CN114807204B - Recombinant vector plasmid, salicylic acid biosensor, construction method and application - Google Patents
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a recombinant vector plasmid, a salicylic acid biosensor, a construction method and application. The vector plasmid comprises a modified NahR protein coding gene, a Pr promoter, a reporter marker protein coding gene, a modified Psal promoter and a plasmid skeleton; the Pr promoter and the modified Psal promoter are opposite in direction; the amino acid sequence of the modified NahR protein is shown as SEQ ID No. 6; the nucleotide sequence of the modified Psal promoter is shown as SEQ ID No. 5. The modified Psal promoter has stronger induction effect compared with the wild type. The modified NahR protein further improves the response sensitivity of the biosensor to salicylic acid. The biosensor has higher sensitivity to salicylic acid, and reduces the detection limit of salicylic acid.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant vector plasmid, a salicylic acid biosensor, a construction method and application.
Background
Salicylic acid is taken as an intermediate product of Polycyclic Aromatic Hydrocarbon (PAHs) metabolism, and has important significance in detection of polycyclic aromatic hydrocarbon. Polycyclic aromatic hydrocarbons are a type of persistent organic pollutants widely existing in the environment and have semi-volatility, bioaccumulation and environmental persistence; and has carcinogenic, mutagenic and teratogenic effects. The existing common polycyclic aromatic hydrocarbon detection method has various defects, so that the search for a simple, quick, efficient and sensitive method is urgent.
Whole cell biosensors have become one of the latest fields in environmental contaminant monitoring molecular tools. Due to the low cost of microorganisms and the suitable pH and temperature ranges, they have been widely used as biosensing elements in the construction of biosensors. The whole cell biosensor obtains a signal that is easily recognized by combining a response promoter with a reporter gene. Wherein the regulatory protein or promoter needs to be able to respond to a change in the concentration of the analyte to be detected and then convert the signal to a reporter protein signal that is readily detectable. Compared with the traditional chemical and electronic detection method of the environmental pollutants, the whole-cell microorganism sensor has the advantages of low cost, small volume, degradability, high analysis speed, on-line or in-situ analysis and the like, and has extremely high application value in the fields of biological manufacturing process monitoring, environmental monitoring and food safety, medical diagnosis and monitoring and the like.
Pseudomonas putida G7 is one of the most widely studied PAHs degrading strains at present, polycyclic aromatic hydrocarbon can be oxidized and opened by an upstream gene cluster of the strain to obtain an intermediate product salicylic acid, and NahR can use the salicylic acid as an effector of the regulatory protein to obtain a signal through a reporter gene which is easy to detect, so that the detection of PAHs is indirectly realized.
However, the current biological sensing method has poor performance in terms of sensitivity to detection of the poly-bad aromatic hydrocarbon, and practical application is not realized yet.
Disclosure of Invention
The invention aims to provide a recombinant vector plasmid, a salicylic acid biosensor, a construction method and application.
The technical scheme for solving the technical problems is as follows:
the invention provides a recombinant vector plasmid of a salicylic acid biosensor, which comprises a modified NahR protein coding gene, a Pr promoter, a report marker protein coding gene, a modified Psal promoter and a plasmid skeleton; the Pr promoter and the modified Psal promoter are opposite in direction; the amino acid sequence of the modified NahR protein is shown as SEQ ID No. 6; the nucleotide sequence of the modified Psal promoter is shown as SEQ ID No. 5.
Further, the Pr promoter and the-35 region nucleotide sequence of the engineered Psal promoter overlap.
Further, the reporter marker protein is green fluorescent protein eGFP, and the amino acid sequence of the reporter marker protein is shown as SEQ ID No. 4.
Further, the plasmid backbone is a high copy vector pUC57 of Escherichia coli.
The invention provides a salicylic acid biosensor, which comprises a host cell and a recombinant vector plasmid positioned in the host cell; the host cell is E.coli.
Further, the E.coli DH 5. Alpha. Is E.coli DH 5. Alpha.
The invention provides a construction method of the salicylic acid biosensor, which comprises the following steps:
firstly constructing a vector containing a coding gene of a wild type NahR protein and a wild type Psal promoter, and respectively modifying the coding gene of the wild type NahR protein and the wild type Psal promoter to obtain a recombinant plasmid vector containing the modified coding gene of the NahR protein and the modified Psal promoter; and finally, transferring the recombinant plasmid vector into a host cell to obtain the salicylic acid biosensor.
Further, the nucleotide sequence of the wild type Psal promoter is shown as SEQ ID No.3, and the amino acid sequence of the wild type NahR protein is shown as SEQ ID No.1.
The invention provides application of the salicylic acid biosensor in detecting salicylic acid.
