CN114133436B - E.coli membrane protein ZraS mutant, gene for encoding mutant, recombinant vector, preparation method and application - Google Patents

E.coli membrane protein ZraS mutant, gene for encoding mutant, recombinant vector, preparation method and application Download PDF

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CN114133436B
CN114133436B CN202111230759.5A CN202111230759A CN114133436B CN 114133436 B CN114133436 B CN 114133436B CN 202111230759 A CN202111230759 A CN 202111230759A CN 114133436 B CN114133436 B CN 114133436B
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刘复荣
张润钊
崔金明
李小明
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Guangzhou Institute Of Advanced Technology
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Abstract

The invention provides a escherichia coli bi-component regulation system membrane protein ZraS mutant, a gene for encoding the mutant, a recombinant vector, a preparation method and application. The mutant comprises one or more of S154T, A214S, Q313R, T A. The preparation method of the mutants comprises the following steps: constructing SP-ZraS phage; the substrate specificity of SP-ZraS phage to lead ions is alternately evolved through a positive sieve and a negative sieve by adopting a phage-assisted continuous directed evolution method (PACE) to obtain an evolved sample; amino acid sequencing analysis is carried out on the evolution sample, and mutation site information is obtained; and constructing an expression vector of the membrane protein ZraS mutant according to mutation site information, marking the expression vector as pZraS, and expressing to obtain the membrane protein ZraS mutant. Compared with a wild type system, the response sensitivity of the mutant to metal lead ions is obviously improved, the regulation and control range of lead is increased, and the detection limit is reduced to 1 mu M; meanwhile, when the ion concentration is in the range of 0-10 mu M, the specific and sensitive detection of heavy metal lead ions can be realized.

Description

E.coli membrane protein ZraS mutant, gene for encoding mutant, recombinant vector, preparation method and application
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an escherichia coli membrane protein ZraS mutant, a gene for encoding the mutant, a recombinant vector, a preparation method and application.
Background
Lead is an environmental heavy metal contaminant widely distributed in nature, and to varying degrees contains some amount of lead in soil, air and water sources, which can pass through the air, water sources and soil into our diet. Lead in foods is mainly carried into food materials by environmental pollution, so that the lead is taken into the body through diet. Therefore, lead in food is out of standard, and most of the reasons are that the production enterprises do not take strict attention to raw materials, and raw materials with out-of-standard lead content are used, so that the possibility of migrating into food from production equipment is not excluded. Lead has a long half-life in vivo, and thus can be accumulated in vivo for a long period. If the lead content in the body exceeds the standard, the lead absorbed into the blood can have different damage effects on various organ tissues in the organism, and especially damage to the hematopoietic system, the nervous system and the kidneys is obvious. The detection of lead in food is particularly important as a heavy metal which has a serious threat to human health.
Along with the severe environmental pollution and the improvement of life quality requirements of people, the food sources and the safety of the food sources are more and more emphasized, and particularly, the harmful components such as heavy metals, antibiotics and the like contained in the food sources are required to be strictly controlled. The detection method commonly used at present generally integrates a sample pretreatment technology and a chromatographic and mass spectrometry separation analysis technology; in addition, the technology of chemo-qualitative detection, enzyme inhibition, ELISA enzyme-linked reaction based on antigen-antibody recognition, surface plasma resonance, optical fiber signal transduction amplification and the like is also used in the detection of agricultural products and food safety at home and abroad. However, the existing detection method is not satisfactory, and has the limitations of not wide application range, complex experimental operation, high large-scale detection cost and the like.
