CN110172498B - Method for rapidly and efficiently analyzing interaction of transcription factor and target DNA binding sequence thereof - Google Patents

Abstract

The invention discloses a method for rapidly and efficiently analyzing interaction of a transcription factor and a target DNA binding sequence thereof. The present invention analyzes a mixture of cell lysates diluted 2-fold in series with ECBS-AFBS probes using either electrophoretic migration assay (EMSA) or filter plate assay, which found that EMSA can detect probes in cell lysates diluted 1:16, whereas filter plate assay can detect probes in cell lysates diluted 1:64, indicating that the filter plate assay is 4-fold more sensitive than EMSA. Thus, filter plate assays are able to rapidly and efficiently analyze the binding of probes to target proteins.

Description

Method for rapidly and efficiently analyzing interaction of transcription factor and target DNA binding sequence thereof

The technical field is as follows:

the invention belongs to the technical field of genetic engineering, and particularly relates to a method for rapidly and efficiently analyzing interaction of a transcription factor and a target DNA binding sequence thereof.

Background art:

the interaction between a Transcription Factor (TF) and its target DNA binding sequence is critical in the regulation of gene transcription. Each eukaryotic TF can bind to a set of similar DNA sequences on chromosomal DNA rather than a single DNA sequence. The base composition determines the binding affinity of the DNA sequence to TF, which can be known from alignment analysis of the target sequence. A statistically simple Position Weight Matrix (PWM) model is typically used to calculate conserved base binding sequences. Within the conserved sequence, some base pairs are conserved in their interaction with TF, while others are more flexible and less important in the interaction of DNA with TF. The contribution of each base pair interacting with TF is assessed by its conservation. In addition, conserved binding sequences may also be determined by a set of DNA fragments or oligonucleotides that specifically bind TF by in vitro methods such as ChIP-seq or SELEX. However, currently more than 40% of TF have unknown target binding sequences. In the prokaryotic cell E.coli, most TF binds to a single site on chromosomal DNA, such as arsenic transcription repressing factor (ArsR). ArsR is a metal-regulated transcription repressor that binds to its operator/promoter (O/P) sequence. However, due to the large number of ArsR binding sequences present in microbial chromosomes, alignment analysis of these binding sequences by PWM models can also identify conserved binding sequences or domains.

Although the PWM method can be widely used to identify DNA binding sequences of TF, recent studies have shown that this model based on the independent contribution of each conserved base pair in protein interactions has been insufficient to account for various complex gene transcriptional regulation such as related di-or trinucleotides that are critical for protein-DNA regulation, significant differences from conserved sequence low-affinity binding sites, new DNA binding specificity that forms multi-protein complexes with TF, and the effect on binding affinity upstream and downstream of conserved sequences. These factors, as well as the interaction of TF cofactors in eukaryotic cells, complicate TF-DNA recognition. In this study, we used a simpler prokaryotic ArsR regulatory system to evaluate recognition between TF and the target DNA sequence.

ArsR belongs to the Smt/ArsR family of proteins, a regulatory protein that controls the expression of genes associated with arsenic resistance by interacting with an arsenic-responsive operon. ArsR binding prevents the RNA polymerase from interacting with the O/P sequence of the target gene in the absence of arsenic compounds. Once ArsR has been arsenic bound, the ArsR protein is isolated from the promoter, and expression of the arsenic resistance-associated gene is subsequently activated. The ArsR protein has been studied more extensively in the chromosome of plasmid R773 and E.coli (E.coli). Both ArsR proteins are dimers containing an arsenic-binding sequence Cys32-Val-Cys-Asp-Leu-Cys at the beginning of their target DNA-binding domain. ArsR from Acidithiobacillus ferrooxidans (Acidithiobacillus ferrooxidans) do not have arsenic binding sequences at this origin, but instead their cysteine residues are located at amino acid residues 95, 96 and 102. The binding properties and conserved sequences of the Smt/ArsR family proteins and of the ArsR proteins of A.ferrooxidans are well defined.

In previous researches, two arsenic reporter vectors pLHPars9 and pLLPars9 (the arsenic reporter vectors pLHPars9 and pLLPars9 are disclosed in the patent number 201780001826.1, the name of the invention is a class of arsenite inhibitor reporter plasmids and a construction method and application thereof), which are plasmids with high copy number and low copy number respectively, are constructed. We added the common ArsR-luciferase fusion element downstream of the ArsR operon of plasmid R773 and two copies of the ArsR target DNA binding sequence (ECBS) from the e.coli chromosome and the a.ferrooxidans chromosome (AFBS), respectively, upstream of the operon. Both of these reporter vectors are highly sensitive to arsenite with a minimum detection limit of 0.04 μ M arsenite (-5 μ g/L), and the two reporter vectors differ in specificity for metals, with pLLPars9 being more specific for arsenite and pLHPars9 being more specific for arsenite and antimonite. pLLPars9 differs from pLHPars9 only in the number of copies of their plasmids.

The invention content is as follows:

the invention aims to overcome the defects in the prior art and provide a method for quickly and efficiently analyzing interaction of a transcription factor and a target DNA binding sequence thereof.

The invention relates to a method for rapidly and efficiently analyzing interaction of a transcription factor and a target DNA binding sequence thereof (filter membrane plate assay), which comprises the following steps: preparing cell lysate containing transcription factors, designing a DNA double-stranded probe capable of being specifically combined with the transcription factors according to a target DNA combination sequence of the transcription factors, wherein biotin is marked at the 5' end of a sense strand of the DNA double-stranded probe, combining the transcription factors in the cell lysate with the DNA double-stranded probe to obtain a protein-probe compound, combining the protein-probe compound on a nitrocellulose membrane, washing off free DNA double-stranded probes, treating the protein in the protein-probe compound combined on the nitrocellulose membrane by SDS (sodium dodecyl sulfate) to denature the protein in the protein-probe compound combined on the nitrocellulose membrane, releasing the combined DNA double-stranded probe, collecting the released DNA double-stranded probe, hybridizing the denatured DNA double-stranded probe with a complementary sequence of the sense strand of the DNA double-stranded probe pre-combined on the nitrocellulose membrane, and sequentially adding horseradish peroxidase-labeled streptavidin and a chemiluminescent substrate, recording the luminescence, and judging the binding strength of the transcription factor and the target DNA binding sequence according to the luminescence.

The method comprises the following specific steps: preparing cell lysate containing transcription factors, designing a DNA double-stranded probe capable of being specifically combined with the transcription factors according to a target DNA combination sequence of the transcription factors, wherein the 5' end of a sense chain of the DNA double-stranded probe is marked with biotin, forming a combination reaction system of the cell lysate containing the transcription factors and the DNA double-stranded probe, culturing at 25 ℃ for 30 minutes to obtain a protein-probe complex, adding the protein-probe complex to a 96-well plate of a nitrocellulose membrane substrate which is pre-cleaned by a cleaning buffer solution, culturing on ice to combine the protein-probe complex to a nitrocellulose membrane, inverting and centrifuging, discarding an effluent, cleaning the 96-well plate by the cleaning buffer solution, adding an elution buffer solution to denature proteins in the protein-probe complex combined to the nitrocellulose membrane, releasing the combined DNA double-stranded probe, and collecting the released DNA double-stranded probe, heating at 95 ℃ to denature the DNA double-stranded probe, adding 2 Xhybridization buffer solution and the denatured DNA double-stranded probe into a nitrocellulose membrane-based 96-well plate pre-combined with a sense strand complementary sequence of the DNA double-stranded probe, hybridizing at 42 ℃, washing the 96-well plate by using a washing buffer solution, then adding streptavidin marked by horseradish peroxidase, incubating for 30 minutes at 37 ℃, washing the 96-well plate by using the washing buffer solution, adding a chemiluminescent substrate Luminol of the horseradish peroxidase, reading the number of photons by using a chemiluminescence instrument, recording the luminescence condition by using a relative light unit, and judging the binding strength of the transcription factor and a target DNA binding sequence thereof according to the luminescence condition.

Preferably, the binding reaction system is 20. mu.L, and comprises 2. mu.L of cell lysate, 10. mu.L of 2 Xbinding buffer mixture, 1. mu.L of LDNA double-stranded probe, and 7. mu.L of ddH ₂ O, said 2 × binding buffer mixture is: 40mM HEPES, 20mM ammonium sulfate, 2mM DTT, 20mM KCl and 0.4% Tween-20, the balance being water, pH 7.6.

Preferably, the washing buffer is: 100mM Tris-HCl, 2.5mM EDTA and 0.1% Tween-20, balance water, pH 7.6.

Preferably, the elution buffer is: 0.5% SDS, 100mM Tris-HCl, 2.5mM EDTA and 0.1% Tween-20, the balance being water, pH 7.6.

Preferably, the hybridization buffer is: 20mM HEPES, 10mM ammonium sulfate, 10mM KCl and 0.2% Tween-20, the balance being water, pH 7.6.

The transcription factor is preferably arsenic transcription repressing factor.

The present invention analyzes a mixture of serial 2-fold dilutions of cell lysate mixed with ECBS-AFBS probe using either electrophoretic migration assay (EMSA) or filter plate assay, and finds that EMSA can detect probes in 1:16 dilutions of cell lysate, whereas filter plate assay can detect probes in 1:64 dilutions of cell lysate, indicating that the filter plate assay is 4-fold more sensitive than EMSA. Thus, filter plate assays are capable of rapidly and efficiently analyzing the binding of probes to target proteins.

Description of the drawings:

FIG. 1 is an assay of luciferase activity of a reporter vector containing different combinations of two binding sequences; wherein, A is the luciferase activity of cells without arsenic treatment (blank) and cells with arsenic treatment (grey) which are respectively converted from E.coli DH5 alpha by report vectors pECBS-AFBS and pAFBS-ECBS, and then treated with or without 10 mu M sodium arsenite for 1 hour; b is report carrier pECBS-AFBS, pAFBS-ECBS, pECBS-smt2/1BS, pECBS-arsrBCBS, psmt2/1BS-AFBS and parsRBcBS-AFBS are respectively transformed into E.coli DH5 alpha, and then treated with or without 10 mu M sodium arsenite for 1 hour, and luciferase activity ratio values between the arsenic-free treated cells and the arsenic-treated cells are measured; c is the sequence of the ArsR binding core part sequence and CS, the underlined nucleotides are non-conserved base pair positions; d is the luciferase activity ratio between the arsenic-free treated cells and the arsenic-treated cells after E.coli DH 5. alpha. was transformed with the reporter vectors pECBS-AFBS, pECBS-CS, pCS-AFBS, pAFBS-CS and parsRBCBS-CS, respectively, and treated with or without 10. mu.M sodium arsenite for 1 hour.