The invention provides a salicylic acid detection method, which adopts the salicylic acid biosensor to detect.
The invention has the beneficial effects that:
(1) The recombinant vector plasmid of the salicylic acid biosensor contains an improved Psal promoter; the-35 region of the modified Psal promoter is changed from TTATCAA to TTGTCA; the-10 region is changed from TATCGT to TATAAT, and has stronger induction effect compared with a wild type Psal promoter.
(2) The recombinant vector plasmid of the salicylic acid biosensor contains the coding gene of the modified NahR protein, the modified NahR protein coded by the coding gene changes the 169 locus from glutamic acid to glycine, and the 248 locus from cysteine to threonine, so that the response sensitivity of the biosensor to salicylic acid is further improved.
(3) Compared with the traditional biosensor, the salicylic acid biosensor has higher sensitivity to salicylic acid, and reduces the detection limit of salicylic acid.
(4) The salicylic acid detection method provided by the invention is used for detecting the salicylic acid biosensor, and has the advantages of rapidness, convenience, accuracy and high sensitivity.
Drawings
FIG. 1 is a schematic structural view of a salicylic acid biosensor according to the present invention;
FIG. 2 shows the relative fluorescence intensities of salicylic acid at different concentrations of mutant W10 and wild-type cells of example 2 in the salicylic acid biosensor of the present invention; wherein the abscissa indicates salicylic acid concentration, and the ordinate indicates fluorescence intensity and OD 600 Is a ratio of (2);
FIG. 3 shows the relative fluorescence intensities of mutant E169GC248T of example 3 and bacterial cells containing only the engineered Psal promoter at different concentrations of salicylic acid in the salicylic acid biosensor of the present invention; wherein the abscissa indicates salicylic acid concentration, and the ordinate indicates fluorescence intensity and OD 600 Is a ratio of (2).
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
The recombinant vector plasmid of the salicylic acid biosensor comprises a modified NahR protein coding gene, a Pr promoter, a reporter marker protein coding gene, a modified Psal promoter and a plasmid skeleton; the Pr promoter and the modified Psal promoter are opposite in direction; the nucleotide sequence of the modified Psal promoter is shown as SEQ ID No.5, and the amino acid sequence of the modified NahR protein is shown as SEQ ID No. 6.
SEQ ID No.5：
tatttgtcaatattgtttgctccgttataattattaacaagtcatcaataaagccatcacgagtaccatag
SEQ ID No.6:
MELRDLDLNLLVVFNQLLVDRRVSITAENLGLTQPAVSNALKRLRTSLQDPLFVRTHQGMEPTPYAAHLAEPVTSAMHALRNALQHHESFDPLTSERTFTLAMTDIGEIYFMPRLMDVLAHQAPNCVISTVRDSSMSLMQALQNGTVDLAVGLLPNLQTGFFQRRLLQGHYVCLCRKDHPVTREPLTLERFCSYGHVRVIAAGTGHGEVDTYMTRVGIRRDIRLEVPHFAAVGHILQRTDLLATVPITLADCCVEPFGLSALPHPVVLPEIAINMFWHAKYHKDLANIWLRQLMFDLFTD*。
Among the above recombinant vector plasmids of the present invention, the modified Psal promoter and the modified NahR protein have a more sensitive response to salicylic acid.
As shown in FIG. 1, in the recombinant vector plasmid of the present invention, the sequence of each of the above-mentioned gene fragments is preferably such that the modified NahR protein-encoding gene (NahR mut ) Pr promoter, engineered Psal promoter (Psal mut ) And a gene encoding a reporter marker protein. Preferably, the Pr promoter overlaps the-35 region nucleotide sequence of the engineered Psal promoter, and both may share the-35 region nucleotide sequence.
The Pr promoter can control the transcription of the coding gene of the modified NahR protein to obtain the modified NahR protein; salicylic acid (SaA) molecules enter cells and bind to the engineered NahR proteins, and the bound molecules are capable of modulating the engineered psa promoter (psa mut ) The coding gene of the report marker protein is transcribed to obtain the report marker protein, and the report marker protein sends out a signal, so that the detection of salicylic acid molecules is indicated.
Preferably, the reporter marker protein is green fluorescent protein eGFP, and the amino acid sequence of the reporter marker protein is shown as SEQ ID No. 4.
SEQ ID No.4：
MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*
Preferably, the Pr promoter of the invention is wild type, and the nucleotide sequence of the Pr promoter is shown as SEQ ID No. 2.
SEQ ID No.2：
tattgacaaatacaccactcgatatataataaatcatcaacatgaatattgcgcccggccgggcaccagcaataacccaagcgaggcccc
Preferably, the plasmid backbone of the present invention is the E.coli high copy vector pUC57.