In prokaryotic cells, as an important mechanism for coupling external stimuli to cellular stress, two-component regulatory systems generally consist of histidine kinase receptors on the membrane (Sensor histidine kinase, HK) and intracellular response regulatory proteins (Response regulator, RR), HK being responsible for sensing external signals and RR for regulating expression of target genes. After HK is activated by external stimulus, phosphorylating one histidine of the HK, transferring phosphate group to one aspartic acid on response regulatory protein RR and activating, and regulating the expression of target genes by the activated RR; this is a natural set of signal transduction systems that rapidly transduce extracellular stimulus signals into intracellular gene expression responses. The method is characterized in that a two-component regulation and control system is used as a core, microbial cells are used as a sensor detection main body, the microbial cells are combined with a physical and chemical detector, color, fluorescence or luminescence signals are output by utilizing the sensitive induction of the microbial cells to heavy metals, and detection results are read by adopting equipment such as a spectrophotometer, an enzyme-labeled instrument and the like. The two-component regulation and control system has two advantages as a signal transduction ring joint: (1) the living cells of the microorganism spontaneously provide each regulatory protein element required by signal transduction, so that additional purchasing reagent is not needed, and the cost is low; (2) the living cells of the microorganism effectively isolate extracellular environment fluctuation from intracellular reporter gene expression through cell membranes, so that the robustness of the system is obviously superior to that of enzymatic reaction, and the requirements on pretreatment or purity of a tested sample are greatly reduced.
The ZraS-ZraR of the escherichia coli dual-component regulation system can identify and sense extracellular metallic lead and zinc ions, when the extracellular lead ions exist, the membrane protein ZraS identifies ion signals, and then the regulation protein ZraR is activated to be phosphorylated, and the phosphorylated ZraR regulation protein is combined with a recognition site at the upstream of a ZraP promoter to induce the expression of a target gene ZraP. The system has high efficiency and specificity for detecting lead ions or zinc ions, and is an ideal microorganism detector. In view of the harm of lead to human bodies, the detection of the content of lead in agricultural products has greater application value and significance. However, in the practical application process, the sample to be measured often has complex components, and two ions may exist at the same time, but the system cannot specifically distinguish lead ions from zinc ions. On the other hand, the detection range of ZraS-ZraR to lead by using a two-component regulation and control system of escherichia coli is reported to be 0.3-1.0 mM; however, the lead content in food is generally lower than 1.0mg/kg (0.0048 mM) according to the national lead limit standard, and it is difficult to achieve the detection objective using the wild type system.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide an escherichia coli membrane protein ZraS mutant, a gene for encoding the mutant, a recombinant vector, a preparation method and application. The specific of the escherichia coli membrane protein ZraS is subjected to directed evolution, so that the recognition sensitivity of the system to lead ions is improved while the recognition capability to the lead ions is reserved, and the recognition capability to zinc ions is reduced or even lost, thereby achieving the aim of specifically detecting the lead ions. The substrate specificity of the membrane protein ZraS is alternately subjected to positive screening and negative screening by using a phage-assisted continuous directed evolution method (PACE), so that the receptor membrane protein capable of sensitively identifying extracellular lead ions is provided, heavy metal lead ions can be sensitively detected, and the evolved membrane protein ZraS can be applied to quantitative detection of lead in agricultural products.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an escherichia coli membrane protein ZraS mutant, which comprises one or more than one of S154T, A214S, Q313R, T A;
wherein, the 154 th amino acid S of the escherichia coli membrane protein ZraS is mutated into T to obtain a mutant S154T;
the 214 th amino acid A of the escherichia coli membrane protein ZraS is mutated into S to obtain mutant A214S;
mutation of 313 th amino acid Q of the escherichia coli membrane protein ZraS into R to obtain mutant Q313R;
the 415 th amino acid T of the escherichia coli membrane protein ZraS is mutated into A to obtain mutant T415A.
The invention also provides a gene for encoding the mutant.
The invention also provides an expression vector containing the gene.
Further, the promoter of the ZraS gene in the expression vector is an endogenous promoter ZraSp.
The invention also provides a recombinant plasmid containing the gene.
The invention also provides a recombinant cell containing the recombinant plasmid.