FIG. 2 is a graph comparing the binding of ArsR protein to different DNA sequences using EMSA; a is EMSA of the cell lysate action after the probe ECBS-AFBS and the report vector pECBS-AFBS transform E.coli DH5 alpha, and EMSA of the cell lysate action after the probe AFBS-ECBS and the report vector pAFBS-ECBS transform E.coli DH5 alpha, B is EMSA of the cell lysate action after the probe ECBS-CS and the report vector pECBS-CS transform E.coli DH5 alpha, and EMSA of the cell lysate action after the probe CS-ECBS and the report vector pCS-ECBS transform E.coli DH5 alpha; where 1 is a biotin-labeled probe, 2 is arsenic-free treated cell lysate, and 3 is arsenic-treated cell lysate (10. mu.M sodium arsenite treatment for 1 hour).

FIG. 3 shows the effect of the 3Ts linker between ECBS and CS on the removal of repressor proteins by arsenic treatment; a is luciferase activity of cells without arsenic treatment (blank) and cells with arsenic treatment (grey) measured after E.coli DH5 alpha cells were transformed with the reporter vectors pECBS-CS and pECBS-CS (-3T), respectively, with or without 10. mu.M sodium arsenite for 1 hour; b, after transforming e.coli DH5 α cells with the reporter vectors pECBS-CS and pECBS-CS (-3T), respectively, and then treating the cells with or without 10 μ M sodium arsenite for 1 hour, the luciferase activity ratio between the arsenic-free treated cells and the arsenic-treated cells thereof was measured; and C is EMSA of the action of the cell lysate after the probe ECBS-CS and the report vector pECBS-CS are transformed into E.coli DH5 alpha, and EMSA of the action of the cell lysate after the probe ECBS-CS (-3T) and the report vector pECBS-CS (-3T) are transformed into E.coli DH5 alpha, wherein 1 is a biotin labeled probe, 2 is an arsenic-free treated cell lysate, and 3 is an arsenic-treated cell lysate (treated by 10 mu M sodium arsenite for 1 hour).

FIG. 4 is a filter plate assay; a is a flow chart of filter assay, comprising 3 steps: mixing probes with cell lysates, adding the mixture to NC-based filter plates, and analyzing the protein-bound probes; after E.coli DH5 alpha is transformed by the report vector, the cell lysate is prepared by treating with or without 10 mu M sodium arsenite for 1 hour, and a filter membrane plate is used for measuring the combination of the probe ECBS-AFBS and the cell lysate after E.coli DH5 alpha is transformed by the report vector pECBS-AFBS, the combination of the probe AFBS-ECBS and the cell lysate after E.coli DH5 alpha is transformed by the report vector pAFBS-ECBS, the combination of the probe ECBS-CS and the cell lysate after E.coli DH5 alpha is transformed by the report vector pECBS-CS, the combination of the probe CS-ECBS and the cell lysate after E.coli DH5 alpha is transformed by the report vector pCS-ECBS, and the combination of the probe ECBS-CS (-3T) and the cell lysate 5 alpha after E.coli DH 32 alpha is transformed by the report vector pECBS-CS (-3T); c is reporter vector pECBS-AFBS after transformation of e.colidh5 α, treatment with 10 μ M sodium arsenite for 1 hour to make cell lysate, serial 2-fold dilution, mixing with ECBS-AFBS probe, and analysis of the mixture by EMSA and filter plate assays, respectively.

FIG. 5 is a filter plate assay and luciferase activity assay for probes substituted at non-conserved base pairs; a is a list of probes that are substituted at non-conserved base pairs; coli DH 5. alpha. transformed with a reporter vector, with or without 10. mu.M sodium arsenite treatment for 1 hour to prepare a cell lysate, and determining the binding strength of the cell lysate to a probe corresponding to the transformed reporter vector by a filter plate assay, wherein probe ECBS-AFBS corresponds to reporter vector pECBS-AFBS, probe ECBS-CS corresponds to reporter vector pECBS-CS, probe ECBS-arsSRBCBS corresponds to reporter vector pECBS-arsSRBCBS, probe ECBS-smt2/1BS corresponds to reporter vector pECBS-smt2/1BS, probe sECBS-CS1M corresponds to reporter vector psECBS-CS1M, probe sECBS-CS2 corresponds to reporter vector psECBS-CS M, probe sECBS-3M corresponds to reporter vector ECBS-ECBS M, probe sECBS-CS4M corresponds to reporter vector psBS-CS 4, probe sECCS 4 corresponds to PSCS M-ECBS M-PSECBS M, probe sECBS-CS6m corresponds to a report carrier psECBS-CS6m, probe sECBS-CS7m corresponds to a report carrier psECBS-CS7m, probe sECBS-CS8m corresponds to a report carrier psECBS-CS8m, probe sECBS-CS9m corresponds to a report carrier psECBS-CS9m, probe sECBS-CS10m corresponds to a report carrier psECBS-CS10m, probe sECBS-CS11m corresponds to a report carrier psECBS-CS11m, probe sECBS-CS12m corresponds to a report carrier psECBS-CS12m, probe sECBS-CS13m corresponds to a report carrier psECBS-CS13m, probe sECBS-CS14m corresponds to a report carrier psECBS-CS14m, probe sECBS-CS15m corresponds to a report carrier psECBS-CS15, and probe sECBS-CS16 corresponds to a report carrier psECBS 5857316-PSECBS 5857316; c after transformation of E.coli DH 5. alpha. with or without treatment with 10. mu.M sodium arsenite for 1 hour to prepare a cell lysate, determining by filter plate assay the ratio of binding of the probe to arsenic-free treated cell lysate and its arsenic-treated cell lysate after transformation of E.coli DH 5. alpha. with probe ECBS-AFBS corresponding to reporter vector pECBS-AFBS, probe ECBS-CS corresponding to reporter vector pECBS-CS, probe ECBS-arsRBBS corresponding to reporter vector pECBS-arsRBCBS, probe ECBS-smT2/1BS corresponding to reporter vector pECBS-smt2/1BS, probe sECBS-CS1M corresponding to reporter vector psecBS-CS1M, probe sECBS-CS2M corresponding to reporter vector ECBS-CS2 psBS M, probe sECBS-CS3M corresponding to reporter vector psECBS-CS M, probe sECBS 4-ECCS M corresponding to reporter vector ECBS M-ECBS, probe sECBS-CS5m corresponds to reporter vector psECBS-CS5m, probe sECBS-CS6m corresponds to reporter vector psECBS-CS6m, probe sECBS-CS7m corresponds to reporter vector psECBS-CS7m, probe sECBS-CS8m corresponds to reporter vector psECBS-CS8m, probe sECBS-CS9m corresponds to reporter vector psECBS-CS9m, probe sECBS-CS10m corresponds to reporter vector psECBS-CS10m, probe sECBS-CS11m corresponds to reporter vector psECBS-CS11m, probe sECBS-CS12m corresponds to reporter vector psECBS-CS12m, probe sECBS-CS13m corresponds to reporter vector psECBS-CS13m, probe sECBS-CS14m corresponds to reporter vector psECBS-CS14m, probe sECBS-CS2 corresponds to reporter vector psECBS-CS 8616, and probe sECBS-CS 8616 corresponds to reporter vector psECBS 828616.

FIG. 6 is an EMSA analysis of sECBS-CS9m, sECBS-CS12m, and sECBS-CS15m and an analysis of luciferase activity of a reporter vector with corresponding binding sequences; a is probes sECBS-CS9M, sECBS-CS12M and sECBS-CS15M which are respectively mixed with arsenic-free treated cells and arsenic-treated cell lysates after report vectors psECBS-CS9M, psECBS-CS12M and psECBS-CS are transformed into E.coli DH5 alpha, and EMSA analysis is carried out, wherein 1 is a biotin labeled probe, 2 is an arsenic-free treated cell lysate, and 3 is an arsenic-treated cell lysate (treated with 10 mu M sodium arsenite for 1 hour); b is luciferase activity of arsenic-free treated cells (blank) and arsenic-treated cells (grey) measured after e.coli DH5 α cells were transformed with the reporter vectors psECBS-CS9M, psECBS-CS12M, and psECBS-CS15M, respectively, with or without 10 μ M sodium arsenite treatment for 1 hour; c is luciferase activity of arsenic-treated cells measured by treating e.coli DH5 α cells with 10 μ M sodium arsenite for 15 min, 30 min, 60 min and 120 min after transformation with e.coli DH5 α cells with the reporter vectors pECBS-CS, pECBS-AFBS and psECBS-CS12M, respectively.

The specific implementation mode is as follows:

the following is a further description of the invention and is not intended to be limiting.

Example 1:

1 materials and methods

1.1 plasmid construction

A series of reporter vectors with different binding sequence order and origin were constructed by modifying the binding sequence of pLLPars9 (renamed as pECBS-AFBS in this study, with the nucleotide sequence shown in SEQ ID NO. 6). First, sense and antisense DNA sequences were synthesized in vitro and annealed to generate a double-stranded fragment containing XbaI and HindIII sticky ends, which was then cloned into pLLPars9The XbaI and HindIII sites of (A) to replace ECBS-AFBS (the nucleotide sequence of which is shown in the specificationCACATTCGTTA AGTCATATATGTTTTTGACTT-TTT-ATCCACGAATATTTCTTGCA, shown in SEQ ID NO.1) Thus, reporter vectors pAFBS-ECBS, pECBS-smt2/1BS, pECBS-arsCBS, psmt2/1BS-AFBS, parsRBBS-AFBS, pCS-AFBS, parsRBBS-CS, pAFBS-CS, pECBS-CS (-3T), psECBS-CS9m, psECBS-CS12m and psECBS-CS15m with different sequence structures were obtained.

