CN115094127A - Method for in-situ detection of binding position of protein and deoxyribonucleotide - Google Patents

Abstract

The invention discloses a method for detecting the binding position of protein and deoxyribonucleotide in situ, which comprises the steps of constructing a target strain for expressing cytosine deoxyribonucleotide deaminase-target protein fusion protein and a control strain for expressing target protein, wherein the target strain and the control strain do not contain uracil DNA glycosidase genes; after inducing the protein expression of the target strain and the control strain, extracting the genome DNA of the target strain and the control strain; and (3) performing high-throughput sequencing on the genome DNA, analyzing to obtain point mutations of the genome DNA of the target strain and the genome DNA of the control strain, removing the point mutation shared by the target strain and the control strain, and screening the point mutation in the non-coding region from the rest point mutations of the target strain to obtain the mutant. The method utilizes the mutation capability of pyrimidine deoxynucleotide deaminase in bacteria on DNA, constructs a target strain for expressing fusion protein, and then obtains a final result only by simple genome sequencing, thereby being convenient and quick.

Description

Method for in-situ detection of binding position of protein and deoxyribonucleotide

Technical Field

The invention belongs to the technical field of synthetic biology, and particularly relates to a method for detecting a binding position of a protein and deoxyribonucleotide in situ.

Background

The binding site of a transcriptional regulator protein on its genomic DNA is usually a conserved nucleotide sequence, and finding the binding site of a target transcription factor on the genomic DNA can help researchers find out which genes the transcription factor regulates. Therefore, the detection method of protein-DNA interaction is a key technology for analyzing gene regulation circuits and signal transduction pathways of living systems.

The most common method for detecting the interaction between protein and DNA at present is chromatin co-immunoprecipitation-sequencing technology, and the specific method of the method in bacteria is to firstly over-express a transcription factor fused with a tag sequence, then break, enrich and separate DNA fragments combined with the transcription factor, and finally obtain the DNA sequences by a sequencing method. In addition, some people have developed the Calling-cards method, which fuses the transcription factor of interest with the transposon guide protein Sir4, guides the transposon to perform transposition insertion in the transcription factor binding region on the genome, and determines the position of the transposon insertion by means of enzyme digestion, circularization and sequencing, thereby determining the binding site of the transcription factor of interest. The Calling-cards method is currently used by a relatively few people.

The defects of the existing chromatin co-immunoprecipitation-sequencing technology are as follows: 1) the operation time is long, and the labor capacity is large; 2) the method has the advantages that the number of steps is large, small errors in some experiments can be accumulated slowly, so that the finally obtained data result cannot be satisfied, and due to the small errors, troubleshooting after the experiments have problems is difficult; 3) many technical details need to be paid attention in the experimental process, for example, how protein is fixed, how DNA is purified, the ultrasonic condition of DNA breaking and the like greatly influence the final result, and for many experimenters, the technical requirements are difficult to master in a short time, so that the method does not have good popularization.

Therefore, it is important to find a method for rapidly detecting the interaction between protein and DNA.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a method for detecting the binding position of a protein and deoxyribonucleotides in situ. According to the invention, through theoretical derivation and optimized maturation of an experimental method, global scanning of a target protein binding site on a genome is realized, a bioinformatics method is utilized to predict a conserved nucleotide sequence of protein-bound deoxyribonucleotide (hereinafter, abbreviated as DNA), and the detection of the interaction between the target protein and the DNA can be realized in a microbial cell. The specific technical scheme is as follows:

the invention provides a method for detecting the binding position of a protein and deoxyribonucleotides in situ, which comprises the following steps:

1) constructing a target strain for expressing the cytosine deoxynucleotide deaminase-target protein fusion protein and a control strain for expressing the target protein;

the target strain and the control strain do not contain uracil DNA glycosidase genes;

2) after inducing the protein expression of the target strain and the control strain, extracting the genome DNA of the target strain and the control strain;

3) and performing high-throughput sequencing on the genomic DNA of the target strain and the genomic DNA of the control strain, analyzing to obtain point mutations of the genomic DNA of the target strain and the genomic DNA of the control strain, removing the point mutation shared by the target strain and the control strain, and screening the point mutation in a non-coding region from the rest point mutations of the target strain to obtain the target protein binding site.

Further, in step 3), setting n groups of genomic DNA of the target strain and the genome DNA of the control strain respectively for high-throughput sequencing, and further obtaining a target protein binding site, wherein the method specifically comprises the following steps: performing high-throughput sequencing on the genomic DNA of the n groups of target strains and the genomic DNA of the n groups of control strains, analyzing to obtain point mutations of the genomic DNA of the target strains and the genomic DNA of the control strains, screening the common point mutations of the n groups of target strains from the rest point mutations of the target strains after removing the common point mutations of the target strains and the control strains, and screening the common point mutations in the non-coding regions to obtain target protein binding sites;

n is more than or equal to 2 and less than or equal to 5.

Further, the construction method of the target strain expressing the cytosine deoxynucleotide deaminase-target protein fusion protein comprises the following steps: constructing a cytosine deoxynucleotide deaminase-target protein fusion protein expression vector, and then introducing the fusion protein expression vector into a strain without uracil DNA glycosidase genes;

the construction method of the target protein-expressing control strain comprises the following steps: a target protein expression vector was constructed, and then the target protein expression vector was introduced into a strain containing no uracil DNA glycosidase gene.

Furthermore, in the cytosine deoxynucleotide deaminase-target protein fusion protein expression vector, the cytosine deoxynucleotide deaminase is positioned at the nitrogen end of the fusion protein, the target protein is positioned at the carbon end of the fusion protein, and the cytosine deoxynucleotide deaminase and the target protein are connected by 8 glycines.

Further, the inducing protein expression of the target strain and the control strain comprises the following steps: recovering bacterial strains, selecting clone bacterial plaques, and shaking the bacterial strains for 6-20 hours at 37 ℃ by using an LB culture medium and adding an inducer;

preferably, the inducer is arabinose;

preferably, the formulation of the LB medium is: 10g/L of sodium chloride, 5g/L of yeast powder and 10g/L of peptone.

Further, the target protein is a transcription factor.

Further, the method for detecting the binding position of the protein and the deoxyribonucleotide in situ also comprises the step of calculating a conserved DNA sequence bound by the target protein, and the specific steps comprise:

with the target protein binding site obtained in the step 3) as a center, respectively extending m base pairs at the upstream and the downstream of the genome DNA to obtain a DNA fragment sequence containing the target protein binding site; m is more than or equal to 10 and less than or equal to 200;

and storing the DNA fragment sequence containing the target protein binding site into a text document, and calculating the conserved DNA sequence bound by the target protein by using a bioinformatics tool MEME Suite.