The invention also provides a preparation method of the membrane protein ZraS mutant, which comprises the following steps: constructing SP-ZraS phage; the substrate specificity of SP-ZraS phage to lead ions is alternately evolved through a positive sieve and a negative sieve by adopting a phage-assisted continuous directed evolution method (PACE) to obtain a mutant sample; amino acid sequencing analysis is carried out on the mutant sample to obtain mutation site information; and constructing an expression vector of the membrane protein ZraS mutant according to mutation site information, marking the expression vector as pZraS, and expressing to obtain the membrane protein ZraS mutant.
Further, the preparation method specifically comprises the following steps:
construction of PACE directed evolution helper plasmids
1.1 construction of the Positive Screen helper plasmid and host
1.11 Construction of gIII protein expression plasmid AP 1: the gIII protein is started by a promoter psp, and a recognition site nucleic acid sequence of ZraR regulatory protein is inserted into the downstream of the promoter psp to obtain an AP1 plasmid;
1.12 construction of regulatory protein ZraR and mutagenesis Gene expression plasmid MP-ZraR: the carrier template is mutagenized plasmid MP4, zraR gene sequence is inserted into the upstream of the mutagenized plasmid MP4 arabinose promoter, and the promoter is J23109, thus obtaining MP-ZraR plasmid;
1.13 E.coli S1030 competent cells are co-transformed by the AP1 and MP-ZraR plasmids to obtain a positive screen Host, which is marked as a Host positive;
1.2 construction of negative Screen helper plasmids and hosts
1.21 Construction of gIII protein expression plasmid AP 2: the gIII protein is started by a promoter J23109, and a recognition site nucleic acid sequence of ZraR regulatory protein is inserted into the downstream of the promoter J23109 to obtain an AP2 plasmid;
1.22 Construction of the gIIIneg protein expression plasmid AP 3: the gIIIneg protein is started by a promoter psp, and a ZraR regulatory protein recognition site nucleic acid sequence is inserted into the downstream of the promoter to obtain an AP3 plasmid;
1.23 E.coli S1030 competent cells were co-transformed with AP2, AP3 and MP-ZraR plasmids to obtain a negative screen Host, designated as Host negative;
1.3 construction of phage SP-ZraS
1.31 amplifying large fragments except gIII using M13 phage genome as template; amplifying a ZraS gene fragment of the membrane protein by taking the E.coli S1030 genome as a template;
1.32 recovering and purifying the two gene fragment gels obtained in the step 1.31, constructing a carrier SP-ZraS by homologous recombination, transferring into competent cells carrying plasmid AP1, recovering for 2 hours, adding 200 mu L of recovery liquid into 0.5% LB soft agar which is subjected to warm bath to about 50 ℃, simultaneously adding zinc ions with the final concentration of 100 mu M, uniformly mixing, plating onto 1.5% LB agar plates, inverting into a 37 ℃ incubator after the upper agar is solidified, and culturing overnight;
1.33, performing PCR amplification and electrophoresis experiments on the obtained plaque, and performing sequencing verification to obtain phage SP-ZraS;
substrate specificity of PACE plus Screen and minus Screen alternate evolution SP-ZraS for lead ions
2.1 Preparation of M9-HEPES Medium: the final concentration composition of the M9-HEPES medium was: naCl 0.25g/L, NH 4 Cl 0.5g/L, maltose 0.8%, mgCl 2 2 mM，CaCl 2 0.1mM, casein hydrolysate 0.8%, vitamin B11%, solvent ddH 2 O; naCl and NH are firstly added 4 Cl is dissolved in ddH 2 Sterilizing in O at 121deg.C for 15min, cooling, and adding maltose solution and MgCl via 0.45 μm sterile filter membrane 2 Solution, caCl 2 The solution, casein hydrolysate solution and vitamin B1 solution;
2.2 positive screen: culturing Host positive with M9-HEPES medium, adding SP-ZraS phage thereto, and adding arabinose and Pb 2+ Culturing, centrifuging the culture solution, and collecting supernatant, and filtering with 0.45 μm sterile filter membrane;