Taking the report vector psECBS-CS12m as an example, the specific construction method comprises the following steps: the sense strand was synthesized in vitro (its nucleotide sequence was:

as shown in SEQ ID NO.7, the bold part is XbaI and HindIII sticky ends) and antisense strand(s) ((II)

As shown in SEQ ID NO.8, the bold part is XbaI and HindIII sticky ends), annealing to generate a double-chain fragment containing XbaI and HindIII sticky ends, double-enzyme digesting the reporter vector pLLPars9 with XbaI and HindIII to obtain a linear vector containing XbaI and HindIII sticky ends, connecting the double-chain fragment containing XbaI and HindIII sticky ends with the linear vector, transforming the connecting product into E.coli DH5 alpha competent cells, coating the competent cells on an LB plate containing 25 mu g/mL chloramphenicol for overnight culture, picking out positive clones, sequencing and verifying sECBS-CS12m (the nucleotide sequence of which is shown as SEQ ID NO. 8) (the nucleotide sequence of the competent cells is SbI and HindIII sticky ends)ttcgTTAAGTCATATATGTTTTTGACTT-TTT-TACAGTCAAATAGCTATTTGACTGTA, as shown in SEQ ID NO.9 Display device) Replaces ECBS-AFBS (the nucleotide sequence of which is shown as the following formula) on the pLLPars9 vectorCACATTCGTTAAGTCATATATGTTTTT GACTT-TTT-ATCCACGAATATTTCTTGCA, shown as SEQ ID NO.1) Obtaining a report vectorpsECBS-CS12m (the nucleotide sequence is shown in SEQ ID NO. 10).

The reporter vectors pAFBS-ECBS (AFBS-ECBS (nucleotide sequence of which is shown asATCCACGAATATTTCTTGCA-TTT-CACATTCGTTAAGTCATATATGTTTTTGACTT, shown in SEQ ID NO.11) Replaces ECBS-AFBS on pLLPars9 vector), pECBS-smt2/1BS (ECBS-smt2/1BS (the nucleotide sequence is shown asCACATTCGTTAAGTCATATATGTTTTTGACTT-TTT-GCTAAACACATGAACAGTTATTCAGATATTCAAAShown as SEQ ID NO. 12) replaces ECBS-AFBS on pLLPars9 vector and pECBS-arsRBCBS (ECBS-arsRBCBS (the nucleotide sequence of which is shown asCACATTCGTTAAGTCATATATGTTTTTGACTT-TTT-TGTGATTAATCATATGCGTTTTTGGTT ATGTGTTShown as SEQ ID NO. 13) replaces ECBS-AFBS on pLLPars9 vector, psmt2/1BS-AFBS (smt2/1BS-AFBS (the nucleotide sequence is shown asGCTAAACACATGAACAGTTATTCAGATATTCAAA-TTT-ATCCACGAATATTTCTTGCAShown as SEQ ID NO. 14) in place of ECBS-AFBS on pLLPars9 vector, and parsrSCBS-AFBS (arsCBS-AFBS (the nucleotide sequence of which is shown asTGTGATTAATCATATGCGTTTTTGGTTATGTG TT-TTT-ATCCACGAATATTTCTTGCAShown as SEQ ID NO. 15) replaces ECBS-AFBS and pCS-AFBS (CS-AFBS (the nucleotide sequence of which is shown as SEQ ID NO. 15) on pLLPars9 vectorTAAAATCAAATACGTATTTGATTATA-TTT-ATCCACGAATATTTCTTGCAShown as SEQ ID NO. 16) substituted for ECBS-AFBS on pLLPars9 vector, and parsrSCBS-CS (arsCBS-CS (the nucleotide sequence is shown asTGTGATTAATCATATGCGTTTTTGGTTATGTGTT-TTT-TAAAATCAAATACGTATTTGATTATAShown as SEQ ID NO. 17) substituted for ECBS-AFBS, pAFBS-CS (AFBS-CS (the nucleotide sequence of which is shown as SEQ ID NO. 32) on pLLPars9 vectorATCCACGAATATTTCTTGCA-TTT-TAAAATCAAATACGTATTTGATTATAShown as SEQ ID NO. 18) replaces ECBS-AFBS on pLLPars9 vector and pECBS-CS (ECBS-CS (the nucleotide sequence of which is shown asCACATTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAAAATCAAATACGTATTTGATTATAShown as SEQ ID NO. 19) replaces ECBS-AFBS on the pLLPars9 vector), pECBS-CS (-3T) (ECBS-CS (-3T) (the nucleotide sequence of which is shown asCACATTCGTTAAGTCATATATGTTTTTGA CTT-TAAAATCAAATACGTATTTGATTATAAs shown in SEQ ID NO. 20) in place of ECBS-AFBS on pLLPars9 vector, psecBS-CS9m (sECBS-CS9m (nucleoside thereof)The sequence isTTCGTTAAGTCATATATGTTTTTGACTT-TTT-TACAATCAAATAGCTATTTGATTGTAShown as SEQ ID NO. 21) to replace ECBS-AFBS on pLLPars9 vector and pseCBS-CS15m (sECBS-CS15m (the nucleotide sequence of which is shown as sECBS-CS15m)TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAGACTCAAATAGCTATTTGAGTCTAShown as SEQ ID NO. 22) in place of ECBS-AFBS).

1.2 luciferase Activity assay

E.coli DH 5. alpha. containing the reporter vector was spread on LB plates containing 25. mu.g/mL of chloramphenicol, cultured at 37 ℃ for 16 hours, and a single colony was picked up and inoculated into 2mL of LB medium containing 25. mu.g/mL of chloramphenicol, and cultured with shaking at 37 ℃ for 12 hours to obtain an overnight culture. The overnight culture was diluted 50-fold in 100mL of pre-warmed and freshly prepared LB medium containing 25. mu.g/mL chloramphenicol. The diluted cells were incubated at 37 ℃ for an additional 3 hours until the o.d. reached 0.5. The cells were treated at 37 ℃ for 60 minutes with or without 10. mu.M sodium arsenite (AsIII) to obtain a cell culture. mu.L of the treated cell culture was mixed with 50. mu.L of luciferase substrate and luciferase activity was measured on a chemiluminescence apparatus (Veritas).

1.3 preparation of cell lysates

1mL of the cell culture, with or without treatment with sodium arsenite, was centrifuged at 10,000rpm for 1 minute, and the cell pellet resuspended in 300. mu.L of lysis buffer (10mM Tris-HCl, pH8.0, 0.1M NaCl, 1mM EDTA and 0.1% [ w/v ] TRITON X-100). 7.5. mu.L of a freshly prepared lysozyme solution (lysozyme solution preparation method: lysozyme solution in pH8.010mM Tris-HCl to obtain a lysozyme concentration of 10 mg/mL; lysozyme solution added to the cell precipitate containing lysis buffer, lysozyme final concentration of 0.25mg/mL) was added and mixed well by tapping the tube to obtain a lysis mixture, which was incubated at room temperature for 10 minutes. After centrifugation, the supernatant (i.e., cell lysate) is used for Electrophoretic Migration Shift Assay (EMSA) or filter plate assay.

1.4 electrophoretic migration Change assay (EMSA)

Mu.g of cell lysate was mixed with 2. mu.L of 5 Xbinding buffer and 1. mu.L of poly d ((I-C), plus ddH ₂ O is complemented to9 μ L, and incubated on ice for 5 minutes. mu.L of biotin-labeled probe was added to the above mixture and incubated at 22 ℃ for 30 minutes. Electrophoresis was carried out in 0.5 XTBE using 6.5% native polyacrylamide gel at 100V at 4 ℃ for about 50-60 minutes. The gel was then transferred to NB membranes, blocked for 20 min at room temperature after addition of 15mL blocking buffer, and the imprinted biotin-labeled probe was detected with streptavidin-HRP and a chemiluminescent substrate (increasing chemiluminescence with luminol, Pierce). An image is acquired with an imager.

1.5 Filter Membrane plate assay

mu.L of cell lysate was mixed with 10. mu.L of 2 Xbinding buffer mixture (40mM HEPES, 20mM ammonium sulfate, 2mM DTT, 20mM KCl and 0.4% Tween-20, balance water, pH7.6), 1. mu.L of biotin-labeled probe (DNA double-stranded probe) and 7. mu.L of ddH ₂ And mixing the O to form a combined reaction system. After incubation at room temperature (25 ℃) for 30 minutes, protein-probe complexes were obtained, and then the binding reaction system was applied to a filter assay plate (nitrocellulose (NC) -based 96-well plate) previously washed with a washing buffer (100mM Tris-HCl, 2.5mM EDTA, and 0.1% Tween-20, the balance water, pH7.6), incubated on ice for 20 minutes to bind the protein-probe complexes to the nitrocellulose membrane, and then centrifuged at 600g upside down for 2 minutes. The effluent was discarded, and the filter assay plate was washed with a washing buffer (100mM Tris-HCl, 2.5mM EDTA, and 0.1% Tween-20, balance water, pH 7.6). mu.L of an elution buffer (0.5% SDS, 100mM Tris-HCl, 2.5mM EDTA and 0.1% Tween-20, the balance water, pH7.6) was added to denature the proteins in the protein-probe complex bound to the nitrocellulose membrane, release the bound DNA double-stranded probe, and the released DNA double-stranded probe was collected. The collected DNA double-stranded probe was heated at 95 ℃ for 3 minutes before hybridization to denature the DNA double-stranded probe. Hybridization was performed by: to a nitrocellulose membrane-based 96-well plate (hybridization plate) to which a complementary sequence to the sense strand of a DNA double-stranded probe had been previously bound, 18. mu.L of a hybridization buffer (40mM HEPES, 20mM ammonium sulfate, 20mM KCl, and 0.4% Tween-20, the balance being water, pH7.6) was added, 2. mu.L of a heat-denatured DNA double-stranded probe was added, and after 12 hours of hybridization at 42 ℃, a washing buffer (100mM Tris-HCl, pH7.6, 2.5mM EDTA, and 0.5 mM EDTA) was added1% Tween-20) washing the hybridization plate. After addition of 20. mu.L of horseradish peroxidase (HRP) -labeled streptavidin and incubation at 37 ℃ for 30 minutes, the hybridization plates were washed with a washing buffer (100mM Tris-HCl, pH7.6, 2.5mM EDTA and 0.1% Tween-20). 20 μ L of HRP chemiluminescent substrate Luminol was added and the number of photons read by a chemiluminescence meter and luminescence was recorded in Relative Light Units (RLUs).

