds into fresh medium and cultured in the dark. When the OD600 reached 0.4-0.6, 1 mM IPTG was added to induce the expression of LacZ gene in the genome. The cells were transferred to dark conditions to induce the expression of SRRz gene cassette after 1.5 h, the cells were harvested with 4000 rpm centrifugation after h, the LacZ activities in the supernatant and precipitate were measured; the percent of LacZ activity in the supernatant to the total LacZ activities in the supernatant and precipitate was calculated. Cells kept in the dark were used as the control and shared the same manipulation with that in light conditions. The results showed that the light-switchable transcription factor LV-L0 could repress cI expression in light conditions, resulting in no effect on P.sub.O12 promoter activity, the high expression of S, R and Rz in SRRz gene cassette enabled lysis of 75% of cells, resulting in release of LacZ to the culture. Only 10% of bacteria lysed in the dark (FIG. 32).

Example 15 Large Scale Production of Sulfhydryl Oxidase Ero1 Using the Light-Switchable Gene Expression System of Prokaryotic Bacteria

(185) Due to the fast multiplication, low culture costs and the ability of high-level expression of exogenous protein, such a light-switchable gene expression system of prokaryotic bacterium was used for large scale production of sulfhydryl oxidase Ero1 in prokaryotic bacterium cells. pB-colE-mCherry-P.sub.O12-Ero1 constructed in example 9 and pALV-L0 constructed in example 3 were co-transformed into JM109(DE3,sulA.sup.,LexA.sup.) strain. The clones were picked into test tube containing 5 ml fresh LB and cultured overnight, the cells were transferred to conical flask containing 100 ml fresh LB and cultured at 37 C. When OD600 reached 0.8, the cells were transferred to conical flask containing 500 ml fresh LB and cultured at 25 C. in dark conditions, there were 9 conical flasks in all. The cells were illuminated with blue light LED when OD600 reached 0.6, cells were harvested with 4000 rpm centrifugation after 18 h and resuspended with Buffer A (0.02 M Na.sub.3PO.sub.4, 10 mM imidazole, 0.5 M NaCl, PH 7.2). The resuspended cells were broken by sonification, the conditions of the sonification are: P=40%, work for 1 s with 4 s interval, 300 s every cycle, 5 cycles in all. Supernatant was collected after 10,000 rpm centrifugation for 30 min at 4 C. The supernatant was loaded onto GE HisTrap HP (5 mL) column, the progress of loading: GE HisTrap HP.fwdarw.control flow rate at 1 mL/min using peristaltic pump, remove the alcohol in the column using deionized water firstly.fwdarw.equilibrate the column using Buffer A.fwdarw.load the 100 ml supernatant onto the column, control flow rate at 1 mL/min. Gradient elution was carried out using AKTA prime in the following elution procedure (1 mL/min of flow rate):

(186) (1) eluting with 10 ml Buffer A;

(187) (2) gradiently increasing the content of Buffer B (0.02 M Na.sub.3PO.sub.4, 500 mM imidazole, 0.5 M NaCl, PH 7.2) from 0% to 100% in the following 50 ml elution buffer;

(188) (3) removing all the proteins on the column using 200 ml 100% Buffer B;

(189) (4) gradiently decreasing the content of Buffer B from 100% to 0% in the following 20 ml elution buffer;

(190) (5) eluting the column using 50 ml deionized water; and

(191) (6) eluting the column using 20 ml 20% alcohol.

(192) The isoelectric point of Ero1 was 4.8 obtained from ExPASy website, ion-exchange chromatography was carried out using anion-exchange column. Ero1 protein solution after affinity column purification was loaded onto 5 ml Hitrap QFF column for ion-exchange chromatography, the experiment precedure: Ero1 protein solution after affinity column purification.fwdarw.dilute into 10 folds using Buffer A (20 mM Na.sub.3PO.sub.4, 10 mM NaCl, PH 7.2).fwdarw.load the solution onto 5 mL Hitrap QFF column.fwdarw.elute with 50 ml Buffer A.fwdarw.elute using AKTA purifier. The elution procedure (1 mL/min of flow rate) is as follows:

(193) (1) eluting with 10 ml Buffer A;

(194) (2) gradiently increasing the content of Buffer B (20 mM Na.sub.3PO.sub.4, 500 mM NaCl, PH 7.2) from 0% to 100% in the following 25 ml elution buffer;

(195) (3) removing all the proteins on the column using 20 ml 100% Buffer B;

(196) (4) gradiently decreasing the content of Buffer B from 100% to 0% in the following 20 ml elution buffer;

(197) (5) eluting the column using 50 ml deionized water; and

(198) (6) eluting the column using 20 ml 20% alcohol.

(199) The result of ion-exchange chromatography using AKTA purifier is shown as FIG. 33. Protein solution in part of the collected tubes was loaded onto 12% SDS-PAGE to identify their purity (FIG. 34), the tubes with high purity of protein were collected together. The purity of the lastly obtained protein solution was determined by 12% SDS-PAGE; the result showed that the Ero1 protein had high purity which reached 96% (FIG. 35). The amount of the Ero1 was 25 mg determined by Brandford.

(200) It will be understood that the dosages, reaction conditions, etc., in the examples are approximate values unless noted otherwise, and they can be exactly changed based on the situations to obtain similar results. All of the professional terms used in the Description, except those specially defined, have identical meanings to those known by persons skilled in the art. All the references referred to are incorporated into the application as a whole. The preferable embodiments are only exemplified for the illustration of the invention. Those skilled in the art can adopt similar methods or materials to obtain similar results. All the changes and modifications are within the scope of the attached claims.

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