2.3 positive screen phage purification: adding polyethylene glycol-NaCl solution into the obtained supernatant, mixing, standing at room temperature, centrifuging, removing supernatant, and adding ddH 2 O resuspended pellet particles and counted the obtained positive screen phage;
2.4 negative sieves: culturing Host negative with M9-HEPES medium, adding positive-screened purified phage, and adding arabinose and Zn 2+ Culturing, centrifuging the culture solution, and collecting supernatant, and filtering with 0.45 μm sterile filter membrane;
2.5 negative screen phage purification: adding polyethylene glycol-NaCl solution into the obtained supernatant, mixing, standing at room temperature, centrifuging, removing supernatant, and adding ddH 2 O re-suspending the precipitated particles and counting the obtained negative screen phage;
2.6 repeating steps 2.2-2.5, and performing alternate evolution, wherein Pb is added 2+ The concentration is decreased by 2 times, zn 2+ Increasing the concentration by 2 times;
2.7, plating the mutant phage heavy suspension obtained by purification after alternate evolution to obtain plaques, and amplifying ZraS gene fragments on the mutant phage by PCR.
Further, the number of alternate evolutions is 20 to 30.
Preferably, the number of alternate evolutions is 22.
The invention also provides application of the mutant or the gene or the expression vector or the recombinant plasmid or the recombinant cell or the mutant obtained by the preparation method in low-concentration lead content detection.
Further, the mutant or the gene or the expression vector or the recombinant plasmid or the recombinant cell or the mutant obtained by the preparation method is applied to lead residue detection of agricultural products.
The beneficial effects of the invention are as follows:
1) The invention constructs the escherichia coli membrane protein ZraS mutant, which has obviously improved response sensitivity to metal lead ions compared with a wild type system, increases the regulation and control range of lead and has the detection limit as low as 1 mu M; meanwhile, when the ion concentration is in the range of 0-10 mu M, the specific and sensitive detection of heavy metal lead ions can be realized.
2) The mutant protein ZraS obtained by the invention is used for detecting heavy metal lead ions, and has the advantages of low cost, simple operation, high flux detection and the like.
Drawings
FIG. 1 is a map of a membrane protein ZraS expression vector.
FIG. 2 is a map of the reporter gene expression plasmid pLuxAB-zraR.
FIG. 3 shows the map of the protein expression plasmid gIII of the positive screen system AP 1.
FIG. 4 is a map of the regulatory protein ZraR and the mutant gene expression plasmid MP-ZraR.
FIG. 5 shows a map of the negative screen system gIII protein expression plasmid AP 2.
FIG. 6 is a map of the negative screen system gIIIneg protein expression plasmid AP 3.
FIG. 7 is a SP-ZraS phage spotting plate.
FIG. 8 is a SP-ZraS phage map.
FIG. 9 is a graph of the response of wild type and three different mutants to lead detection signals.
FIG. 10 is a graph showing the quadratic dependence of the optical signal intensity on ion concentration for three mutants at 3h and 6 h.
FIG. 11 is a graph showing the response of wild type and three different mutants to zinc detection signals, wherein FIG. 11-1 shows the response of wild type to zinc detection signals, and FIGS. 11-2, 11-3, and 11-4 show the response of three different mutants to zinc detection signals.
FIG. 12 is a schematic of a phage-assisted continuous directed evolution (PACE) process.
FIG. 13 is a schematic and process diagram of the detection of heavy metal lead ions using mutant membrane proteins.
Detailed Description
The mutagenized pellet MP4 used in the examples of the present invention was purchased from addgene, cat# 69652.