In a second aspect, the present invention provides a method for predicting the intracellular concentration of a protein transcriptionally expressed from a promoter, comprising: based on the method for detecting the combination position of the protein and the deoxyribonucleotide in situ, the transcription factor target promoter is mutated, and the intracellular concentration of the protein transcribed and expressed by the promoter is predicted according to the following formula;

x＝1-exp(-K ₅ ·[Protein]·t)

wherein the content of the first and second substances,

x represents the proportion of the mutant DNA on the transcription factor target promoter;

[ Protein ] represents the intracellular concentration of a Protein expressed by transcription of a promoter;

t represents the total time of occurrence of the mutation process;

K ₅ denotes the equilibrium constant, K ₅ Is represented as follows:

k _TIC1 、k _TIC2 is a constant number, k _translation To translation Rate, γ _mRNA Is the mRNA decomposition rate, γ _Protein The protein degradation rate.

Further, the proportion x of the mutant DNA on the transcription factor target promoter is expressed as follows:

x＝1-exp(-k _m ·t)

wherein, the first and the second end of the pipe are connected with each other,

k _m representing transcription factor binding sitesThe mutation rate of (c);

t represents the total time of occurrence of the mutation process.

Further, the proportion x of the mutant DNA on the transcription factor target promoter is expressed as follows:

x＝1-exp(-K ₃ ·θ·t)

wherein the content of the first and second substances,

θ represents the ratio occupied by the DNA bound to the transcription factor protein, and θ is expressed as follows:

[LasR]represents the concentration of the transcription factor protein, k _d Represents an equilibrium constant;

k ₃ is a constant number, k ₃ Is represented as follows:

K ₃ ＝[RNAP]·[DNA _all ]·K ₁ ·K ₂ ·k _TIC1

[RNAP]indicates the concentration of RNA polymerase, [ DNA ] _all ]Denotes the total target DNA concentration, K, in the bacterial cell ₁ And K ₂ The equilibrium constants during the recruitment of ribonucleic acid polymerase by the following transcription factors, respectively:

RNAP denotes RNA polymerase, LasR denotes transcription factor protein, RNAP-LasR ₂ DNA represents a complex of RNA polymerase with transcription factors and DNA, and LasR-TIC represents a transcription initiation complex in an open loop state to which transcription factor proteins are bound;

preferably, the θ is determined by the method for detecting the binding position of the protein to the deoxyribonucleotide in situ according to the present invention.

The invention has the beneficial effects that:

(1) the invention provides a method for detecting the binding position of protein and deoxyribonucleotide in situ, which utilizes the mutation ability of pyrimidine deoxyribonucleotide deaminase in bacteria to DNA, constructs a target strain expressing cytosine deoxyribonucleotide deaminase-target protein fusion protein, and then obtains the final result by simple genome sequencing. The method completes the construction of the cytosine deoxynucleotide deaminase-target protein fusion protein expression plasmid through basic experimental conditions such as PCR, simple molecular cloning technology and the like, is convenient, quick and time-saving, and does not need fussy experimental design. The method has low operation threshold, and only needs bacterial culture and sequencing after the construction of the strain is completed.

(2) Based on the method for detecting the binding position of the protein and the deoxyribonucleotide in situ, the obtained target protein binding site can also help to understand the transcription level of a target promoter combined by the transcription factor protein.

Drawings

FIG. 1 is a schematic diagram of an experiment for detecting binding sites of a target protein and DNA by fused cytosine deoxynucleotide deaminase;

FIG. 2 shows the base mutation effect on the rhlI gene promoter region after 3 hours, 6 hours, 9 hours and 12 hours of shake-induced bacteria induction;

FIG. 3 shows that the LasR protein can bind to multiple promoters detected by sequencing in a gel electrophoresis migration assay;

FIG. 4 is a graph of the ratio of promoter mutations at different transcription levels as a function of the transcription level of the corresponding promoter;

FIG. 5 shows the conserved nucleotide sequence prediction for LasR binding.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

The principle of the invention is that cytosine deoxynucleotide deaminase and target protein are fused together, DNA near the fused target protein is mutated by utilizing the mutation capability of the cytosine deoxynucleotide deaminase to the DNA in bacteria, and the position of gene mutation is found by means of sequencing, namely the binding site of the target protein on genome. The specific implementation of the invention comprises the steps of designing fusion protein expression, constructing a target bacterial strain, culturing bacteria, sequencing analysis and the like. The method comprises the following specific steps:

1) design of fusion protein expression: the design of fusion protein expression is a key link of the invention, and specifically comprises the expression optimization of cytosine deoxynucleotide deaminase gene and the selection of expression vectors. First, according to the codon usage of the target strain, the web page tool Jcat is used (http://www.jcat.de/) The coding sequence of the original cytosine deoxynucleotide deaminase gene was optimized to ensure that the gene used the most commonly used codons in the target strain and was synthesized by commercial companies. Then, primers of polymerase chain reaction are respectively designed for amplifying the optimized cytosine deoxynucleotide deaminase gene, the target protein gene and a plasmid vector for inducing expression of the fusion protein, and the primers are prepared to be applied to the next Gibbson assembly reaction. Preferably, the cytosine deoxynucleotide deaminase is placed at the nitrogen terminus of the fusion protein and the target protein is placed at the carbon terminus of the fusion protein, with 8 glycines in between.

2) Construction of the target Strain: after the target protein gene segment and the cytosine deoxynucleotide deaminase gene after codon optimization are obtained by amplification, two DNA segments are connected by using a method of overlap extension polymerase chain reaction, and a DNA segment fusing the two genes and an intermediate connecting sequence is obtained after purification. And connecting the DNA fragment with a linearized fragment of a plasmid vector for inducing and expressing the fusion protein through Gibbson assembly reaction, transferring the DNA fragment into an escherichia coli competent strain Top10 by a chemical method, paving a gentamicin resistant plate on the bacteria, selecting clone sequencing and verifying to finally obtain a target plasmid with correct connection.

Meanwhile, the gene fragment of the target protein is connected with the linearized fragment of a plasmid vector for inducing and expressing the fusion protein through Gibbson assembly reaction, and then the gene fragment is transferred into an escherichia coli competent strain Top10 by a chemical conversion method, a gentamicin resistant plate is paved on the bacteria, and the bacteria are selected, cloned and sequenced to verify to finally obtain the plasmid which is correctly connected and expresses the target protein.