The salicylic acid biosensor comprises a host cell and the recombinant vector plasmid positioned in the host cell; the host cell is E.coli.
Preferably, E.coli DH 5. Alpha. Is used.
As shown in FIG. 1, the salicylic acid biosensor of the present invention senses that NahR transcription regulator can specifically bind to Psal promoter region, thereby inducing the expression of activating reporter marker protein.
The construction method of the salicylic acid biosensor comprises the steps of firstly constructing a vector containing a coding gene of a wild type NahR protein and a wild type Psal promoter, respectively modifying the coding gene of the wild type NahR protein and the wild type Psal promoter to obtain a recombinant plasmid vector containing the modified coding gene of the NahR protein and the modified Psal promoter, and finally transferring the recombinant plasmid vector into host cells to obtain the salicylic acid biosensor.
In the construction method, the specific plasmid vector construction process, gene transformation and host bacterium transfer modes can be carried out by adopting a conventional genetic engineering method.
The method specifically comprises the following steps:
(1) Constructing a first vector plasmid containing a coding gene of a wild type NahR protein and a wild type Psal promoter.
Specifically, a gene encoding a wild type NahR protein, the Pr promoter, a gene encoding a reporter marker protein, and a wild type psa promoter are ligated to a vector to obtain a first vector plasmid containing the wild type psa promoter.
(2) Modifying a wild type Psal promoter;
designing a Psal point mutation primer according to the nucleotide sequence of a wild type Psal promoter, carrying out PCR (polymerase chain reaction) amplification by using a first vector plasmid as a template and adopting the point mutation primer to mutate the nucleotide sequence of the wild type Psal promoter into the nucleotide sequence of an improved Psal promoter, and obtaining a second vector plasmid containing the improved Psal promoter.
(3) The coding gene of the wild NahR protein is modified.
Designing a NahR point mutation primer according to the coding gene of the wild NahR protein, and preparing a mutant fragment of the coding gene of the NahR protein; and connecting the mutant fragment to a second carrier plasmid through homologous recombination to obtain a recombinant carrier plasmid.
The coding gene of the wild type NahR protein is reported by literature (Park HH, lee H Y, lim W K, et al NahR: effects of replacements at Asn 169and Arg 248on promoter binding and inducer recognition[J ]. Archives of Biochemistry & Biophysics,2005,434 (1): 67-74.) that the N169E/R248C mutant shows low background and high induction activity, so that the amino acid sequence of the mutant NahR protein is taken as an initial amino acid sequence when the sequence is synthesized, namely the coding gene of the wild type NahR protein.
(4) And transforming the plasmid containing the recombinant vector into a host cell to obtain the salicylic acid biosensor.
In the above construction method of the present invention, the sequence of steps for modifying the wild-type Psal promoter and modifying the gene encoding the wild-type NahR protein may be interchanged, as long as the finally obtained recombinant vector plasmid contains both modified genes.
Preferably, the nucleotide sequence of the wild type Psal promoter is shown as SEQ ID No.3, and the amino acid sequence of the wild type NahR protein is shown as SEQ ID No.1.
SEQ ID No.3：
tatttatcaatattgtttgctccgttatcgttattaacaagtcatcaataaagccatcacgagtaccatag。
SEQ ID No.4：
MELRDLDLNLLVVFNQLLVDRRVSITAENLGLTQPAVSNALKRLRTSLQDPLFVRTHQGMEPTPYAAHLAEPVTSAMHALRNALQHHESFDPLTSERTFTLAMTDIGEIYFMPRLMDVLAHQAPNCVISTVRDSSMSLMQALQNGTVDLAVGLLPNLQTGFFQRRLLQEHYVCLCRKDHPVTREPLTLERFCSYGHVRVIAAGTGHGEVDTYMTRVGIRRDIRLEVPHFAAVGHILQRTDLLATVPICLADCCVEPFGLSALPHPVVLPEIAINMFWHAKYHKDLANIWLRQLMFDLFTD*
The salicylic acid biosensor can be applied to detection of salicylic acid.
The salicylic acid detection method adopts the salicylic acid biosensor to detect.
Specifically, the salicylic acid biosensor can detect the salicylic acid content in an aqueous solution. The salicylic acid biosensor is added into the aqueous solution to be detected, and the salicylic acid content in the aqueous solution can be qualitatively or quantitatively detected by measuring the fluorescence intensity of the salicylic acid sensor.
The invention will be further illustrated by the following examples in order to provide a better understanding of the invention, but without limiting the invention thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 construction of a first vector plasmid containing the wild type Psal promoter
The coding gene of the wild type NahR protein, the Pr promoter, the coding gene of the report marker protein and the wild type Psal promoter are connected on a vector to obtain a first vector plasmid containing the wild type Psal promoter.