In the description of the present invention, it is to be noted that the specific conditions are not specified in examples, and the description is performed under conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides an escherichia coli membrane protein ZraS mutant, which comprises one or more than one of S154T, A214S, Q313R, T A;
wherein, the 154 th amino acid S of the escherichia coli membrane protein ZraS is mutated into T to obtain a mutant S154T;
the 214 th amino acid A of the escherichia coli membrane protein ZraS is mutated into S to obtain mutant A214S;
mutation of 313 th amino acid Q of the escherichia coli membrane protein ZraS into R to obtain mutant Q313R;
the 415 th amino acid T of the escherichia coli membrane protein ZraS is mutated into A to obtain mutant T415A.
The wild ZraS expression vector gene sequence is shown in SEQ ID NO.1, wherein the ZraS sequence is a wild ZraS sequence, the expression vector map is shown in figure 1, and the corresponding sites of the vector sequences aiming at different mutant types are replaced by the mutated sequences.
Compared with the amino acid sequence (see SEQ ID NO. 2) and the nucleic acid sequence (see SEQ ID NO. 3) of the wild-type ZraS, each amino acid mutation site and the corresponding nucleic acid sequence of the present invention are shown in Table 1:
table 1 mutation sites of amino acids and corresponding nucleic acid sequences
Mutation site numbering 1 2 3 4
Amino acid mutation S154T A214S Q313R T415A
Nucleic acid mutation TCA-ACA GCA-TCA CAG-CGG ACT-GCT
The invention also provides a gene for encoding the mutant.
The invention also provides an expression vector containing the gene. In the present invention, the promoter of the ZraS gene in the expression vector is an endogenous promoter ZraSp.
The invention also provides a recombinant plasmid containing the gene.
The invention also provides a recombinant cell containing the recombinant plasmid.
The invention also provides a preparation method of the mutant, which comprises the following steps: constructing SP-ZraS phage; the substrate specificity of SP-ZraS phage to lead ions is alternately evolved through a positive sieve and a negative sieve by adopting a phage-assisted continuous directed evolution method (PACE) to obtain an evolved sample; amino acid sequencing analysis is carried out on the evolution sample, and mutation site information is obtained; and constructing an expression vector of the membrane protein ZraS mutant according to mutation site information, marking the expression vector as pZraS, and expressing to obtain the membrane protein ZraS mutant.
In the invention, the preparation method specifically comprises the following steps:
1.1 construction of the Positive Screen helper plasmid and host
1.11 Construction of gIII protein expression plasmid AP 1: the gIII protein is started by a promoter psp, and a recognition site nucleic acid sequence of ZraR regulatory protein is inserted into the downstream of the promoter psp to obtain an AP1 plasmid;
1.12 construction of regulatory protein ZraR and mutagenesis Gene expression plasmid MP-ZraR: the carrier template is mutagenized plasmid MP4, zraR gene sequence is inserted into the upstream of the mutagenized plasmid MP4 arabinose promoter, and the promoter is J23109, thus obtaining MP-ZraR plasmid;
1.13 E.coli S1030 competent cells are co-transformed by the AP1 and MP-ZraR plasmids to obtain a positive screen Host, which is marked as a Host positive;
1.2 construction of negative Screen helper plasmids and hosts
1.21 Construction of gIII protein expression plasmid AP 2: the gIII protein is started by a promoter J23109, and a recognition site nucleic acid sequence of ZraR regulatory protein is inserted into the downstream of the promoter J23109 to obtain an AP2 plasmid;
1.22 Construction of the gIIIneg protein expression plasmid AP 3: the gIIIneg protein is started by a promoter psp, and a ZraR regulatory protein recognition site nucleic acid sequence is inserted into the downstream of the promoter to obtain an AP3 plasmid;
1.23 E.coli S1030 competent cells were co-transformed with AP2, AP3 and MP-ZraR plasmids to obtain a negative screen Host, designated as Host negative;
1.3 construction of phage SP-ZraS
1.31 amplifying large fragments except gIII using M13 phage genome as template; amplifying a ZraS gene fragment of the membrane protein by taking an E.coli S1030 genome as a template;
1.32 recovering and purifying the two gene fragment gels obtained in the step 1.31, constructing a carrier SP-ZraS by homologous recombination, transferring into competent cells carrying plasmid AP1, recovering for 2 hours, adding 200 mu L of recovery liquid into 0.5% LB soft agar which is subjected to warm bath to about 50 ℃, simultaneously adding zinc ions with the final concentration of 100 mu M, uniformly mixing, plating onto 1.5% LB agar plates (ampicillin resistance), standing the upper agar after solidification, inverting into a 37 ℃ incubator, and culturing overnight;
1.33 PCR amplification of the obtained plaques, wherein the forward primer is M13-vF (GTTCCGATGCTGTCTTTCG), the reverse primer is M13-vR (ACCCAAAAGAACTGGCATG), the target band size is 1849bp, electrophoresis experiments and sequencing verification are carried out on the PCR products, and phage SP-ZraS is obtained.