2 results

2.1 promoters containing ECBS and any other ArsR binding sequences have high arsenic-responsive transcriptional induction

In previous studies, we found that a luciferase reporter vector pLLPars9 (designated as pECBS-AFBS in this study) containing two copies of the ArsR Binding Sequence (BS), one from the ArsR binding sequence (ECBS) of the E.coli (EC) chromosomal DNA and the other from the ArsR binding sequence (AFBS) of the A.ferrooxidans (AF) chromosomal DNA, showed better response to arsenic treatment than a reporter vector containing only one copy or two copies of the same ECBS or AFBS. In this study, we exchanged the positions of ECBS and AFBS, generating the pAFBS-ECBS report carrier. Luciferase activities of pECBS-AFBS and pAFBS-ECBS were measured and compared after transformation of the reporter vector into e.coli DH5 α (fig. 1A), and it was found that the luciferase gene transcription inducing activity of pAFBS-ECBS arsenic-treated cells was only 2-fold higher than that of untreated control cells compared to the 9-fold transcription inducing effect formed with pECBS-AFBS (fig. 1B). The fold of arsenic response transcriptional induction of pAFBS-ECBS was reduced by more than 70% compared to pECBS-AFBS. This result indicates that the order of these two binding sequences is critical in reporting the response of the vector to arsenic.

Furthermore, we replaced the AFBS part in ECBS-AFBS with the binding sequence of smt2/1 (smt2/1BS) or the binding sequence of arsRBC (arsRBBS) to form reporter vectors pECBS-smt2/1BS and pECBS-arsRBBS, and compared the luciferase activity of their transformed cells with and without arsenic treatment. As shown in FIG. 1B, the arsenic response to transcriptional induction was slightly reduced for pECBS-smt2/1BS and pECBS-arsCBBS, by a factor of approximately 15-25% compared to pECBS-AFBS. This indicates that AFBS at the second location is not critical for arsenic-responsive transcriptional induction and can be replaced by other ArsR binding sequences. When the ECBS in the ECBS-AFBS was replaced with the binding sequence of smt2/1 (smt2/1BS) or the binding sequence of arsRBC (arsRBCBS) to form the reporter vectors psmt2/1BS-AFBS and parsrSRBCBS-AFBS, we found that the luciferase activity ratio of the reporter vectors was significantly reduced with and without arsenic treatment, by about 70% compared to pECBS-AFBS, as shown in FIG. 1B. This indicates that ECBS needs to be the first binding sequence to obtain a better response to arsenic.

Based on the conserved sequences of arsRBC and cadCA ca, we designed a binding sequence CS (nucleotide sequence of the binding sequence CS is:TAAAATCAAATACGTATTTGATTATAshown in SEQ ID NO.23, FIG. 1C) and replaced AFBS or an ECBS moiety, respectively, to construct pECBS-CS (replacing ECBS-AFBS on the reporter vector pLLPars9 with ECBS-CS (having the nucleotide sequence of SEQ ID NO: 23)CACATTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAAAATCAAATACGTATTTGATTATAShown as SEQ ID NO. 19) and pCS-AFBS (ECBS-AFBS on the reporter vector pLLPars9 was replaced by CS-AFBS (the nucleotide sequence of which is shown as SEQ ID NO. 19)TAAAATCAAATACGTATTTGATTATA-TTT-ATCCACGAATATTTCTTGCAAs shown in SEQ ID NO. 16). Luciferase activity assays showed no significant difference in the response to arsenic for pECBS-CS compared to pECBS-AFBS. However, pCS-AFBS showed significant changes (fig. 1D). Furthermore, when we replaced ECBS in ECBS-CS with arsCBBS or AFBS to construct parsCBBS-CS (replacing ECBS-AFBS on the reporter vector pLLPars9 with arsCBBS-CS (whose nucleotide sequence is shown as arsCBBS-CS)TGTGATTAATCATATGCGTTTTTGGTTATGTGTT-TTT-TAAAATCAAATACGTATTTGATTATAShown as SEQ ID NO. 17) and pAFBS-CS (replacing ECBS-AFBS on the reporter vector pLLPars9 with AFBS-CS (the nucleotide sequence of which is shown as AFBS-CS)ATCCACGAATATTTCTTGCA-TTT-TAAAATCAAATACGTATTTGATTATAAs shown in SEQ ID No. 18), their arsenic response transcription induction was significantly reduced as was shown above for any of the other reporter vectors that did not contain ECBS at the first position. The results of these CS-bearing reporter vectors indicate that ECBS must be the first binding sequence.

2.2 arsenic failure to remove the repressor ArsR from AFBS-ECBS and CS-ECBS probes

To examine ECBS-AFBS (nucleotide sequence:CACATTCGTTAAGTCATATATGTTTTTGACTT-TTT-ATCCACGAATATTTCTTGCAshown as SEQ ID NO. 1) and AFBS-ECBS (the nucleotide sequence is:ATCCACGAATATTTCTTGCA-TTT-CACATTCGTTAAGTCATATATGTTTTTGACTTas shown in SEQ ID No. 11) whether there was any difference in binding of the ArsR protein, we performed EMSA experiments. The biotin-labeled probe ECBS-AFBS (the sense strand of the ECBS-AFBS probe is 5-CACATTCGTTAAGTCATATATGTTTTTGACTT-TTT-ATCCACGAATATTTCTTGCA-3' (as shown in SEQ ID No. 1), and the antisense strand is: 5'-TGCAAGAAATATTCGTGGATAAAAAGTCAAAAACATATATGACTTAACGAATGTG-3' (shown in SEQ ID NO. 24); the 5' end of the sense strand was labeled with biotin) was mixed with cell lysates of E.coli DH 5a cells transformed with the reporter vector pECBS-AFBS, with or without arsenic treatment; a biotin-labeled probe AFBS-ECBS (the sense strand of the AFBS-ECBS probe is 5-ATCCACGAATATTTCTTGCA-TTT-CACATTCGTTAAGTCATATATGTTTTTGACTT-3' (as shown in SEQ ID No. 11), the antisense strand is: 5'-AAGTCAAAAACATATATGACTTAACGAATGTGAAATGCAAGAAATATTCGTGGAT-3' (shown in SEQ ID NO. 25); the 5' end of the sense strand is labeled with biotin) was mixed with cell lysates of E.coli DH5 alpha cells transformed with the reporter vector pAFBS-ECBS, with or without arsenic treatment. As previously reported, two bands of probe displacement were observed with the ECBS-AFBS probe in control cell lysates (without arsenic treatment), and the intensity of the probe displacement band was significantly reduced in arsenic-treated cell lysates (fig. 2A), indicating that arsenic treatment resulted in a reduction in the interaction between the ECBS-AFBS probe and the binding ArsR repressor protein. However, when performing EMSA experiments using AFBS-ECBS probes, we found no difference in the number and intensity of the probe displacement bands between arsenic treated cell lysates and untreated control cell lysates. This indicates that arsenic treatment failed to remove the repressor protein from the AFBS-ECBS probe.

Next, we compared the ECBS-CS probes (sense strand of ECBS-CS probes: 5-CACA TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAAAATCAAATACGTATTTGATTATA-3' (as shown in SEQ ID No. 19), the antisense strand is: 5' -TATAATCAAATACGTATTTGATTTTAAAAAAGTCAAAAACATATATGACTTAACGAATGTG-3' (shown in SEQ ID NO. 26); biotin labeled at the 5' end of the sense strand) and the CS-ECBS probe (sense strand of CS-ECBS probe: 5' -TAAAATCAAATACGTATTTGATTATA-TTT-CACATTCGTTAAGTCATATATGTTTTTGAC TT-3' (as shown in SEQ ID No. 27), the antisense strand is: 5'-AAGTCAAAAACATATATGACTTAACGAATGTGAAATATAATCAAATACGTATTTGATTTTA-3' (shown in SEQ ID NO. 28); the 5' end of the sense strand is labeled with biotin). The ECBS-CS probe was found to show two bands of displacement in control cell lysates, and the intensity of the probe bands in arsenic-treated cell lysates was very weak (FIG. 2B). But there was no significant difference in the intensity of the probe displacement band between control cells and arsenic-treated cell lysates for the CS-ECBS probe. This result is consistent with the AFBS-ECBS probe, indicating that arsenic treatment failed to remove the repressor protein from the CS-ECBS probe.

2.3 arsenic treatment to remove repressor protein requires a 3Ts linker between ECBS and CS

The 3Ts linker was located between ECBS and CS, and to examine whether it was necessary, we removed the 3Ts linker from pECBS-CS, thereby forming a pECBS-CS (-3T) reporter vector (replacing ECBS-AFBS on reporter vector pLLPars9 with ECBS-CS (-3T) (the nucleotide sequence of which is shown as ECBS-CS (-3T))CACATTCGTTAAGTCATATATGTTTTTGACTT-TAAAATCAAATACGTATTTGATTATAAs shown in SEQ ID NO. 20). Arsenic-responsive luciferase transcription-inducing activity was determined after treatment of pECBS-CS (-3T) transformed cells with or without sodium arsenite. As a result, no significant decrease in luciferase activity was found in arsenic-treated pECBS-CS transformed cells compared to untreated control cells; however, this significant reduction in luciferase activity was observed in arsenic-treated pECBS-CS (-3T) cells (fig. 3A). This result indicates that the 3Ts linker between ECBS and CS is required for arsenic-mediated transcription induction of luciferase genes.