Wherein the amino acid sequence of the wild NahR protein is shown as SEQ ID No.1, the nucleotide sequence of the Pr promoter is shown as SEQ ID No.2, and the nucleotide sequence of the Psal promoter is shown as SEQ ID No. 3.
Specifically, the above sequence is synthesized by biology and constructed on pUC57 vector to obtain vector pUC57-KanR-Pr-nahR-Psal.
PCR was performed using pDG1730-egfp plasmid as template and primers shown as SEQ ID No.7 and SEQ ID No. 8.
Egfp-F:5’tcggatcccggatggtgagcaagggcg3’(SEQ ID No.7)；
Egfp-R:5’agtcgacgggccaaaaaacccctcaagacccg3’(SEQ ID No.8)。
PCR reaction system:
PCR reaction conditions: pre-denaturation at 98℃for 30s, then denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 5s,25 cycles, and extension at 72℃for 30s.
The resulting PCR product was checked for yield and specificity by 0.7% agarose gel electrophoresis and purified using a DNA purification kit. The vector pUC57-KanR-Pr-nahR-Psal and egfp fragments were treated with BamHI and SalI double enzyme cuts and the two were subjected to enzyme ligation.
Wherein the enzyme digestion system is that the recovered product is 25 mu L, buffer is 5 mu L, bamHI is 1.5 mu L, salI is 1.5 mu L, water is added to 50 mu L, water bath is carried out at 37 ℃ for 2 hours, and enzyme ligation is carried out after gel recovery.
The enzyme ligation system was as follows: 3. Mu.L of treated fragment, 1. Mu.L of treated carrier, 0.5. Mu.L of buffer and 0.5. Mu.L of solution I ligase were mixed uniformly and ligated at 22℃for two hours.
Then, transferring the connection product into E.coli DH5 alpha competent cells to obtain pUC57-KanR-Pr-nahR-Psal-egfp, and completing the construction of the first vector plasmid by sequencing verification.
Example 2: modification of the Psal promoter
The strength of the promoter directly affects the transcription level of the reporter gene, and the dynamic range is mainly controlled by the sequences at the-35 and-10 sites of the promoter.
In the salicylic acid biosensor, TTATCAA of a-35 region of a Psal promoter is changed into TTGTCA; the-10 region TATCGT is changed into TATAAT, and the specific modification process is as follows:
psal point mutation primers shown as SEQ ID No. 9and SEQ ID No.10 were designed based on the nucleotide sequence of the wild-type Psal, and PCR amplification was performed using the first vector plasmid pUC57-KanR-Pr-nahR-Psal-egfp obtained in example 1 as a template.
psal-F:5’tgtcaatattgtttgctccgttaaattattaac3’(SEQ ID No.9)；
psal-R:5’ataattataacggagcaaacaatattgacaaatac3’(SEQ ID No.10)。
PCR reaction system:
PCR reaction conditions: pre-denaturation at 98℃for 30s, then denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 25s,25 cycles, and extension at 72℃for 30s.
The resulting PCR product was checked for yield and specificity by 0.7% agarose gel electrophoresis and purified using a DNA purification kit. The purified PCR product was demethylated with DpnI in a system of 25. Mu.L of recovered product; dpn I1. Mu.L; buffer 5. Mu.L; adding water to 50 mu L; the Dpn I enzyme is inactivated by heating at 80 ℃ for 10min after two hours of treatment in a water bath kettle at 37 ℃.
After demethylation, the solution is recovered to 10 mu L, 3-5 mu L is transformed into E.coli DH5 alpha clone competent cells, and two transformants are selected for sequencing to obtain a recombinant vector with correct point mutation, namely a second vector plasmid containing the modified Psal promoter.
And transferring the second vector plasmid into E.coli DH5 alpha competent cells, and marking the grown transformant on a plate to obtain a mutant thallus containing the second vector plasmid, which is named as W10.
Single colonies were picked, induced with salicylic acid at final concentrations of 0, 10, 20, 50, 100. Mu.M for 10h, usingThe relative fluorescence intensity is measured by a multifunctional enzyme-labeled instrument, wherein the excitation wavelength is 488nm, the emission wavelength is 511nm, and the OD of the thallus is measured 600 Value, fluorescence intensity and OD 600 Is defined as the relative fluorescence intensity.
As a result of measurement, as shown in FIG. 2, it was found that the lower limit of induction of salicylic acid by mutant bacterial cells W10 containing the modified Psal promoter (Psal mutant) was increased to 20. Mu.M, and the induction effect was enhanced 4-fold when induced with 100. Mu.M salicylic acid as compared with the wild type (Wt).