Substrate specificity of PACE plus Screen and minus Screen alternate evolution SP-ZraS for lead ions
2.1 Preparation of M9-HEPES Medium: the final concentration composition of the M9-HEPES medium was: naCl 0.25g/L, NH 4 Cl 0.5g/L, maltose 0.8%, mgCl 2 2 mM，CaCl 2 0.1mM, casein hydrolysate 0.8%, vitamin B11%, solvent ddH 2 O; naCl and NH are firstly added 4 Cl is dissolved in ddH 2 Sterilizing in O at 121deg.C for 15min, cooling, and adding maltose solution and MgCl via 0.45 μm sterile filter membrane 2 Solution, caCl 2 The solution, casein hydrolysate solution and vitamin B1 solution;
2.2 positive screen: culturing Host positive with M9-HEPES medium, adding SP-ZraS phage thereto, and adding arabinose and Pb 2+ Culturing, centrifuging the culture solution, and collecting supernatant, and filtering with 0.45 μm sterile filter membrane;
2.3 positive screen phage purification: adding polyethylene glycol-NaCl solution into the obtained supernatant, mixing, standing at room temperature, centrifuging, removing supernatant, and adding ddH 2 O resuspended pellet particles and counted the obtained positive screen phage;
2.4 negative sieves: culturing Host negative with M9-HEPES medium, adding positive-screened purified phage, and adding arabinose and Zn 2+ Culturing, centrifuging the culture solution, and collecting supernatant, and filtering with 0.45 μm sterile filter membrane;
2.5 negative screen phage purification: adding polyethylene glycol-NaCl solution into the obtained supernatant, mixing, standing at room temperature, centrifuging, removing supernatant, and adding ddH 2 O re-suspending the precipitated particles and counting the obtained negative screen phage;
2.6 repeating steps 2.2-2.5, and performing alternate evolution, wherein Pb is added 2+ The concentration is decreased by 2 times, zn 2+ Increasing the concentration by 2 times;
2.7, plating the mutant phage heavy suspension obtained by purification after alternate evolution to obtain plaques, carrying out PCR (polymerase chain reaction) amplification on ZraS gene fragments on the mutant phage by using primers M13-vF and M13-vR, and sequencing to obtain mutation information of the membrane protein ZraS.
In the present invention, the number of alternate evolutions is 20 to 30. Preferably 22 times.
The invention also provides application of the mutant or the gene or the expression vector or the recombinant plasmid or the recombinant cell or the mutant obtained by the preparation method in detection of lead residues of agricultural products.
The invention will now be described in further detail with reference to the drawings and to specific examples, which are given by way of illustration and not limitation.
Example 1
ZraS mutation site obtained by directed evolution aiming at lead ion substrate recognition specificity
Construction of PACE directed evolution helper plasmids
1.1 construction of the Positive Screen helper plasmid and host
1) Construction of gIII protein expression plasmid AP 1: the gIII protein is started by a promoter psp, and a ZraR regulatory protein recognition site nucleic acid sequence is inserted into the downstream of the promoter. The AP1 map is shown in figure 3.