On the other hand, the construction of a target strain in which the intracellular uracil DNA glycosidase gene of bacteria is deleted is initiated. Firstly, uracil DNA glycosidase gene on bacterial genome is found, and the gene is knocked out by means of homologous recombination to prevent the coding protein of the gene from repairing the mutant DNA base.

After obtaining the target strain with deleted bacterial intracellular uracil DNA glycosidase gene, respectively introducing the target plasmid with cloned cytosine deoxynucleotide deaminase gene and target protein gene fusion fragment and the plasmid expressing target protein into the target strain by electric transfer, selecting a single clone on a gentamycin resistant plate, identifying the correct clone by polymerase chain reaction, shaking the strain and preserving the strain to obtain the target strain containing the target plasmid and the control strain expressing the target protein without the fused cytosine deoxynucleotide deaminase gene. The stored strains were stored in 35% final glycerol and stored in a freezer at-80 ℃.

3) And (3) bacterial culture: the target strain containing the target plasmid constructed above and the control strain expressing the target protein without the fused cell pyrimidine deoxynucleotide deaminase gene were first streaked and cultured in a constant temperature incubator at 37 ℃ for 20 hours. The clone bacterial plaque is picked and shaken at 37 ℃, an LB culture medium (10 g/L of sodium chloride, 5g/L of yeast powder and 10g/L of peptone) is used, the final concentration of an inducer arabinose is 0.4 percent (mass fraction), the bacteria are shaken every time for 1 ml, and the total time of the bacteria shaking induction is 6-20 hours. The bacteria were collected by centrifugation, and genomic DNA of the bacteria was extracted using a commercial genome extraction kit.

4) Sequencing and analysis: three groups of target strain samples and three groups of control strains were set up for sequencing. Genome sequencing of bacteria was done by commercial companies. After obtaining the point mutation result of the whole genome, the point mutation common to the target strain and the control strain is first eliminated, and then the common point mutation contained in all of the three groups of target strains is found from the remaining point mutations. Further, screening out the common point mutation in the non-coding region of the gene, namely the target protein combinationThe site of (1). Then, the DNA fragment was extended forward and backward by 50 base pairs on the genome centering around each mutation site, and the DNA fragment was cut out to a length of 100 base pairs for each mutation site. Then the sequences are stored in a text document in batches and then a bioinformatics tool MEME Suite (Introduction-MEME Suite(meme-suite.org)) The conserved DNA sequence bound by the transcription factor protein is calculated.

Example 1

The feasibility of the method of the present invention will be described in detail below by taking the intracellular detection of the DNA binding site of the transcription factor LasR protein in Pseudomonas aeruginosa as an example. The experimental scheme is shown in figure 1, and the specific scheme is as follows:

1) design of expression of cytosine deoxynucleotide deaminase-LasR fusion protein

When designing the fusion protein, cytosine deoxynucleotide deaminase is placed at the nitrogen end of the fusion protein, LasR is placed at the carbon end of the fusion protein, and the middle is connected by 8 glycines.

The amino acid sequence of the transcription factor LasR protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO. 2.

The amino acid sequence of the cytosine deoxynucleotide deaminase is shown as SEQ ID NO.3, and according to the codon usage of a target strain Pseudomonas aeruginosa, a webpage tool Jcat (Jcat)http://www.jcat.de/) The coding sequence of the original cytosine deoxynucleotide deaminase gene is optimized, and the nucleotide sequence of the optimized cytosine deoxynucleotide deaminase is shown as SEQ ID NO.4 and synthesized by a commercial company.

The plasmid vector for inducing expression of the fusion protein is pJN105 plasmid vector, which is a tool plasmid capable of inducing expression of a target gene through arabinose, an arabinose-responsive promoter on the tool plasmid is used for controlling the expression of the target gene, the nucleotide sequence of the arabinose-responsive promoter is shown in SEQ ID NO.5, wherein the bold mark is an arabinose promoter sequence region, and the black triangle indicates the position of the target gene insertion.

Primers for polymerase chain reaction are respectively designed for amplifying the cytosine deoxynucleotide deaminase gene and the LasR gene segment after codon optimization and a plasmid vector pJN105 for inducing expression of the fusion protein.

2) Construction of target and control strains

The primers designed for polymerase chain reaction are used for amplifying the cytosine deoxynucleotide deaminase gene after codon optimization, the LasR gene segment and a plasmid vector pJN105 for inducing expression of the fusion protein. After amplification to obtain LasR gene segment and codon optimized cytosine deoxynucleotide deaminase gene, two segments of DNA are connected by using a method of overlap extension polymerase chain reaction, and a DNA segment fusing two gene segments and middle 8 glycine connecting sequences is obtained after purification.

The DNA fragment is connected with a linearized fragment of an pJN105 plasmid vector through Gibbson assembly reaction, then the DNA fragment is transferred into an escherichia coli competent strain Top10 by a chemical conversion method, a gentamycin resistant plate is paved on the bacteria, and clone sequencing is selected for verification, so that a target plasmid which is correctly connected and expresses the cytosine deoxynucleotide deaminase-target protein fusion protein is finally obtained.

Meanwhile, the LasR gene fragment is connected with the linearized fragment of the pJN105 plasmid vector through Gibbson assembly reaction, and then the LasR gene fragment is transferred into an escherichia coli competent strain Top10 by a chemical method, the bacteria are paved with gentamicin resistant plates, and cloning sequencing is selected for verification, so that the plasmid which is connected with the correct expression LasR protein is finally obtained.

A No. 750 gene PA0750 gene on a pseudomonas aeruginosa genome is knocked out by utilizing a homologous recombination mode, and the pseudomonas aeruginosa strain with the intracellular uracil DNA glycosidase gene deleted is obtained.

Respectively introducing the obtained target plasmid for expressing the cytosine deoxynucleotide deaminase-target protein fusion protein and the obtained plasmid for expressing the LasR protein into a pseudomonocell strain in which intracellular uracil DNA glycosidase genes are deleted through electrotransformation, selecting a monoclonal on a gentamicin resistant plate, identifying the correct clone by using polymerase chain reaction, shaking the strain and preserving the strain to obtain a target strain containing the target plasmid and a control strain which does not contain the fusion cytosine deoxynucleotide deaminase gene and expresses the LasR protein. The stored strains were stored in 35% final glycerol and stored in a freezer at-80 ℃.