The measurement results of the embodiment show that the modified Psal promoter can effectively improve the induction effect of the biosensor on salicylic acid.
Example 3: transformation of transcription regulatory factor NahR protein and verification of effect thereof
This example continued the engineering of NahR protein on the basis of the second vector plasmid obtained in example 2.
The transcription factor comprises two large functional domains, namely a ligand binding domain and a DNA binding domain, and directly interacts with the ligand and the DNA, thereby indirectly influencing the binding of RNA polymerase and playing a key role in a whole-cell microbial sensor. Therefore, the affinity of the transcription factor with the ligand and DNA can be effectively changed by modifying the transcription factor through protein engineering means such as random/point saturation mutation, rational design and the like, so that the sensing performance parameters of the whole-cell microbial sensor can be regulated.
The N169 and R248 sites of NahR proteins may be involved in DNA and inducer recognition and binding. Therefore, two-point combined mutant libraries are made for 169and 248 sites, and then mutants with the strongest sensing efficiency are screened out from the mutant libraries through high-throughput screening.
The method specifically comprises the following steps:
1) Method for constructing 169/248 saturated mutant library:
first, a 169-248 mutant fragment was prepared.
A pair of primers is designed according to 169 locus and 248 locus on the coding gene of the wild NahR protein, and coding design is carried out by NNK degenerate codons at mutated amino acid positions. Wherein the front primer design comprises a 169 site mutation and the rear primer design comprises a 248 site mutation.
PCR amplification was performed using the above primers and the second vector plasmid pUC57-KanR-Pr-nahR-Psal-egfp obtained in example 2 as a template to obtain gene fragments 169-248 containing 169and 248 site mutations, which were correctly confirmed by running gel, and then recovered as gel.
Wherein, the nucleotide sequence of the wild NahR is used for designing a point mutation primer pair as shown in SEQ ID No.11 and SEQ ID No.12, s is g or c, and n is any base.
1-F:5’gcagcagtccgccagsnngatcggaacggtcgccagcag3’(SEQ ID No.11)；
2-R:5’cgccgtctgctgcagnnscactacgtttgcctgtgccg3’(SEQ ID No.12)。
The PCR reaction system is as follows:
PCR reaction conditions: pre-denaturation at 98℃for 30s, then denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 5s,25 cycles, and extension at 72℃for 30s.
The resulting PCR products were checked for yield and specificity by 0.7% agarose gel electrophoresis and purified using DNA purification kit.
Preparing a recombinant vector plasmid containing the gene of the modified NahR protein: the mutant fragment is connected to a second carrier plasmid through homologous recombination to obtain a recombinant carrier plasmid, and the primers are shown as SEQ ID No.13 and SEQ ID No. 14:
2-F:5’ctgcagcagacggcgctggaagaagccggtctg3’(SEQ ID No.13)；
2-R:5’ctggcggactgctgcgttgaaccgttcggtc3’(SEQ ID No.14)。
the method comprises the following specific steps: PCR amplification was performed using the second vector plasmid as template and the above primers, and DpnI template removal was also performed, and the primer design required attention to have a 20bp homology arm with the fragment. The method and system are consistent with the DpnI demethylation process in example 2. The vector and fragment were then ligated by homologous recombination, ligation system: 5 XCE II; buffer 1. Mu.L; 1 mu L of Exnase II; the molar concentration ratio of linearized fragment to carrier is 2:1, a step of; connecting at 72 ℃ for 30min; the ligation product was then transferred into E.coli DH 5. Alpha. Clone competent cells, giving approximately 1400 transformant cells.
Theoretically, there should be 400 mutants in the 169and 248 two site mutant libraries of NahR proteins; around 1400 were actually obtained, approximately three times the theoretical value, indicating that the library was in sufficient quantity to cover all mutation combinations.
Then 10 transformants are randomly selected for extracting plasmid sequencing, and when the 9 plasmids contain 169/248 mutant amino acids and are inconsistent, the mutant library is successfully constructed, and the mutant library is the 169/248 saturated mutant library.
2) Screening mutants with high sensitivity in the obtained saturated mutant library:
all transformants on the plates were washed with sterile water, applied to 0.1mM salicylic acid solid plates, incubated overnight at 37℃and selected for high fluorescence intensity transformants using a blue light instrument, then induced with 100. Mu.M salicylic acid at final concentration, incubated at 37℃with shaking at 200rpm for 10h.
And (3) measuring the fluorescence value of the mutant through an enzyme-labeled instrument, and re-screening to obtain the mutant with high sensitivity.