2) Construction of regulatory protein ZraR and mutagenesis gene expression plasmid MP-ZraR: the carrier template is mutagenized grain MP4, zraR gene sequence is inserted in the upstream of the mutagenized grain MP4 arabinose promoter, and the promoter is J23109. The plasmid can express regulatory protein ZraR and provides higher mutation rate under the induction of arabinose during the DNA replication process. MP-ZraR patterns are shown in FIG. 4.
3) E.coli S1030 competent cells were co-transformed with the AP1 and MP-ZraR plasmids to obtain a positive Host, designated as Host positive.
1.2 construction of negative Screen helper plasmids and hosts
1) Construction of gIII protein expression plasmid AP 2: the gIII protein was started by promoter J23109, and the AP2 map is shown in FIG. 5.
2) Construction of the gIIIneg protein expression plasmid AP 3: the gIIIneg protein is started by a promoter psp, and a ZraR regulatory protein recognition site nucleic acid sequence is inserted into the downstream of the promoter. The AP3 map is shown in FIG. 6.
3) E.coli S1030 competent cells were co-transformed with AP2, AP3 and MP-ZraR plasmids to obtain a negative screen Host, designated as Host negative.
1.3 construction of phage SP-ZraS
1) Amplifying large fragments except gIII by using M13 phage genome as a template; amplifying a ZraS gene fragment of the membrane protein by taking the E.coli S1030 genome as a template;
2) The two gene fragment gels are recovered and purified, then the vector SP-ZraS is constructed by homologous recombination, the vector SP-ZraS is transferred into competent cells carrying plasmid AP1, after recovery for 2 hours, 200 mu L of recovery liquid is added into 0.5% LB soft agar which is subjected to a warm bath to about 50 ℃, meanwhile zinc ions with the final concentration of 100 mu M are added, the mixture is uniformly mixed and plated onto 1.5% LB agar plates (ampicillin resistance), and after the upper agar is solidified, the mixture is inverted into a 37 ℃ incubator for overnight culture.
3) And (3) observing the condition of the plaque the next day, carrying out PCR amplification and electrophoresis verification on the plaque, and sequencing verification to obtain the correct phage SP-ZraS. Plaque plates are shown in FIG. 7 and phage maps are shown in FIG. 8.
The substrate specificity of PACE positive and negative sieves to lead ions was evolved alternately SP-ZraS (process is shown in FIG. 12)
1) Preparation of M9-HEPES Medium (1L)
Accurately weigh NaCl 0.25g and NH 4 Cl 0.50g was dissolved in 800mL of ultra pure water, autoclaved at 121℃for 15min, cooled, and maltose, 2mM MgCl, 0.8% final concentration through a 0.45 μm sterile filter membrane, were added thereto, respectively 2 0.1mM CaCl2, 0.8% casein hydrolysate, 1% vitamin B1, and ddH was added last 2 O makes up the volume to 1L.