3) Bacterial culture

The target strain containing the target plasmid and the control strain expressing the LasR protein without the fused cell pyrimidine deoxynucleotide deaminase gene constructed above were first streaked and cultured in a constant temperature incubator at 37 ℃ for 20 hours. The clone bacterial plaque is picked and shaken at 37 ℃, LB culture medium (10 g/L of sodium chloride, 5g/L of yeast powder and 10g/L of peptone) is used, the final concentration of the inducer arabinose is 0.4 percent (mass fraction), 1 ml of bacteria is shaken every time, and the total time of the induction of the bacteria is 12 hours. The bacteria were collected by centrifugation, and genomic DNA of the bacteria was extracted using a commercial genome extraction kit.

4) Sequencing and analysis: three groups of target strain samples and three groups of control strains were set up for sequencing. Genome sequencing of bacteria was done by commercial companies. After obtaining the point mutation result of the whole genome, the point mutation common to the target strain and the control strain is first eliminated, and then the common point mutation contained in all of the three groups of target strains is found from the remaining point mutations. Furthermore, the common point mutation in the non-coding region of the gene is screened out, and the target transcription factor binding site is obtained. Then, the DNA fragment is extended forward and backward by 50 base pairs on the genome, centered at each mutation site, and the DNA fragment sequence is cut out to a length of 100 base pairs for each mutation. Then the sequences are stored in a text document in batches and then a bioinformatics tool MEME Suite (Introduction-MEMESuite(meme-suite.org)) The conserved DNA sequence bound by the transcription factor protein was calculated.

Example 2

After streaking recovery of the target strain and the control strain constructed in example 1, the clone bacterial plaque is picked and shaken at 37 ℃, an LB culture medium (10 g/L of sodium chloride, 5g/L of yeast powder and 10g/L of peptone) is used, 1 ml of the clone bacterial plaque is shaken every time, the final concentration of the inducer arabinose is added to be 0.4% (mass fraction), and the total time of shaking is 3, 6, 9 and 12 hours. The bacteria were collected by centrifugation, and genomic DNA of the bacteria was extracted using a commercial genome extraction kit. And analyzed by the method of example 1.

As shown in FIG. 2, in the case of mutation in the promoter region known to bind to LasR (the promoter of rhlI gene) detected by shake at different times, the boxed position is the DNA base in which the mutation occurs, and the mutation rate in the promoter region increases significantly with the increase of the induction time until the mutation is close to the complete mutation.

Example 3

After streaking and recovering the target strain and the control strain constructed in example 1, colony plaques were picked and shaken at 37 ℃ and induced with LB medium (10 g/L sodium chloride, 5g/L yeast powder, 10g/L peptone) for 12 hours using 0.4% arabinose and 10 μm/L3-oxododecanoylhomoserine lactone, and then the bacteria were collected for genome sequencing. And analyzed by the method of example 1.

Mutations were obtained in a total of 16 promoter regions, of which 10 known promoters, including PA1003, PA1431, PA2426, PA2763, PA2769, PA3104, PA3326, PA3384, rhlI, PA3904, were available. In addition, 6 promoters which have not been found in the past are found, including PA0717, PA0727, PA0861, PA1131, PA3347 and PA 5295. The binding sites obtained after genome sequencing were verified by gel electrophoresis migration assay (EMSA), as shown in FIG. 3, which indicates that all the new promoters have LasR binding activity.

Example 4

Based on the fact that the ability of cytosine deoxynucleotide deaminase to mutate DNA depends on the transcription activity of the target DNA, the transcription at the corresponding position is estimated from the mutation ratio at the measured mutation position. Based on the method, a theoretical model based on steady-state assumption is established and verified through experiments. The model is as follows:

(1) the formula is a combined equilibrium reaction formula of the transcription factor and the target promoter, and the equilibrium constant is k _d . LasR represents a transcription factor protein. Let θ be the proportion occupied by DNA binding to the transcription factor protein, then θ can be expressed as:

let the total target DNA concentration in the bacterial cell be [ DNA ] _all ]At a constant value, have

[LasR ₂ -DNA]＝DNA _all ·θ (3)

The process of transcription factor recruitment to ribonucleic acid (hereinafter abbreviated as RNA) polymerase can be written as

Wherein RNAP represents RNA polymerase, RNAP-LasR ₂ DNA represents a complex of RNA polymerase with a transcription factor and DNA, and LasR-TIC represents a transcription initiation complex in an open loop state to which a transcription factor protein is bound. K ₁ And K ₂ The equilibrium constants for the two-step reactions are respectively. From equation (4), the following relationship can be obtained:

[LasR-TIC]＝[RNAP]·[LasR ₂ -DNA]·K ₁ ·K ₂ (5)

the mutation process is a primary reaction that must be mediated by LasR-TIC and can be written as:

wherein the mutation rate k _m Proportional to the concentration of LasR-TIC, i.e. k _m ＝k _TIC1 ·[LasR-TIC]Where k is _TIC1 Is a constant. Then the expression of the ratio x of the mutated DNA can be derived from the expression (6):

x＝1-exp(-k _m ·t)

wherein t is the total time of occurrence of the mutation process, i.e., the time of inducing the protein expression of the target strain and the control strain of the present invention, in combination with formulas (3) and (5), the base ratio of the mutation is finally expressed as:

x＝1-exp(-K ₃ ·θ·t),(K ₃ ＝[RNAP]·[DNA _all ]·K ₁ ·K ₂ ·k _TIC1 constant) (7)

The process of expressing protein by transcription and translation of DNA can be written as the following reaction group according to the central rule:

wherein mRNA represents messenger RNA and Protein represents Protein. k is a radical of _{transcription} Is the transcription rate constant, k _translation To translation Rate, γ _mRNA Is the mRNA decomposition rate, γ _Protein The protein degradation rate. According to published work by the Xieliang et al in 2006 (DOI:10.1103/PhysRevLett.97.168302), the average concentration of the bacterial intracellular proteins was

The transcription process can be considered as a zero order reaction mediated by LasR-TIC, and the transcription rate constant can be expressed as:

k _{transcription} ＝k _TIC2 ·[LasR-TIC] (9)

where k is _TIC2 Is a constant. According to formulae (8), (9), combining formulae (3) and (5) to give

[Protein]＝K ₄ ·θ (10)

Wherein K ₄ ＝k _translation ·k _TIC2 ·[RNAP]·DNA _all ·K ₁ ·K ₂ /(γ _mRNA ·γ _Protein ) And is constant.