Furthermore, to examine whether the absence of the 3Ts linker affects the steric hindrance of binding of the dimeric repressor ArsR to the binding sequence or affects the removal of the repressor from the binding sequence, we used the ECBS-CS probe (sense strand of the ECBS-CS probe: 5' -CACATTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAAAATCAAATACGTATTTGATTATA-3' (e.g. SE)Q ID NO. 19), and the antisense strand is: 5'-TATAATCAAATACGTATTTGATTTTAAAAAAGTCAAAAACATATATGACTTAACGAATGTG-3' (shown in SEQ ID NO. 26); the 5' end of the sense strand is marked with biotin) and an ECBS-CS (-3T) probe (the sense strand of the ECBS-CS (-3T) probe is: 5' -CACATTCGTTAAGTCATATATGTTTTTGACTT-TAAAATCAAATACGTATTTGATTATA-3' (as shown in SEQ ID No. 20), and the antisense strand is: 5'-TATAATCAAATACGTATTTGATTTTAAAGTCAAAAACATATATGACTTAACGAATGTG-3' (shown in SEQ ID NO. 29); the 5' end of the sense strand was labeled with biotin) the EMSA experiment was performed. Results the results using the ECBS-CS probe show that there are two bands of displacement in the control cell lysate identical to the ECBS-AFBS probe, but the probe bands of displacement are significantly reduced in the arsenic treated cell lysate, as shown in figure 3B. The presence of two probe displacement bands for the ECBS-CS (-3T) probe without the 3Ts linker in both control and arsenic treated cell lysates indicates that the absence of the 3Ts linker does not interfere with binding of repressor protein to the binding sequence nor is steric hindrance present. However, the results also indicate that the absence of the 3Ts linker prevents arsenic treatment from removing the repressor protein from the binding sequence.

2.4 Rapid analysis of DNA binding sequences by Filter plate assay

Although pECBS-arsRBBS and pECBS-CS contain the same conserved sequences, they respond with significant intermediate differences to arsenic treatment. We hypothesize that this difference can only be caused by the contribution of non-conserved base pairs of the second binding sequence. To clarify the contribution of non-conserved base pairs in sequence binding and transcription induction of arsenic-responsive luciferase gene, we only performed non-conserved base pair substitutions in the ECBS-arsRBCBS second binding sequence, thereby constructing a series of probes and reporter vectors. According to the conserved sequence of arsRBCBS, only 4 base pairs are non-conserved. When we investigate different combinations of these 4 base pairs, a series of probes is required for testing.

EMSA is commonly used to monitor protein/DNA interactions. Because of the lower throughput, EMSA analysis may be time consuming in cases of larger sample sizes. To this end we developed a rapid filter binding assay (filter plate assay) that can effectively monitor the interaction of several probes with their binding proteins simultaneously (the assay scheme is shown in figure 4A). In this assay, after the probes were separately mixed with sodium arsenite-treated or non-treated e.coli cell lysates and cultured, the mixtures were added to Nitrocellulose (NC) -based 96-well plates. Only the protein-bound probe (protein-probe complex) can stay on the plate, and the free probe is filtered from the NC membrane 96-well plate by centrifugation. After washing the 96-well plate, the protein bound to the plate is denatured by SDS treatment, and the probe is released from the bound denatured protein. These probes were collected and hybridized to a plate pre-bound with complementary sequences, followed by streptavidin and chemiluminescent substrate for further monitoring of luminescence.

To test the feasibility of the filter plate assay, we used probes ECBS-AFBS, AFBS-ECBS, ECBS-CS, CS-ECBS and ECBS-CS (-3T) and mixed them with arsenic treated and untreated cell lysates, respectively. The cell lysis mixture containing the probe was verified by EMSA prior to assay with filter plates. Filter plate assays showed that the ECBS-AFBS probes bound far more strongly to cell lysates without arsenic treatment than to cell lysates with arsenic treatment, with the ratio of the binding strength of the probes to control cells and arsenic treated cell lysates being about 5: 1 (fig. 4B). The binding of the AFBS-ECBS probe to both control cells and arsenic treated cell lysates was strong and no significant difference was observed in the two bindings. The results with the ECBS-CS probe were similar to the ECBS-AFBS probe, with a binding ratio of approximately 3: 1. furthermore, both ECBS-CS (-3T) and CS-ECBS probes showed no difference in binding to two different cell lysates (FIG. 4B). These results, measured with filter plates, are consistent with the results of the EMSA assay. The only difference between the filter plate assay and the EMSA assay is that it does not exhibit two distinct probe displacement bands as does EMSA.

Next, we used two assays to serially dilute the E.coli DH 5. alpha. cells transformed with the reporter vector pECBS-AFBS using 2-fold dilutions of cell lysates without arsenic treatment and mixing with ECBS-AFBS probes. As shown in FIG. 4C, EMSA was able to detect probes in cell lysates at 1:16 dilution, whereas filter plate assays were able to detect probes in cell lysates at 1:64 dilution, indicating that the filter plate assays were 4 times more sensitive than EMSA. Thus, filter plate assays are capable of rapidly and efficiently analyzing the binding of several probes to a target protein.

2.5 identification of non-conserved base pairs crucial in protein binding Using Filter plate assay

Original e.coli ArsR binding sequence ECBS CACA TTCG TT AA GT CA TA TA (TG) TT TT TG AC TT (cShown as SEQ ID NO.5). Based on comparison with other ArsR binding sequences, it was found that 8 base pairs at the 5' end may not be involved in arsenic-mediated gene transcription induction. To reduce the cost of oligonucleotide synthesis, we removed 4 base pairs at the 5' end, changing ECBS to TTCG TT AA GT CA TA TA (TG) TT TT TG AC TT (sECBS, shown as SEQ ID No. 30). The reporter vector psECBS-AFBS was constructed using a short sequence of 4 bases removed scecbs instead of ECBS, and functional analysis showed no difference between psECBS-AFBS and pECBS-AFBS (data not shown).

The core binding sequence of ArsR (nucleotide sequence: T.sub.g.: core binding sequence of ArsR) was obtained by aligning the ArsR binding sequences among the O/P sequences of arsRBC, cadCA, smtS2/S1, smtS4/S3, ziaA, czrAB and nmtAAXAXTCAAATAXXTATTTGAXTXTA, underlined base pairs are non-conserved base pairs, fig. 1C), of the 4 non-conserved base pairs, 2 base pairs are located on each side of the inverted repeat region, taxaxtaata xx tattttgaxtxta. To investigate the contribution of these non-conserved base pairs in ArsR binding, we systematically designed different nucleotides (T A, TT, TC, TG, AA, AT, AC, AG, CA, CT, CC, CG, GA, GT, GC, and GG) to the left of the repeat region, and generated corresponding complementary nucleotides to the right of the repeat region, to construct the sequence sECBS-CS1m (having the nucleotide sequence of sECBS-CS1 m), respectively

Shown as SEQ ID N O.31), sECBS-CS2m (the nucleotide sequence is

Shown as SEQ ID NO. 32), sECBS-CS3m (the nucleotide sequence is

Shown as SEQ ID NO. 33), sECBS-CS4m (the nucleotide sequence is

Shown as SEQ ID NO. 34), sECBS-CS5m (the nucleotide sequence is

Shown as SEQ ID NO. 35), sECBS-CS6m (the nucleotide sequence is

As shown in SEQ ID NO. 36), sECBS-CS7m (the nucleotide sequence is

Shown as SEQ ID NO. 37), sECBS-CS8m (the nucleotide sequence is

As shown in SEQ ID NO. 38), sECBS-CS9m (which isThe nucleotide sequence is

Shown as SEQ ID NO. 21), sECBS-CS10m (the nucleotide sequence is shown as

As shown in SEQ ID NO. 39), sECBS-CS11m (the nucleotide sequence is shown as

Shown as SEQ ID NO. 40), sECBS-CS12m (the nucleotide sequence is

Shown as SEQ ID NO. 9), sECBS-CS13m (the nucleotide sequence is

As shown in SEQ ID NO. 41), sECBS-CS14m (the nucleotide sequence of which is

Shown as SEQ ID NO. 42), sECBS-CS15m (the nucleotide sequence is

Shown as SEQ ID NO. 22) and sECBS-CS16m (the nucleotide sequence of which is shown as