It was found by sequencing that in the resulting mutant with high sensitivity, the 169 site of the NahR protein had been changed from glutamic acid to glycine and the 248 site had been changed from cysteine to threonine. This mutant was designated E169G/C248T.
Based on the 169 glycine at position and threonine at position 248, primers can be designed for engineering nahR proteins as shown in sequences SEQ ID No.15, SEQ ID No.16, SEQ ID No.17 and SEQ ID No. 18:
169 site-directed mutagenesis primer:
169-F:5’gtctgctgcagGGGcactacgtttgcctg3’(SEQ ID No.15)；
169-R:5’gtagtgCCCctgcagcagacggcgctggaag3’(SEQ ID No.16)。
248 site-directed mutagenesis primer:
248-F:5’tccgatcACCctggcggactgctgcg3’(SEQ ID No.17)；
248-R:5’agtccgccagGGTgatcggaacggtcgc3’(SEQ ID No.18)。
combining mutant E169G/C248T containing an engineered NahR protein and an engineered Psal promoter with a cell containing a wild-type NahR protein and an engineered Psal promoter(Psal mutant) separately performing cell OD 600 Value measurement and calculation of fluorescence intensity and OD 600 Is defined as the ratio of the relative fluorescence intensities compared to the value having a significantly enhanced salicylic acid response sensitivity.
The results of the measurement are shown in fig. 3. As can be seen from FIG. 3, mutant E169G/C248T exhibited a more excellent linear response at salicylic acid concentrations of 0-1. Mu.M.
Thus, the response sensitivity of the salicylic acid biosensor containing the engineered NahR protein and the engineered psa promoter to salicylic acid is significantly better than that of the salicylic acid biosensor containing only the engineered psa promoter.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> university of Hubei
<120> a recombinant vector plasmid, salicylic acid biosensor, construction method and application
<141> 2022-04-08
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 300
<212> PRT
<213> Artificial Sequence
<400> 1
Met Glu Leu Arg Asp Leu Asp Leu Asn Leu Leu Val Val Phe Asn Gln
1 5 10 15
Leu Leu Val Asp Arg Arg Val Ser Ile Thr Ala Glu Asn Leu Gly Leu
20 25 30
Thr Gln Pro Ala Val Ser Asn Ala Leu Lys Arg Leu Arg Thr Ser Leu
35 40 45
Gln Asp Pro Leu Phe Val Arg Thr His Gln Gly Met Glu Pro Thr Pro
50 55 60
Tyr Ala Ala His Leu Ala Glu Pro Val Thr Ser Ala Met His Ala Leu
65 70 75 80
Arg Asn Ala Leu Gln His His Glu Ser Phe Asp Pro Leu Thr Ser Glu
85 90 95
Arg Thr Phe Thr Leu Ala Met Thr Asp Ile Gly Glu Ile Tyr Phe Met
100 105 110
Pro Arg Leu Met Asp Val Leu Ala His Gln Ala Pro Asn Cys Val Ile
115 120 125
Ser Thr Val Arg Asp Ser Ser Met Ser Leu Met Gln Ala Leu Gln Asn
130 135 140
Gly Thr Val Asp Leu Ala Val Gly Leu Leu Pro Asn Leu Gln Thr Gly
145 150 155 160
Phe Phe Gln Arg Arg Leu Leu Gln Glu His Tyr Val Cys Leu Cys Arg
165 170 175
Lys Asp His Pro Val Thr Arg Glu Pro Leu Thr Leu Glu Arg Phe Cys
180 185 190
Ser Tyr Gly His Val Arg Val Ile Ala Ala Gly Thr Gly His Gly Glu
195 200 205
Val Asp Thr Tyr Met Thr Arg Val Gly Ile Arg Arg Asp Ile Arg Leu
210 215 220
Glu Val Pro His Phe Ala Ala Val Gly His Ile Leu Gln Arg Thr Asp
225 230 235 240
Leu Leu Ala Thr Val Pro Ile Cys Leu Ala Asp Cys Cys Val Glu Pro
245 250 255
Phe Gly Leu Ser Ala Leu Pro His Pro Val Val Leu Pro Glu Ile Ala
260 265 270
Ile Asn Met Phe Trp His Ala Lys Tyr His Lys Asp Leu Ala Asn Ile
275 280 285
Trp Leu Arg Gln Leu Met Phe Asp Leu Phe Thr Asp
290 295 300
<210> 2
<211> 90
<212> DNA/RNA
<213> Artificial Sequence
<400> 2
tattgacaaa tacaccactc gatatataat aaatcatcaa catgaatatt gcgcccggcc 60
gggcaccagc aataacccaa gcgaggcccc 90
<210> 3
<211> 71
<212> DNA/RNA
<213> Artificial Sequence
<400> 3
tatttatcaa tattgtttgc tccgttatcg ttattaacaa gtcatcaata aagccatcac 60
gagtaccata g 71