Further, the relationship between the intracellular concentration [ Protein ] of a target Protein transcribed and expressed by a transcription factor Protein-targeted promoter DNA and the mutation ratio x on the corresponding promoter DNA can be expressed as follows:

as can be seen from the formula (11), the mutation ratio of the transcription factor protein-binding target promoter DNA is in a negative exponential relationship with the concentration of the protein expressed, and after an indefinite period of time, the mutation ratio tends to 1.

Based on examples 1 and 4 of the present invention, the theoretical curve of the mutation ratio (y) of the target promoter DNA to which the transcription factor protein LasR binds and the concentration (x) of the protein expressed thereby was determined to be y ═ 1-exp (-K ×), K ═ 2.075.

In this example, LasR-binding promoters with different transcription levels were further constructed, and the concentration of promoter-expressed proteins was characterized by fluorescent proteins, and the frequency of mutations at corresponding times was calculated from the results of first-generation sequencing, as shown in fig. 4. The theoretical curve can be well fitted to the experimental data, which indicates that the predicted relationship between the transcription level of the promoter DNA and the ratio of the transcription factor protein to its mutation is correct.

Example 5

Further based on the promoters found in example 3, these sequences were stored in bulk in text documents, followed by the bioinformatics tool MEME Suite (R) ((R))Introduction-MEME Suite(meme-suite.org)) Conserved DNA sequences to which LasR binds are shown in FIG. 5.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

The invention relates to amino acid and nucleotide sequences as follows:

amino acid sequence of the transcription factor protein LasR for testing (SEQ ID NO.1)

MALVDGFLELERSSGKLEWSAILQKMASDLGFSKILFGLLPKDSQDYENAFIVGNYPAAWREHYDRAGYARVDPTVSHCTQSVLPIFWEPSIYQTRKQHEFFEEASAAGLVYGLTMPLHGARGELGALSLSVEAENRAEANRFMESVLPTLWMLKDYALQSGAGLAFEHPVSKPVVLTSREKEVLQWCAIGKTSWEISVI CNCSEANVNFHMGNIRRKFGVTSRRVAAIMAVNLGLITL

Nucleotide sequence of transcription factor protein LasR for testing (SEQ ID NO.2)

ATGGCCTTGGTTGACGGTTTTCTTGAGCTGGAACGCTCAAGTGGAAAATTGGAGTGGAGCGCCATCCTGCAGAAGATGGCGAGCGACCTTGGATTCTCGAAGATCCTGTTCGGCCTGTTGCCTAAGGACAGCCAGGACTACGAGAACGCCTTCATCGTCGGCAACTACCCGGCCGCCTGGCGCGAGCATTACGACCGGGCTGGCTACGCGCGGGTCGACCCGACGGTCAGTCACTGTACCCAGAGCGTACTGCCGATTTTCTGGGAACCGTCCATCTACCAGACGCGAAAGCAGCACGAGTTCTTCGAGGAAGCCTCGGCCGCCGGCCTGGTGTATGGGCTGACCATGCCGCTGCATGGTGCTCGCGGCGAACTCGGCGCGCTGAGCCTCAGCGTGGAAGCGGAAAACCGGGCCGAGGCCAACCGTTTCATGGAGTCGGTCCTGCCGACCCTGTGGATGCTCAAGGACTACGCACTGCAGAGCGGTGCCGGACTGGCCTTCGAACATCCGGTCAGCAAACCGGTGGTTCTGACCAGCCGGGAGAAGGAAGTGTTGCAGTGGTGCGCCATCGGCAAGACCAGTTGGGAGATATCGGTTATCTGCAACTGCTCGGAAGCCAATGTGAACTTCCATATGGGAAATATTCGGCGGAAGTTCGGTGTGACCTCCCGCCGCGTAGCGGCCATTATGGCCGTTAATTTGGGTCTTATTACTCTCTGA

Amino acid sequence of cytosine deoxynucleotide deaminase (SEQ ID NO.3)

MDSLLMNRREFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFISWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHGRTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRT

Optimized nucleotide sequence of cytosine deoxynucleotide deaminase (SEQ ID NO.4)

ATGGACAGCCTGCTGATGAACCGCCGCGAGTTCCTGTACCAGTTCAAGAACGTGCGCTGGGCCAAGGGCCGCCGCGAGACCTACCTGTGCTACGTGGTGAAGCGCCGCGACAGCGCCACCAGCTTCAGCCTGGACTTCGGCTACCTGCGCAACAAGAACGGCTGCCACGTGGAGCTGCTGTTCCTGCGCTACATCAGCGACTGGGACCTGGACCCGGGCCGCTGCTACCGCGTGACCTGGTTCATCAGCTGGAGCCCGTGCTACGACTGCGCCCGCCACGTGGCCGACTTCCTGCGCGGCAACCCGAACCTGAGCCTGCGCATCTTCACCGCCCGCCTGTACTTCTGCGAGGACCGCAAGGCCGAGCCGGAGGGCCTGCGCCGCCTGCACCGCGCCGGCGTGCAGATCGCCATCATGACCTTCAAGGACTACTTCTACTGCTGGAACACCTTCGTGGAGAACCACGGCCGCACCTTCAAGGCCTGGGAGGGCCTGCACGAGAACAGCGTGCGCCTGAGCCGCCAGCTGCGCCGCATCCTGCTGCCGCTGTACGAGGTGGACGACCTGCGCGACGCCTTCCGCACCTAA

Construction of the nucleotide sequence of pJN105 plasmid vector for cloning, wherein the bold symbol is the arabinose promoter sequence region and the black triangle indicates the position of the target gene insertion (SEQ ID NO.5)

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> method for detecting binding position of protein and deoxyribonucleotide in situ

<130> CP122010004C

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 239

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala Leu Val Asp Gly Phe Leu Glu Leu Glu Arg Ser Ser Gly Lys