As shown in SEQ ID No. 43), as shown in FIG. 5A. We constructed a series of probes, the sECBS-CS1m probe (the sense strand of the sECBS-CS1m probe: 5-TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TATAATCAAATAGCTATTTGATTATA-3' (shown in SEQ ID No. 31), and the antisense strand is: 5'-TATAATCAAATAGCTATTTGATTATAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 44); biotin labeled at the 5' end of the sense strand), the scecbs-CS 2m probe (the sense strand of the scecbs-CS 2m probe is: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TATATTCAAATAGCTATTTGAATATA-3' (shown in SEQ ID NO. 32), and the antisense strand is: 5'-TATATTCAAATAGCTATTTGAATATAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 45); biotin labeled at the 5' end of the sense strand), the scecbs-CS 3m probe (the sense strand of the scecbs-CS 3m probe is: 5' -TTCGTTAAGTCATATATGTTTTTGACTTTTT-TATACTCAAATAGCTATTTGAGTATA-3' (SEQ ID NO. 33), the antisense strand being: 5'-TATACTCAAATAGCTATTTGAGTATAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 46); biotin labeled at the 5' end of the sense strand), the scecbs-CS 4m probe (the sense strand of the scecbs-CS 4m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TATAGTCAAATAGCTATTTGACTATA-3' (shown in SEQ ID NO. 34), and the antisense strand is: 5'-TATAGTCAAATAGCTATTTGACTATAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 47); biotin labeled at the 5' end of the sense strand), the scecbs-CS 5m probe (the sense strand of the scecbs-CS 5m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAAAATCAAATAGCTATTTGATTTTA-3' (shown in SEQ ID NO. 35), and the antisense strand is: 5'-TAAAATCAAATAGCTATTTGATTTTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 48); biotin labeled at the 5' end of the sense strand), the scecbs-CS 6m probe (the sense strand of the scecbs-CS 6m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAAATTCAAATAGCTATTTGAATTTA-3' (shown in SEQ ID NO. 36), and the antisense strand is: 5'-TAAATTCAAATAGCTATTTGAATTTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 49); biotin labeled at the 5' end of the sense strand), the scecbs-CS 7m probe (the sense strand of the scecbs-CS 7m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAAACTCAAATAGCTATTTGAGTTTA-3' (shown in SEQ ID NO. 37), and the antisense strand is: 5'-TAAACTCAAATAGCTATTTGAGTTTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown as SEQ ID NO. 50); biotin labeled at the 5' end of the sense strand), the scecbs-CS 8m probe (the sense strand of the scecbs-CS 8m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTTTTT-TAAAGTCAAATAGCTATTTGACTTTA-3' (SEQ ID NO. 38) and the antisense strand: 5'-TAAAGTCAAATAGCTATTTGACTTTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 51); biotin labeled at the 5' end of the sense strand), the scecbs-CS 9m probe (sense strand of the scecbs-CS 9m probe: 5' -TTCGTTAAGTCATATATGTTTTTGACTTTTT-TACAATCAAATAGCTATTTGATTGTA-3' (SEQ ID NO. 21), the antisense strand being: 5'-TACAATCAAATAGCTATTTGATTGTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 52); biotin labeled at the 5' end of the sense strand), the scecbs-CS 10m probe (the sense strand of the scecbs-CS 10m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TACATTCAAATAGCTATTTGAATGTA-3' (shown in SEQ ID NO. 39), and the antisense strand is: 5'-TACATTCAAATAGCTATTTGAATGTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 53); biotin labeled at the 5' end of the sense strand), the scecbs-CS 11m probe (the sense strand of the scecbs-CS 11m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TACACTCAAATAGCTATTTGAGTGTA-3' (shown in SEQ ID NO. 40), and the antisense strand is: 5'-TACACTCAAATAGCTATTTGAGTGTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 54); biotin labeled at the 5' end of the sense strand), the scecbs-CS 12m probe (the sense strand of the scecbs-CS 12m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TACAGTCAAATAGCTATTTGACTGTA-3' (shown in SEQ ID NO. 9), and the antisense strand is: 5'-TACAGTCAAATAGCTATTTGACTGTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown as SEQ ID NO. 55); biotin-labeled at the 5' end of the sense strand), sECBS-CS13m ProbeNeedle (sense strand of sECBS-CS13m probe: 5-TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAGAATCAAATAGCTATTTGATTCTA-3' (shown in SEQ ID No. 41), and the antisense strand is: 5'-TAGAATCAAATAGCTATTTGATTCTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown as SEQ ID NO. 56); biotin labeled at the 5' end of the sense strand), the scecbs-CS 14m probe (the sense strand of the scecbs-CS 14m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAGATTCAAATAGCTATTTGAATCTA-3' (shown in SEQ ID NO. 42), and the antisense strand is: 5'-TAGATTCAAATAGCTATTTGAATCTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 57); biotin labeled at the 5' end of the sense strand), the scecbs-CS 15m probe (the sense strand of the scecbs-CS 15m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTT-TTT-TAGACTCAAATAGCTATTTGAGTCTA-3' (shown in SEQ ID NO. 22), and the antisense strand is: 5'-TAGACTCAAATAGCTATTTGAGTCTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown as SEQ ID NO. 58); biotin labeled at the 5' end of the sense strand) and the scecbs-CS 16m probe (the sense strand of the scecbs-CS 16m probe was: 5' -TTCGTTAAGTCATATATGTTTTTGACTTTTT-TAGAGTCAAATAGCTATTTGACTCTA-3' (SEQ ID NO. 43), the antisense strand being: 5'-TAGAGTCAAATAGCTATTTGACTCTAAAAAAGTCAAAAACATATATGACTTAACGAA-3' (shown in SEQ ID NO. 59); the 5' end of the sense strand is labeled with biotin). These probes labeled with biotin were mixed with arsenic-treated or non-arsenic cell lysate of e.coli DH5 α and subjected to filter plate assay. As shown in FIG. 5B, probes sECBS-CS9m and sECBS-CS10m bound far more strongly to arsenic-treated or non-treated cell lysates than probes ECBS-AFBS, ECBS-arsRBBS, ECBS-sm 2/1BS or ECBS-CS, while the other probe sECBS-CS15m bound less strongly to arsenic-treated or non-treated cell lysates than probes ECBS-AFBS, ECBS-arsRBBS, ECBS-sm 2/1BS or ECBS-CS. Thus, non-conserved bases can alter the binding affinity of the sequence to ArsR. Furthermore, we found that one probe, sECBS-CS12m, bound strongly to proteins in control cell lysates, but weakly to proteins in arsenic-treated cell lysates, and bound to cell lysates with or without arsenic treatment more differentially than any other probe (FIG. 5C).

2.6 contribution of non-conserved nucleotides to ArsR protein binding and luciferase Gene transcription Induction

Based on the results of the filter plate assay, we selected three probes, the strongest sECBS-CS9 m-binding probe, the weakest sECBS-CS15 m-binding probe, and the sECBS-CS12 m-probe with the highest luciferase gene transcription induction, for EMSA analysis. As shown in FIG. 6A, the intensity of the displacement band in the EMSA corresponds to the binding intensity in the filter assay, with the strongest sECBS-CS9m probe and the weakest sECBS-CS15m probe. The difference in the binding strength of the shift band of the sECBS-CS12m probe between control cells and arsenic-treated cell lysates was greatest. These results again demonstrate that changes in non-conserved base pairs can lead to differences in ArsR protein binding. Furthermore, changes in non-conserved base pairs can enhance removal of the ArsR binding protein by arsenic treatment, a function beyond binding itself.

To investigate the effect of the minority binding sequences on the transcription induction of arsenic-responsive luciferase genes, we replaced the ECBS-AFBS sequence in the pECBS-AFBS reporter vector with the sequences sECBS-CS12m, sECBS-CS9m and sECBS-CS15m to construct the report vectors psECBS-CS12m (whose nucleotide sequence is shown in SEQ ID NO.4, replacing ECBS-AFBS on the reporter vector pLLPars9 with sECBS-CS12m), psECBS-CS9m (replacing ECBS-AFBS on the reporter vector pLLPars9 with sECBS-CS9m), and psECBS-CS15m (replacing ECBS-AFBS on the reporter vector pLLPars9 with sECBS-CS15 m). As expected, the transcriptional induction of the arsenic-responsive luciferase gene by psECBS-CS12m was superior to that of the other two reporter vectors, psECBS-CS9m and psECBS-CS15m (FIG. 6B). In addition, we compared pseCBS-CS12m with pECBS-AFBS and pECBS-CS. Coli cells transformed with the reporter vector were treated with 10 μ M sodium arsenite for 15, 30, 60 and 120 minutes. As shown in FIG. 6C, pseCBS-CS12m was significantly superior to pECBS-AFBS and pECBS-CS for arsenic-mediated transcription induction of luciferase genes.

Discussion of 3

The metal-induced operon generally contains an inverted repeat region of 12-2-12 structure, except that the smt operon of S2/S1 and S4/S3 has two inverted repeats. Each repeat region is occupied by an ArsR homodimer. In our previous studies of arsenic response reporter vectors, we designed two binding sequences ECBS-AFBS like the smt operon and found that the transcription induction of the luciferase gene under arsenic treatment was stronger than that of the luciferase gene under arsenic treatment with a single binding sequence. We also found that the induction of gene transcription in response to arsenic with these two different binding sequences was superior to the induction of the same sequence of two ECBS or two AFBS. In this study we found that ECBS must be located at the position of the first binding sequence, and its gene transcription induction on arsenic response is significantly reduced if ECBS is replaced by other binding sequences such as smt2/1BS or arsRBCBS. Furthermore, AFBS at the position of the second binding sequence can be replaced by other binding sequences, but the induction is not significantly affected. This indicates that strong gene transcription induction requires the two binding sequences to be arranged in the proper order. Since the two binding sequences bind to the two dimers, respectively, the protein-DNA complex can be stabilized by dimer-dimer interactions. After changing the order of the two binding sequences, i.e., AFBS-ECBS, binding between the two dimers is still possible, but the order may affect the interaction of arsenic with the repressor protein or affect the removal of the repressor protein from the binding sequence.

protein-DNA recognition is now more complex than previously recognized. A binding moiety in a DNA sequence can be analyzed using a simple PWM model. Recent studies have shown, however, that PWM models fail to account for more complex gene transcription regulation, such as nucleotides flanking conserved sequences that affect their affinity for protein binding. In this study, we found that the base pair at the non-conserved position within the binding sequence such as sECBS-CS15m can lead to lower binding to ArsR protein, while the base pair at the non-conserved position within the binding sequence such as sECBS-CS9m can lead to higher binding to ArsR protein. Although both binding sequences still retain the conserved sequences within the binding sequence, this cannot be explained using the PWM model. More interestingly, our studies demonstrated that non-conserved site base pairs can influence the gene transcription induction of arsenic response beyond the DNA binding function itself. We found that sECBS-CS12m binds as strongly to ArsR as ECBS-CS, but sECBS-CS12m responds much better to arsenic than ECBS-CS. They bind ArsR at similar levels but the arsenic response gene transcription induction rate is different, indicating that arsenic treatment removes the binding protein ArsR from the DNA binding sequence of CS12m faster than from the DNA binding sequence of CS. Thus, as with AFBS-ECBS, interaction of these non-conserved base pairs with a repressor protein may affect binding of arsenic to the repressor protein or arsenic-induced conformational changes in the repressor protein, thereby blocking removal of the repressor protein from the binding sequence. This is shown by AFBS-ECBS being insensitive to arsenic, while sECBS-CS12m becomes more sensitive to arsenic.

Arsenic is widely distributed throughout the environment as a naturally occurring element. Arsenic from long term exposure to drinking water and food can cause a variety of diseases in humans. To prevent further arsenic exposure, rapid, cost-effective field techniques are needed to monitor arsenic in the feed water. Bacteria-based assays are an emerging technology that can monitor arsenic-induced gene expression in cases of arsenic contamination. Bacteria-based assays are both reliable and inexpensive for detecting arsenic in the field, compared to traditional, equipment-based methods that are not suitable for field detection. More significantly, they can measure the difference in arsenic bioavailability at different exposures and doses. The key components of the bacteria-based assay are a reporter vector, including a promoter/operator (or operon) and a reporter gene. A good reporter vector should exhibit high sensitivity and specificity, low endogenous background, and a wide dynamic response range. In previous studies, we constructed the pLLPars9 reporter vector (i.e., pECBS-AFBS in this study) and demonstrated that it was the best reporter vector for arsenic response constructed to date. In this study, we demonstrated that the reporter vector psECBS-CS12m is significantly superior to pLLPars 9. The study also provided innovative strategies to construct better reporter vectors, which facilitated the development of more sensitive biosensors that monitor environmental arsenic by inducing reporter gene expression.