<210> 4
<211> 239
<212> PRT
<213> Artificial Sequence
<400> 4
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Val Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 5
<211> 71
<212> DNA/RNA
<213> Artificial Sequence
<400> 5
tatttgtcaa tattgtttgc tccgttataa ttattaacaa gtcatcaata aagccatcac 60
gagtaccata g 71
<210> 6
<211> 300
<212> PRT
<213> Artificial Sequence
<400> 6
Met Glu Leu Arg Asp Leu Asp Leu Asn Leu Leu Val Val Phe Asn Gln
1 5 10 15
Leu Leu Val Asp Arg Arg Val Ser Ile Thr Ala Glu Asn Leu Gly Leu
20 25 30
Thr Gln Pro Ala Val Ser Asn Ala Leu Lys Arg Leu Arg Thr Ser Leu
35 40 45
Gln Asp Pro Leu Phe Val Arg Thr His Gln Gly Met Glu Pro Thr Pro
50 55 60
Tyr Ala Ala His Leu Ala Glu Pro Val Thr Ser Ala Met His Ala Leu
65 70 75 80
Arg Asn Ala Leu Gln His His Glu Ser Phe Asp Pro Leu Thr Ser Glu
85 90 95
Arg Thr Phe Thr Leu Ala Met Thr Asp Ile Gly Glu Ile Tyr Phe Met
100 105 110
Pro Arg Leu Met Asp Val Leu Ala His Gln Ala Pro Asn Cys Val Ile
115 120 125
Ser Thr Val Arg Asp Ser Ser Met Ser Leu Met Gln Ala Leu Gln Asn
130 135 140
Gly Thr Val Asp Leu Ala Val Gly Leu Leu Pro Asn Leu Gln Thr Gly
145 150 155 160
Phe Phe Gln Arg Arg Leu Leu Gln Gly His Tyr Val Cys Leu Cys Arg
165 170 175
Lys Asp His Pro Val Thr Arg Glu Pro Leu Thr Leu Glu Arg Phe Cys
180 185 190
Ser Tyr Gly His Val Arg Val Ile Ala Ala Gly Thr Gly His Gly Glu
195 200 205
Val Asp Thr Tyr Met Thr Arg Val Gly Ile Arg Arg Asp Ile Arg Leu
210 215 220
Glu Val Pro His Phe Ala Ala Val Gly His Ile Leu Gln Arg Thr Asp
225 230 235 240
Leu Leu Ala Thr Val Pro Ile Thr Leu Ala Asp Cys Cys Val Glu Pro
245 250 255
Phe Gly Leu Ser Ala Leu Pro His Pro Val Val Leu Pro Glu Ile Ala
260 265 270
Ile Asn Met Phe Trp His Ala Lys Tyr His Lys Asp Leu Ala Asn Ile
275 280 285
Trp Leu Arg Gln Leu Met Phe Asp Leu Phe Thr Asp
290 295 300
<210> 7
<211> 27
<212> DNA/RNA
<213> Artificial Sequence
<400> 7
tcggatcccg gatggtgagc aagggcg 27
<210> 8
<211> 32
<212> DNA/RNA
<213> Artificial Sequence
<400> 8
agtcgacggg ccaaaaaacc cctcaagacc cg 32
<210> 9
<211> 33
<212> DNA/RNA
<213> Artificial Sequence
<400> 9
tgtcaatatt gtttgctccg ttaaattatt aac 33
<210> 10
<211> 35
<212> DNA/RNA
<213> Artificial Sequence
<400> 10
ataattataa cggagcaaac aatattgaca aatac 35
<210> 13
<211> 39
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (16)..(16)
<223> s is g or c
<220>
<221> misc_feature
<222> (17)..(18)
<223> n is a, t, c or g
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (18)..(18)
<223> n is a, c, g, t or u
<400> 13
gcagcagtcc gccagsnnga tcggaacggt cgccagcag 39
<210> 14
<211> 38
<212> DNA/RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, t, c or g
<220>
<221> misc_feature
<222> (18)..(18)
<223> s is g or c
<220>
<221> misc_feature
<222> (16)..(16)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is a, c, g, t or u
<400> 14
cgccgtctgc tgcagnnsca ctacgtttgc ctgtgccg 38
<210> 13
<211> 33
<212> DNA/RNA
<213> Artificial Sequence
<400> 13
ctgcagcaga cggcgctgga agaagccggt ctg 33
<210> 14
<211> 31
<212> DNA/RNA
<213> Artificial Sequence
<400> 14
ctggcggact gctgcgttga accgttcggt c 31
<210> 15
<211> 29
<212> DNA/RNA
<213> Artificial Sequence
<400> 15
gtctgctgca ggggcactac gtttgcctg 29
<210> 16
<211> 31
<212> DNA/RNA
<213> Artificial Sequence
<400> 16
gtagtgcccc tgcagcagac ggcgctggaa g 31
<210> 17
<211> 26
<212> DNA/RNA
<213> Artificial Sequence
<400> 17
tccgatcacc ctggcggact gctgcg 26
<210> 18
<211> 28
<212> DNA/RNA
<213> Artificial Sequence
<400> 18
agtccgccag ggtgatcgga acggtcgc 28
Claims (8)
1. The recombinant vector plasmid of the salicylic acid biosensor is characterized by comprising a coding gene of a modified NahR protein, a Pr promoter, a coding gene of a report marker protein, a modified Psal promoter and a plasmid skeleton; the Pr promoter and the modified Psal promoter are opposite in direction;