1 5 10 15

Leu Glu Trp Ser Ala Ile Leu Gln Lys Met Ala Ser Asp Leu Gly Phe

20 25 30

Ser Lys Ile Leu Phe Gly Leu Leu Pro Lys Asp Ser Gln Asp Tyr Glu

35 40 45

Asn Ala Phe Ile Val Gly Asn Tyr Pro Ala Ala Trp Arg Glu His Tyr

50 55 60

Asp Arg Ala Gly Tyr Ala Arg Val Asp Pro Thr Val Ser His Cys Thr

65 70 75 80

Gln Ser Val Leu Pro Ile Phe Trp Glu Pro Ser Ile Tyr Gln Thr Arg

85 90 95

Lys Gln His Glu Phe Phe Glu Glu Ala Ser Ala Ala Gly Leu Val Tyr

100 105 110

Gly Leu Thr Met Pro Leu His Gly Ala Arg Gly Glu Leu Gly Ala Leu

115 120 125

Ser Leu Ser Val Glu Ala Glu Asn Arg Ala Glu Ala Asn Arg Phe Met

130 135 140

Glu Ser Val Leu Pro Thr Leu Trp Met Leu Lys Asp Tyr Ala Leu Gln

145 150 155 160

Ser Gly Ala Gly Leu Ala Phe Glu His Pro Val Ser Lys Pro Val Val

165 170 175

Leu Thr Ser Arg Glu Lys Glu Val Leu Gln Trp Cys Ala Ile Gly Lys

180 185 190

Thr Ser Trp Glu Ile Ser Val Ile Cys Asn Cys Ser Glu Ala Asn Val

195 200 205

Asn Phe His Met Gly Asn Ile Arg Arg Lys Phe Gly Val Thr Ser Arg

210 215 220

Arg Val Ala Ala Ile Met Ala Val Asn Leu Gly Leu Ile Thr Leu

225 230 235

<210> 2

<211> 720

<212> DNA

<213> Artificial sequence

<400> 2

atggccttgg ttgacggttt tcttgagctg gaacgctcaa gtggaaaatt ggagtggagc 60

gccatcctgc agaagatggc gagcgacctt ggattctcga agatcctgtt cggcctgttg 120

cctaaggaca gccaggacta cgagaacgcc ttcatcgtcg gcaactaccc ggccgcctgg 180

cgcgagcatt acgaccgggc tggctacgcg cgggtcgacc cgacggtcag tcactgtacc 240

cagagcgtac tgccgatttt ctgggaaccg tccatctacc agacgcgaaa gcagcacgag 300

ttcttcgagg aagcctcggc cgccggcctg gtgtatgggc tgaccatgcc gctgcatggt 360

gctcgcggcg aactcggcgc gctgagcctc agcgtggaag cggaaaaccg ggccgaggcc 420

aaccgtttca tggagtcggt cctgccgacc ctgtggatgc tcaaggacta cgcactgcag 480

agcggtgccg gactggcctt cgaacatccg gtcagcaaac cggtggttct gaccagccgg 540

gagaaggaag tgttgcagtg gtgcgccatc ggcaagacca gttgggagat atcggttatc 600

tgcaactgct cggaagccaa tgtgaacttc catatgggaa atattcggcg gaagttcggt 660

gtgacctccc gccgcgtagc ggccattatg gccgttaatt tgggtcttat tactctctga 720

<210> 3

<211> 195

<212> PRT

<213> Artificial sequence

<400> 3

Met Asp Ser Leu Leu Met Asn Arg Arg Glu Phe Leu Tyr Gln Phe Lys

1 5 10 15

Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val

20 25 30

Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr

35 40 45

Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr

50 55 60

Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp

65 70 75 80

Phe Ile Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp

85 90 95

Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg

100 105 110

Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg

115 120 125

Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr

130 135 140

Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Gly Arg Thr Phe Lys

145 150 155 160

Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu

165 170 175

Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala

180 185 190

Phe Arg Thr

195

<210> 4

<211> 588

<212> DNA

<213> Artificial sequence

<400> 4

atggacagcc tgctgatgaa ccgccgcgag ttcctgtacc agttcaagaa cgtgcgctgg 60

gccaagggcc gccgcgagac ctacctgtgc tacgtggtga agcgccgcga cagcgccacc 120

agcttcagcc tggacttcgg ctacctgcgc aacaagaacg gctgccacgt ggagctgctg 180

ttcctgcgct acatcagcga ctgggacctg gacccgggcc gctgctaccg cgtgacctgg 240

ttcatcagct ggagcccgtg ctacgactgc gcccgccacg tggccgactt cctgcgcggc 300

aacccgaacc tgagcctgcg catcttcacc gcccgcctgt acttctgcga ggaccgcaag 360

gccgagccgg agggcctgcg ccgcctgcac cgcgccggcg tgcagatcgc catcatgacc 420

ttcaaggact acttctactg ctggaacacc ttcgtggaga accacggccg caccttcaag 480

gcctgggagg gcctgcacga gaacagcgtg cgcctgagcc gccagctgcg ccgcatcctg 540

ctgccgctgt acgaggtgga cgacctgcgc gacgccttcc gcacctaa 588

<210> 5

<211> 5970

<212> DNA

<213> Artificial sequence

<400> 5

gcccggaatg ccgggctggc tgggcggctc ctcgccgggg ccggtcggta gttgctgctc 60

gcccggatac agggtcggga tgcggcgcag gtcgccatgc cccaacagcg attcgtcctg 120

gtcgtcgtga tcaaccacca cggcggcact gaacaccgac aggcgcaact ggtcgcgggg 180

ctggccccac gccacgcggt cattgaccac gtaggccgac acggtgccgg ggccgttgag 240

cttcacgacg gagatccagc gctcggccac caagtccttg actgcgtatt ggaccgtccg 300

caaagaacgt ccgatgagct tggaaagtgt cttctggctg accaccacgg cgttctggtg 360

gcccatctgc gccacgaggt gatgcagcag cattgccgcc gtgggtttcc tcgcaataag 420

cccggcccac gcctcatgcg ctttgcgttc cgtttgcacc cagtgaccgg gcttgttctt 480

ggcttgaatg ccgatttctc tggactgcgt ggccatgctt atctccatgc ggtagggtgc 540

cgcacggttg cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg acaacaatta 600