Sequence listing

<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)

<120> a method for rapidly and efficiently analyzing interaction of transcription factor and target DNA binding sequence thereof

<160> 59

<170> SIPOSequenceListing 1.0

<210> 1

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cacattcgtt aagtcatata tgtttttgac tttttatcca cgaatatttc ttgca 55

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atccacgaat atttcttgca 20

<210> 3

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gctaaacaca tgaacagtta ttcagatatt caaa 34

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgtgattaat catatgcgtt tttggttatg tgtt 34

<210> 5

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cacattcgtt aagtcatata tgtttttgac tt 32

<210> 6

<211> 5654

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 60

gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 120

ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 180

tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 240

aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 300

ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 360

ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 420

ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 480

gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 540

acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact 600

ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa 660

aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc 720

actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc 780

ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa 840

agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc 900

agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc 960

tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc 1020

gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac 1080

tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt 1140

gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt 1200

agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg 1260

tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt 1320

cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc 1380

aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga acacattcgt 1440

taagtcatat atgtttttga ctttttatcc acgaatattt cttgcagtat tgaaagcttt 1500

gtgattaatc atatgcgttt ttggttatgt gttgtttgac ttaatatcag agccgagaga 1560

tacttgtttt ctacaaagga gagggaaatg ttgcaactaa caccacttca gttatttaaa 1620

aacctgtccg atgaaacccg tttgggtatc gtgttgttgc tcagggagat gggagagttg 1680

tgcgtgtgtg atctttgcat ggcactggat caatcacagc ccaaaatatc ccgtcatctg 1740

gcgatgctac gggaaagtgg aatccttctg gatcgtaaac agggaaaatg ggttcactac 1800

cgcttatcac cgcatattcc ttcatgggct gcccagatta ttgagcaggc ctggttaagc 1860

caacaggacg acgttcaggt catcgcacgc aagccggatc ctggaagacg ccaaaaacat 1920

aaagaaaggc ccggcgccat tctatccgct ggaagatgga accgctggag agcaactgca 1980

taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat 2040

cgaggtggac atcacttacg ctgagtactt cgaaatgtcc gttcggttgg cagaagctat 2100

gaaacgatat gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca 2160

attctttatg ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga 2220

catttataat gaacgtgaat tgctcaacag tatgggcatt tcgcagccta ccgtggtgtt 2280

cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa aaaaagctcc caatcatcca 2340

aaaaattatt atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt 2400

cgtcacatct catctacctc ccggttttaa tgaatacgat tttgtgccag agtccttcga 2460

tagggacaag acaattgcac tgatcatgaa ctcctctgga tctactggtc tgcctaaagg 2520

tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg catgccagag atcctatttt 2580

tggcaatcaa atcattccgg atactgcgat tttaagtgtt gttccattcc atcacggttt 2640

tggaatgttt actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag 2700

atttgaagaa gagctgtttc tgaggagcct tcaggattac aagattcaaa gtgcgctgct 2760

ggtgccaacc ctattctcct tcttcgccaa aagcactctg attgacaaat acgatttatc 2820

taatttacac gaaattgctt ctggtggcgc tcccctctct aaggaagtcg gggaagcggt 2880

tgccaagagg ttccatctgc caggtatcag gcaaggatat gggctcactg agactacatc 2940

agctattctg attacacccg agggggatga taaaccgggc gcggtcggta aagttgttcc 3000

attttttgaa gcgaaggttg tggatctgga taccgggaaa acgctgggcg ttaatcaaag 3060

aggcgaactg tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca atccggaagc 3120

gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag cttactggga 3180

cgaagacgaa cacttcttca tcgttgaccg cctgaagtct ctgattaagt acaaaggcta 3240

tcaggtggct cccgctgaat tggaatccat cttgctccaa caccccaaca tcttcgacgc 3300

aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt 3360

ggagcacgga aagacgatga cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac 3420

aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac gaagtaccga aaggtcttac 3480

cggaaaactc gacgcaagaa aaatcagaga gatcctcata aaggccaaga agggcggaaa 3540

gatcgccgtg taagtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg 3600

tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc 3660

gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc 3720

gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc 3780

ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg 3840

gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc 3900

cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg 3960

tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc 4020

agcctaactt cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc 4080

acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg 4140

ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg 4200

ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg 4260

cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc 4320

ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt 4380

tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac 4440

gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa 4500

tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcccct acgtgctgct 4560

gaagttgccc gcaacagaga gtggaaccaa ccggtgatac cacgatacta tgactgagag 4620

tcaacgccat gagcggcctc atttcttatt ctgagttaca acagtccgca ccgctgtccg 4680

gtagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct 4740

ttatcatgca actcgtagga caggtgccgg cagcgcccaa cagtcccccg gccacggggc 4800

ctgccaccat acccacgccg aaacaagcgc cctgcaccat tatgttccgg atctgcatcg 4860

caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctaaccgt 4920

ttttatcagg ctctgggagg cagaataaat gatcatatcg tcaattatta cctccacggg 4980

gagagcctga gcaaactggc ctcaggcatt tgagaagcac acggtcacac tgcttccggt 5040

agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga 5100

accgacgacc gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt 5160

attcaggcgt agcaaccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc 5220

ccgccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag 5280

ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc 5340

gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt 5400

aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata 5460

aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg 5520

tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt 5580

tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct 5640

ttcattgcca tacg 5654

<210> 7

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctagaattcg ttaagtcata tatgtttttg acttttttac agtcaaatag ctatttgact 60

gtagtattga a 71

<210> 8

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agctttcaat actacagtca aatagctatt tgactgtaaa aaagtcaaaa acatatatga 60

cttaacgaat t 71

<210> 9

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttcgttaagt catatatgtt tttgactttt ttacagtcaa atagctattt gactgta 57

<210> 10

<211> 5656

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 60

gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 120

ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 180

tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 240

aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 300

ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 360

ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 420

ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 480

gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 540

acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact 600

ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa 660

aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc 720

actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc 780

ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa 840

agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc 900

agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc 960

tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc 1020

gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac 1080

tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt 1140

gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt 1200

agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg 1260

tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt 1320

cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc 1380

aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga attcgttaag 1440

tcatatatgt ttttgacttt tttacagtca aatagctatt tgactgtagt attgaaagct 1500

ttgtgattaa tcatatgcgt ttttggttat gtgttgtttg acttaatatc agagccgaga 1560

gatacttgtt ttctacaaag gagagggaaa tgttgcaact aacaccactt cagttattta 1620

aaaacctgtc cgatgaaacc cgtttgggta tcgtgttgtt gctcagggag atgggagagt 1680

tgtgcgtgtg tgatctttgc atggcactgg atcaatcaca gcccaaaata tcccgtcatc 1740

tggcgatgct acgggaaagt ggaatccttc tggatcgtaa acagggaaaa tgggttcact 1800

accgcttatc accgcatatt ccttcatggg ctgcccagat tattgagcag gcctggttaa 1860

gccaacagga cgacgttcag gtcatcgcac gcaagccgga tcctggaaga cgccaaaaac 1920

ataaagaaag gcccggcgcc attctatccg ctggaagatg gaaccgctgg agagcaactg 1980

cataaggcta tgaagagata cgccctggtt cctggaacaa ttgcttttac agatgcacat 2040

atcgaggtgg acatcactta cgctgagtac ttcgaaatgt ccgttcggtt ggcagaagct 2100

atgaaacgat atgggctgaa tacaaatcac agaatcgtcg tatgcagtga aaactctctt 2160

caattcttta tgccggtgtt gggcgcgtta tttatcggag ttgcagttgc gcccgcgaac 2220

gacatttata atgaacgtga attgctcaac agtatgggca tttcgcagcc taccgtggtg 2280

ttcgtttcca aaaaggggtt gcaaaaaatt ttgaacgtgc aaaaaaagct cccaatcatc 2340

caaaaaatta ttatcatgga ttctaaaacg gattaccagg gatttcagtc gatgtacacg 2400

ttcgtcacat ctcatctacc tcccggtttt aatgaatacg attttgtgcc agagtccttc 2460

gatagggaca agacaattgc actgatcatg aactcctctg gatctactgg tctgcctaaa 2520

ggtgtcgctc tgcctcatag aactgcctgc gtgagattct cgcatgccag agatcctatt 2580

tttggcaatc aaatcattcc ggatactgcg attttaagtg ttgttccatt ccatcacggt 2640

tttggaatgt ttactacact cggatatttg atatgtggat ttcgagtcgt cttaatgtat 2700

agatttgaag aagagctgtt tctgaggagc cttcaggatt acaagattca aagtgcgctg 2760

ctggtgccaa ccctattctc cttcttcgcc aaaagcactc tgattgacaa atacgattta 2820

tctaatttac acgaaattgc ttctggtggc gctcccctct ctaaggaagt cggggaagcg 2880

gttgccaaga ggttccatct gccaggtatc aggcaaggat atgggctcac tgagactaca 2940

tcagctattc tgattacacc cgagggggat gataaaccgg gcgcggtcgg taaagttgtt 3000

ccattttttg aagcgaaggt tgtggatctg gataccggga aaacgctggg cgttaatcaa 3060

agaggcgaac tgtgtgtgag aggtcctatg attatgtccg gttatgtaaa caatccggaa 3120

gcgaccaacg ccttgattga caaggatgga tggctacatt ctggagacat agcttactgg 3180

gacgaagacg aacacttctt catcgttgac cgcctgaagt ctctgattaa gtacaaaggc 3240

tatcaggtgg ctcccgctga attggaatcc atcttgctcc aacaccccaa catcttcgac 3300

gcaggtgtcg caggtcttcc cgacgatgac gccggtgaac ttcccgccgc cgttgttgtt 3360

ttggagcacg gaaagacgat gacggaaaaa gagatcgtgg attacgtcgc cagtcaagta 3420

acaaccgcga aaaagttgcg cggaggagtt gtgtttgtgg acgaagtacc gaaaggtctt 3480

accggaaaac tcgacgcaag aaaaatcaga gagatcctca taaaggccaa gaagggcgga 3540

aagatcgccg tgtaagtcga ccgatgccct tgagagcctt caacccagtc agctccttcc 3600

ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac 3660

tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga 3720

gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag 3780

ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca 3840

tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct 3900

tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc 3960

tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc gcggctctta 4020

ccagcctaac ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc gcctcggcga 4080

gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc tgcctccccg 4140

cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct 4200

cgctaacgga ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa 4260

tgcgcaaacc aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac 4320

gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc 4380

gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat 4440

acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg 4500

aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtccc ctacgtgctg 4560

ctgaagttgc ccgcaacaga gagtggaacc aaccggtgat accacgatac tatgactgag 4620

agtcaacgcc atgagcggcc tcatttctta ttctgagtta caacagtccg caccgctgtc 4680

cggtagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt 4740

ctttatcatg caactcgtag gacaggtgcc ggcagcgccc aacagtcccc cggccacggg 4800

gcctgccacc atacccacgc cgaaacaagc gccctgcacc attatgttcc ggatctgcat 4860

cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctaacc 4920

gtttttatca ggctctggga ggcagaataa atgatcatat cgtcaattat tacctccacg 4980

gggagagcct gagcaaactg gcctcaggca tttgagaagc acacggtcac actgcttccg 5040

gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct 5100

gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc attcatccgc ttattatcac 5160

ttattcaggc gtagcaacca ggcgtttaag ggcaccaata actgccttaa aaaaattacg 5220

ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga 5280

agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt 5340

gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt 5400

ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa 5460

taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata 5520

tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag 5580

tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt 5640

ctttcattgc catacg 5656

<210> 11

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atccacgaat atttcttgca tttcacattc gttaagtcat atatgttttt gactt 55