the amino acid sequence of the modified NahR protein is shown as SEQ ID No. 6;
the nucleotide sequence of the modified Psal promoter is shown as SEQ ID No. 5;
the Pr promoter and the-35 region nucleotide sequence of the modified Psal promoter overlap; the report marker protein is green fluorescent protein eGFP, and the amino acid sequence of the report marker protein is shown as SEQ ID No. 4.
2. The recombinant vector plasmid of a salicylic acid biosensor according to claim 1, wherein the plasmid backbone is a high copy vector pUC57 of escherichia coli.
3. A salicylic acid biosensor comprising a host cell and the recombinant vector plasmid of claim 1 or 2 positioned within the host cell; the host cell is E.coli.
4. A salicylic acid biosensor according to claim 3, wherein the escherichia coli is e.coli dh5α.
5. A method of constructing a salicylic acid biosensor according to claim 3 or 4, comprising the steps of:
firstly constructing a vector containing a coding gene of a wild type NahR protein and a wild type Psal promoter, and respectively modifying the coding gene of the wild type NahR protein and the wild type Psal promoter to obtain a recombinant plasmid vector containing the modified coding gene of the NahR protein and the modified Psal promoter; and finally, transferring the recombinant plasmid vector into a host cell to obtain the salicylic acid biosensor.
6. The construction method of the salicylic acid biosensor according to claim 5, wherein the nucleotide sequence of the wild type Psal promoter is shown in SEQ ID No.3, and the amino acid sequence of the wild type NahR protein is shown in SEQ ID No.1.
7. Use of a salicylic acid biosensor according to claim 3 or 4 for detecting salicylic acid.
8. A method for detecting salicylic acid, wherein the salicylic acid biosensor according to claim 3 or 4 is used.
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Citations (5)
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|---|---|---|---|---|
| CN1446260A (en) * | 2000-07-13 | 2003-10-01 | 辛根塔参与股份公司 | Lipoxygenase genes, promoters, transit peptides and protein thereof |
| WO2005054477A1 (en) * | 2003-12-04 | 2005-06-16 | Universidad Pablo De Olavide | Method of regulating heterologous protein expression controlled by salicylic derivatives in micro-organisms associated with higher organisms |
| EP1801212A1 (en) * | 2005-12-21 | 2007-06-27 | Eidgenössische Technische Hochschule Zürich | Selection of biocatalysts for chemical synthesis |
| CN101193906A (en) * | 2005-06-06 | 2008-06-04 | 陶氏环球技术公司 | Mannitol induced promoter systems in bacterial host cells |
| WO2015044456A1 (en) * | 2013-09-30 | 2015-04-02 | Danmarks Tekniske Universitet | Improved biosensor-based detection system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446260A (en) * | 2000-07-13 | 2003-10-01 | 辛根塔参与股份公司 | Lipoxygenase genes, promoters, transit peptides and protein thereof |
| WO2005054477A1 (en) * | 2003-12-04 | 2005-06-16 | Universidad Pablo De Olavide | Method of regulating heterologous protein expression controlled by salicylic derivatives in micro-organisms associated with higher organisms |
| CN101193906A (en) * | 2005-06-06 | 2008-06-04 | 陶氏环球技术公司 | Mannitol induced promoter systems in bacterial host cells |
| EP1801212A1 (en) * | 2005-12-21 | 2007-06-27 | Eidgenössische Technische Hochschule Zürich | Selection of biocatalysts for chemical synthesis |
| WO2015044456A1 (en) * | 2013-09-30 | 2015-04-02 | Danmarks Tekniske Universitet | Improved biosensor-based detection system |
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