tgcgttgcgt aaaagtggca gtcaattaca gattttcttt aacctacgca atgagctatt 660

gcggggggtg ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt gatttttaag 720

tctttcgcat ttcgccctat atctagttct ttggtgccca aagaagggca cccctgcggg 780

gttcccccac gccttcggcg cggctccccc tccggcaaaa agtggcccct ccggggcttg 840

ttgatcgact gcgcggcctt cggccttgcc caaggtggcg ctgccccctt ggaacccccg 900

cactcgccgc cgtgaggctc ggggggcagg cgggcgggct tcgccttcga ctgcccccac 960

tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc 1020

gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg 1080

ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc 1140

agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat ccctgtggtc 1200

aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt ccacgggccg 1260

agcgaagcga gccagccggt ggccgctcgc ggccatcgtc cacatatcca cgggctggca 1320

agggagcgca gcgaccgcgc agggcgaagc ccggagagca agcccgtagg gcgccgcagc 1380

cgccgtaggc ggtcacgact ttgcgaagca aagtctagtg agtatactca agcattgagt 1440

ggcccgccgg aggcaccgcc ttgcgctgcc cccgtcgagc cggttggaca ccaaaaggga 1500

ggggcaggca tggcggcata cgcgatcatg cgatgcaaga agctggcgaa aatgggcaac 1560

gtggcggcca gtctcaagca cgcctaccgc gagcgcgaga cgcccaacgc tgacgccagc 1620

aggacgccag agaacgagca ctgggcggcc agcagcaccg atgaagcgat gggccgactg 1680

cgcgagttgc tgccagagaa gcggcgcaag gacgctgtgt tggcggtcga gtacgtcatg 1740

acggccagcc cggaatggtg gaagtcggcc agccaagaac agcaggcggc gttcttcgag 1800

aaggcgcaca agtggctggc ggacaagtac ggggcggatc gcatcgtgac ggccagcatc 1860

caccgtgacg aaaccagccc gcacatgacc gcgttcgtgg tgccgctgac gcaggacggc 1920

aggctgtcgg ccaaggagtt catcggcaac aaagcgcaga tgacccgcga ccagaccacg 1980

tttgcggccg ctgtggccga tctagggctg caacggggca tcgagggcag caaggcacgt 2040

cacacgcgca ttcaggcgtt ctacgaggcc ctggagcggc caccagtggg ccacgtcacc 2100

atcagcccgc aagcggtcga gccacgcgcc tatgcaccgc agggattggc cgaaaagctg 2160

ggaatctcaa agcgcgttga gacgccggaa gccgtggccg accggctgac aaaagcggtt 2220

cggcaggggt atgagcctgc cctacaggcc gccgcaggag cgcgtgagat gcgcaagaag 2280

gccgatcaag cccaagagac ggcccgagac cttcgggagc gcctgaagcc cgttctggac 2340

gccctggggc cgttgaatcg ggatatgcag gccaaggccg ccgcgatcat caaggccgtg 2400

ggcgaaaagc tgctgacgga acagcgggaa gtccagcgcc agaaacaggc ccagcgccag 2460

caggaacgcg ggcgcgcaca tttccccgaa aagtgccacc tggcggcgtt gtgacaattt 2520

accgaacaac tccgcggccg ggaagccgat ctcggcttga acgaattgtt aggtggcggt 2580

acttgggtcg atatcaaagt gcatcacttc ttcccgtatg cccaactttg tatagagagc 2640

cactgcggga tcgtcaccgt aatctgcttg cacgtagatc acataagcac caagcgcgtt 2700

ggcctcatgc ttgaggagat tgatgagcgc ggtggcaatg ccctgcctcc ggtgctcgcc 2760

ggagactgcg agatcataga tatagatctc actacgcggc tgctcaaacc tgggcagaac 2820

gtaagccgcg agagcgccaa caaccgcttc ttggtcgaag gcagcaagcg cgatgaatgt 2880

cttactacgg agcaagttcc cgaggtaatc ggagtccggc tgatgttggg agtaggtggc 2940

tacgtctccg aactcacgac cgaaaagatc aagagcagcc cgcatggatt tgacttggtc 3000

agggccgagc ctacatgtgc gaatgatgcc catacttgag ccacctaact ttgttttagg 3060

gcgactgccc tgctgcgtaa catcgttgct gctgcgtaac atcgttgctg ctccataaca 3120

tcaaacatcg acccacggcg taacgcgctt gctgcttgga tgcccgaggc atagactgta 3180

caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc gccgttacca ccgctgcgtt 3240

cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta cttgcattac agtttacgaa 3300

ccgaacaggc ttatgtcaac tgggttcgtg ccttcatccg tttccacggt gtgcgtccat 3360

gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca 3420

tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtttttatg 3480

catgcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 3540

gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 3600

gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 3660

aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg 3720

cgcaattaac cctcactaaa gggaacaaaa gctgggtacc gggccccccc tcgaggtcga 3780

cggtatcgat gcataatgtg cctgtcaaat ggacgaagca gggattctgc aaaccctatg 3840

ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat tatgacaact tgacggctac 3900

atcattcact ttttcttcac aaccggcacg gaactcgctc gggctggccc cggtgcattt 3960

tttaaatacc cgcgagaaat agagttgatc gtcaaaacca acattgcgac cgacggtggc 4020

gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg 4080

ccagcttaag acgctaatcc ctaactgctg gcggaaaaga tgtgacagac gcgacggcga 4140

caagcaaaca tgctgtgcga cgctggcgat atcaaaattg ctgtctgcca ggtgatcgct 4200

gatgtactga caagcctcgc gtacccgatt atccatcggt ggatggagcg actcgttaat 4260

cgcttccatg cgccgcagta acaattgctc aagcagattt atcgccagca gctccgaata 4320

gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga aatgcggctg 4380

gtgcgcttca tccgggcgaa agaaccccgt attggcaaat attgacggcc agttaagcca 4440

ttcatgccag taggcgcgcg gacgaaagta aacccactgg tgataccatt cgcgagcctc 4500

cggatgacga ccgtagtgat gaatctctcc tggcgggaac agcaaaatat cacccggtcg 4560

gcaaacaaat tctcgtccct gatttttcac caccccctga ccgcgaatgg tgagattgag 4620

aatataacct ttcattccca gcggtcggtc gataaaaaaa tcgagataac cgttggcctc 4680

aatcggcgtt aaacccgcca ccagatgggc attaaacgag tatcccggca gcaggggatc 4740

attttgcgct tcagccatac ttttcatact cccgccattc agagaagaaa ccaattgtcc 4800

atattgcatc agacattgcc gtcactgcgt cttttactgg ctcttctcgc taaccaaacc 4860

ggtaaccccg cttattaaaa gcattctgta acaaagcggg accaaagcca tgacaaaaac 4920

gcgtaacaaa agtgtctata atcacggcag aaaagtccac attgattatt tgcacggcgt 4980

cacactttgc tatgccatag catttttatc cataagatta gcggatccta cctgacgctt 5040

tttatcgcaa ctctctactg tttctccata cccgtttttt tgggctagcg aattcataac 5100

actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 5160

ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 5220

cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg 5280

ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgactgc 5340

gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc ttaaacgcct 5400

ggtgctacgc ctgaataagt gataataagc ggatgaatgg cagaaattcg aaagcaaatt 5460

cgacccggtc gtcggttcag ggcagggtcg ttaaatagcc gcttatgtct attgctggtt 5520

taccggttta ttgactaccg gaagcagtgt gaccgtgtgc ttctcaaatg cctgaggcca 5580

gtttgctcag gctctccccg tggaggtaat aattgacgat atgatcattt attctgcctc 5640

ccagagcctg ataaaaacgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc 5700

gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg 5760

gcgaggcggc tacagccgat agtctggaac agcgcactta cgggttgctg cgcaacccaa 5820

gtgctaccgg cgcggcagcg tgacccgtgt cggcggctcc aacggctcgc catcgtccag 5880

aaaacacggc tcatcgggca tcggcaggcg ctgctgcccg cgccgttccc attcctccgt 5940

ttcggtcaag gctggcaggt ctggttccat 5970

Claims (10)