<210> 12

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cacattcgtt aagtcatata tgtttttgac tttttgctaa acacatgaac agttattcag 60

atattcaaa 69

<210> 13

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cacattcgtt aagtcatata tgtttttgac ttttttgtga ttaatcatat gcgtttttgg 60

ttatgtgtt 69

<210> 14

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctaaacaca tgaacagtta ttcagatatt caaatttatc cacgaatatt tcttgca 57

<210> 15

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tgtgattaat catatgcgtt tttggttatg tgtttttatc cacgaatatt tcttgca 57

<210> 16

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

taaaatcaaa tacgtatttg attatattta tccacgaata tttcttgca 49

<210> 17

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tgtgattaat catatgcgtt tttggttatg tgttttttaa aatcaaatac gtatttgatt 60

ata 63

<210> 18

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atccacgaat atttcttgca ttttaaaatc aaatacgtat ttgattata 49

<210> 19

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cacattcgtt aagtcatata tgtttttgac ttttttaaaa tcaaatacgt atttgattat 60

a 61

<210> 20

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cacattcgtt aagtcatata tgtttttgac tttaaaatca aatacgtatt tgattata 58

<210> 21

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ttcgttaagt catatatgtt tttgactttt ttacaatcaa atagctattt gattgta 57

<210> 22

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ttcgttaagt catatatgtt tttgactttt ttagactcaa atagctattt gagtcta 57

<210> 23

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

taaaatcaaa tacgtatttg attata 26

<210> 24

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tgcaagaaat attcgtggat aaaaagtcaa aaacatatat gacttaacga atgtg 55

<210> 25

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

aagtcaaaaa catatatgac ttaacgaatg tgaaatgcaa gaaatattcg tggat 55

<210> 26

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tataatcaaa tacgtatttg attttaaaaa agtcaaaaac atatatgact taacgaatgt 60

g 61

<210> 27

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

taaaatcaaa tacgtatttg attatatttc acattcgtta agtcatatat gtttttgact 60

t 61

<210> 28

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

aagtcaaaaa catatatgac ttaacgaatg tgaaatataa tcaaatacgt atttgatttt 60

a 61

<210> 29

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tataatcaaa tacgtatttg attttaaagt caaaaacata tatgacttaa cgaatgtg 58

<210> 30

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ttcgttaagt catatatgtt tttgactt 28

<210> 31

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ttcgttaagt catatatgtt tttgactttt ttataatcaa atagctattt gattata 57

<210> 32

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ttcgttaagt catatatgtt tttgactttt ttatattcaa atagctattt gaatata 57

<210> 33

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ttcgttaagt catatatgtt tttgactttt ttatactcaa atagctattt gagtata 57

<210> 34

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ttcgttaagt catatatgtt tttgactttt ttatagtcaa atagctattt gactata 57

<210> 35

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

ttcgttaagt catatatgtt tttgactttt ttaaaatcaa atagctattt gatttta 57

<210> 36

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ttcgttaagt catatatgtt tttgactttt ttaaattcaa atagctattt gaattta 57

<210> 37

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ttcgttaagt catatatgtt tttgactttt ttaaactcaa atagctattt gagttta 57

<210> 38

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ttcgttaagt catatatgtt tttgactttt ttaaagtcaa atagctattt gacttta 57

<210> 39

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ttcgttaagt catatatgtt tttgactttt ttacattcaa atagctattt gaatgta 57

<210> 40

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

ttcgttaagt catatatgtt tttgactttt ttacactcaa atagctattt gagtgta 57

<210> 41

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

ttcgttaagt catatatgtt tttgactttt ttagaatcaa atagctattt gattcta 57

<210> 42

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ttcgttaagt catatatgtt tttgactttt ttagattcaa atagctattt gaatcta 57

<210> 43

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

ttcgttaagt catatatgtt tttgactttt ttagagtcaa atagctattt gactcta 57

<210> 44

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

tataatcaaa tagctatttg attataaaaa agtcaaaaac atatatgact taacgaa 57

<210> 45

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

tatattcaaa tagctatttg aatataaaaa agtcaaaaac atatatgact taacgaa 57

<210> 46

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

tatactcaaa tagctatttg agtataaaaa agtcaaaaac atatatgact taacgaa 57

<210> 47

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

tatagtcaaa tagctatttg actataaaaa agtcaaaaac atatatgact taacgaa 57

<210> 48

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

taaaatcaaa tagctatttg attttaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 49

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

taaattcaaa tagctatttg aatttaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 50

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

taaactcaaa tagctatttg agtttaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 51

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

taaagtcaaa tagctatttg actttaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 52

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

tacaatcaaa tagctatttg attgtaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 53

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

tacattcaaa tagctatttg aatgtaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 54

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

tacactcaaa tagctatttg agtgtaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 55

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

tacagtcaaa tagctatttg actgtaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 56

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

tagaatcaaa tagctatttg attctaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 57

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

tagattcaaa tagctatttg aatctaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 58

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

tagactcaaa tagctatttg agtctaaaaa agtcaaaaac atatatgact taacgaa 57

<210> 59

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

tagagtcaaa tagctatttg actctaaaaa agtcaaaaac atatatgact taacgaa 57

Claims (6)

1. A method for rapidly and efficiently screening interaction of a transcription factor and a target DNA binding sequence thereof is characterized by comprising the following steps: preparing cell lysate containing transcription factors, designing a DNA double-strand probe capable of being specifically combined with the transcription factors according to a target DNA combination sequence of the transcription factors, marking biotin at the 5' end of a sense strand of the DNA double-strand probe, combining the transcription factors in the cell lysate with the DNA double-strand probe to obtain a protein-probe compound, combining the protein-probe compound on a nitrocellulose membrane, washing off free DNA double-strand probe, denaturing the protein in the protein-probe compound combined on the nitrocellulose membrane by SDS treatment, releasing the combined DNA double-strand probe, collecting the released DNA double-strand probe, hybridizing the denatured DNA double-strand probe with a complementary sequence of the sense strand of the DNA double-strand probe pre-combined on the nitrocellulose membrane, and sequentially adding streptavidin marked by horseradish peroxidase and a chemiluminescent substrate, recording the luminescence condition, and judging the binding strength of a transcription factor and a target DNA binding sequence thereof according to the luminescence condition, wherein the transcription factor is arsenic transcription inhibiting factor arsR protein, the target DNA binding sequence of the screened arsR protein is ESBS-CS12m, the sequence of a sense strand of ESBS-CS12m is shown as SEQ ID No.9, and the sequence of an antisense strand of ESBS-CS12m is shown as SEQ ID No. 55.

2. The method of claim 1, comprising the steps of: preparing cell lysate containing transcription factor, designing DNA double-strand probe capable of specifically binding with the transcription factor according to the target DNA binding sequence of the transcription factor, labeling biotin at the 5' end of the sense strand of the DNA double-strand probe, forming a binding reaction system of the cell lysate containing transcription factor and the DNA double-strand probe, culturing at 25 ℃ for 30 minutes to obtain a protein-probe complex, adding the protein-probe complex to a 96-well plate of a nitrocellulose membrane substrate pre-cleaned by a cleaning buffer solution, culturing on ice to bind the protein-probe complex to the nitrocellulose membrane, inverting and centrifuging, discarding an effluent, cleaning the 96-well plate by the cleaning buffer solution, adding an elution buffer solution to denature protein in the protein-probe complex bound to the nitrocellulose membrane, releasing the bound DNA double-strand probe, collecting the released DNA double-strand probe, heating at 95 ℃ to denature the DNA double-strand probe, adding a hybridization buffer solution and the denatured DNA double-strand probe into a nitrocellulose membrane-based 96-well plate pre-combined with a sense strand complementary sequence of the DNA double-strand probe, hybridizing at 42 ℃, washing the 96-well plate by using a washing buffer solution, then adding streptavidin marked by horseradish peroxidase, incubating for 30 minutes at 37 ℃, washing the 96-well plate by using the washing buffer solution, adding a chemiluminescent substrate Luminol of the horseradish peroxidase, reading the number of photons by using a chemiluminescence instrument, recording the luminescence condition by using a relative light unit, and judging the binding strength of the transcription factor and a target DNA binding sequence thereof according to the luminescence condition.

3. The method of claim 2, wherein the binding reaction system is 20 μ L comprising 2 μ L of cell lysate, 10 μ L of 2 Xbinding buffer mixture, 1 μ L of DNA double-stranded probe, and 7 μ L of ddH ₂ O, said 2 × binding buffer mixture is: 40mM HEPES, 20mM ammonium sulfate, 2mM DTT, 20mM KCl and 0.4% Tween-20, the balance being water, pH 7.6.

4. The method of claim 2, wherein the wash buffer is: 100mM Tris-HCl, 2.5mM EDTA and 0.1% Tween-20, the balance being water, pH 7.6.

5. The method of claim 2, wherein the elution buffer is: 0.5% SDS, 100mM Tris-HCl, 2.5mM EDTA and 0.1% Tween-20, the balance water, pH 7.6.

6. The method of claim 2, wherein the hybridization buffer is: 20mM HEPES, 10mM ammonium sulfate, 10mM KCl and 0.2% Tween-20, the balance being water, pH 7.6.

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