1. A method for detecting the binding position of a protein and deoxyribonucleotides in situ, comprising:

1) constructing a target strain for expressing the cytosine deoxynucleotide deaminase-target protein fusion protein and a control strain for expressing the target protein;

the target strain and the control strain do not contain uracil DNA glycosidase genes;

2) after inducing the protein expression of the target strain and the control strain, extracting the genome DNA of the target strain and the control strain;

3) and performing high-throughput sequencing on the genomic DNA of the target strain and the genomic DNA of the control strain, analyzing to obtain point mutations of the genomic DNA of the target strain and the genomic DNA of the control strain, removing the point mutations common to the target strain and the control strain, and screening the point mutations in the non-coding region from the rest point mutations of the target strain to obtain the target protein binding sites.

2. The method according to claim 1, wherein the genomic DNAs of the target strain and the control strain in step 3) are respectively provided with n groups for high-throughput sequencing, and the target protein binding sites are further obtained, and the method specifically comprises the following steps: performing high-throughput sequencing on the genomic DNA of n groups of target strains and n groups of control strains, analyzing to obtain point mutations of the genomic DNA of the target strains and the genomic DNA of the control strains, screening the common point mutations of the n groups of target strains from the rest point mutations of the target strains after removing the common point mutations of the target strains and the control strains, and screening the common point mutations in non-coding regions to obtain target protein binding sites;

n is more than or equal to 2 and less than or equal to 5.

3. The method of claim 1, wherein the target strain expressing the cytosine deoxynucleotide deaminase-target protein fusion protein is constructed by a method comprising: constructing a cytosine deoxynucleotide deaminase-target protein fusion protein expression vector, and then introducing the fusion protein expression vector into a strain containing no uracil DNA glycosidase gene;

the construction method of the control strain reaching the target protein comprises the following steps: a target protein expression vector was constructed, and then the target protein expression vector was introduced into a strain containing no uracil DNA glycosidase gene.

4. The method of claim 3, wherein in the cytosine deoxynucleotide deaminase-target protein fusion protein expression vector, the cytosine deoxynucleotide deaminase is located at the nitrogen terminus of the fusion protein, the target protein is located at the carbon terminus of the fusion protein, and the cytosine deoxynucleotide deaminase and the target protein are linked by 8 glycines.

5. The method of claim 1, wherein the inducing protein expression of the target strain and the control strain comprises: recovering bacterial strains, selecting clone bacterial plaques, and shaking the bacterial strains for 6-20 hours at 37 ℃ by using an LB culture medium and adding an inducer;

preferably, the inducer is arabinose;

preferably, the formulation of the LB medium is: 10g/L of sodium chloride, 5g/L of yeast powder and 10g/L of peptone.

6. The method of claim 1, wherein the protein of interest is a transcription factor.

7. The method of claim 1, further comprising calculating the conserved DNA sequence bound by the target protein, comprising the steps of:

with the target protein binding site obtained in the step 3) as a center, respectively extending m base pairs at the upstream and the downstream of the genome DNA to obtain a DNA fragment sequence containing the target protein binding site; m is more than or equal to 10 and less than or equal to 200;

and storing the DNA fragment sequence containing the target protein binding site into a text document, and calculating the conserved DNA sequence bound by the target protein by using a bioinformatics tool MEME Suite.

8. A method for predicting the intracellular concentration of a protein transcriptionally expressed from a promoter, comprising: mutating a transcription factor-targeted promoter based on the method of claim 1, and predicting the intracellular concentration of a protein transcriptionally expressed by the promoter according to the following formula;

x＝1-exp(-K ₅ ·[Protein]·t)

wherein the content of the first and second substances,

x represents the proportion of the mutant DNA on the transcription factor target promoter;

[ Protein ] represents the intracellular concentration of a Protein expressed by transcription of a promoter;

t represents the total time of occurrence of the mutation process;

K ₅ denotes the equilibrium constant, K ₅ Is represented as follows:

k _TIC1 、k _TIC2 is a constant number k _translation To translation Rate, γ _mRNA Is the mRNA decomposition rate, gamma _Protein The protein degradation rate.

9. The prediction method of claim 8, wherein the ratio x of the mutated DNA on the transcription factor target promoter is expressed as follows:

x＝1-exp(-k _m ·t)

wherein the content of the first and second substances,

k _m represents the mutation rate of the transcription factor binding site;

t represents the total time of occurrence of the mutation process.

10. The prediction method of claim 8, wherein the proportion x of the mutated DNA on the transcription factor target promoter is represented as follows:

x＝1-exp(-K ₃ ·θ·t)

wherein the content of the first and second substances,

θ represents the ratio occupied by the DNA bound to the transcription factor protein, and θ is expressed as follows:

[LasR]represents the concentration of the transcription factor protein, k _d Represents an equilibrium constant;

k ₃ is a constant number, k ₃ Is represented as follows:

K ₃ ＝[RNAP]·[DNA _all ]·K ₁ ·K ₂ ·k _TIC1

[RNAP]indicates the concentration of RNA polymerase, [ DNA ] _all ]Indicates the total target DNA concentration, K, in the bacterial cell ₁ And K ₂ The equilibrium constants in the process of recruiting ribonucleic acid polymerase by the following transcription factors are respectively:

RNAP denotes RNA polymerase, LasR denotes transcription factor protein, RNAP-LasR ₂ DNA represents a complex of RNA polymerase with transcription factors and DNA, and LasR-TIC represents a transcription initiation complex in an open loop state to which transcription factor proteins are bound;

preferably, the θ is determined using the method of claim 1.

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