2) And (3) positive screening: host positive was cultured to OD600 of about 0.2 with M9-HEPES medium, to which was added a final concentration of about 10 7 PFU/mL SP-ZraS phage, and 5. Mu.M Pb and arabinose were added at a final concentration of 1% 2+ Shaking culture at 37deg.C and 200rpm for 3 hr, taking out culture solution, centrifuging, and collecting supernatant, and filtering with 0.45 μm sterile filter membrane;
3) Positive screen phage purification: adding 1/5 volume of 20% polyethylene glycol (dissolved in 2.5M NaCl) into the supernatant, mixing, standing at room temperature for 15min under mild shaking, centrifuging at 12000rpm for 5min at 4deg.C, discarding supernatant, and adding small amount of ddH 2 O resuspended phage pellet particles and phage counted;
4) Negative screen: culturing Host positive to OD600 of about 0.2 with M9-HEPES medium, and adding final concentration of about 10 7 PFU/mL of phage purified after the previous positive screen and added with arabinose at a final concentration of 1% and Zn at 5. Mu.M 2 + Shaking culture at 37deg.C and 200rpm for 3 hr, taking out culture solution, centrifuging, and collecting supernatant, and filtering with 0.45 μm sterile filter membrane;
5) Negative screen phage purification: adding polyethylene glycol-NaCl solution into the obtained supernatant, mixing, standing at room temperature, centrifuging, removing supernatant, and adding ddH 2 O re-suspending the precipitated particles and counting the obtained negative screen phage;
6) Repeating the steps 2) -5), and performing alternate evolution, wherein Pb 2+ The concentration is decreased by 2 times, zn 2+ The concentration was increased by a factor of 2.
7) The obtained mutant phage supernatant was plated to obtain plaques, and ZraS gene fragments (PCR primer sequences and amplification conditions are shown in the following table) on the phage were PCR amplified using primers M13-vF and M13-vR, and the ZraS nucleic acid sequences (see SEQ ID No.4, 6, 8) and amino acid sequences (see SEQ ID No.5, 7, 9) were sequenced and analyzed, and compared with the wild-type ZraS gene sequences to identify base or amino acid mutation. The primers, reaction system and procedure for PCR amplification are shown in tables 2 to 4
TABLE 2 primers for PCR amplification
TABLE 3 PCR System
TABLE 4 PCR procedure
Results were obtained by the above directed evolution: through 22 rounds of alternating evolution of positive sieves and negative sieves, the accumulated mutation site is S154T, A214S, Q313R, T A.
In a positive screen evolution system, adding lead ions into a culture solution, infecting host cells by phage SP-ZraS, expressing and phosphorylating membrane protein ZraS, then transferring phosphate groups to regulatory protein ZraR, combining the phosphorylated ZraR with a recognition site at the upstream of gIII, and expressing pIII protein; the expression quantity of the pIII protein is regulated and controlled by the concentration of extracellular lead ions, the intensity of lead ion activated ZraS is indirectly coupled with the progeny package of SP-ZraS phage, and the screening pressure is improved by continuously reducing the addition quantity of lead ions, so that the ZraS mutant evolves towards the direction of improving the sensitivity of lead ions. Similarly, zinc ions are added in the negative screening process, the ZraS mutant sensitive to zinc activates the expression of pIII-neg protein, and the phage packaged by the pIII-neg protein cannot release extracellular, so that the mutant insensitive to zinc at the concentration is reserved, and the sensitivity of SP-ZraS phage to zinc can be gradually reduced by continuously increasing the addition amount of the zinc ions. The membrane protein ZraS is evolved in the direction of improving the sensitivity to lead ions and weakening the sensitivity to zinc ions by alternately carrying out positive screening and negative screening.
Example 2
Construction of engineering strains
1. The mutation sites or combinations obtained in the example 1 are selected, different ZraS mutant expression vectors pZraS are constructed, and the ZraS promoter is taken as an endogenous promoter ZraSp.
2. The expression vectors pLuxAB-ZraR of the reporter gene luxAB and the regulatory protein ZraR are constructed, and the plasmid map is shown in figure 2. Wherein the reporter gene luxAB and the regulatory protein ZraR are expressed in series, and are started by a promoter zrapP, and two regulatory protein ZraR binding sites are arranged at the upstream of the promoter.
3. E.coli S1030 competent cells were co-transformed with different pZraS vectors and pLuxAB-ZraR plasmids, respectively, to obtain different recombinant mutant strains.
The expression host used in the present invention is E.coli S1030 carrying the F plasmid, which is derived from E.coli DH10B and supplied by David R Liu laboratory, and has been reported in the relevant literature to have genetic information (Nat Chem biol.2014March;10 (3): 216-222) of the genotype F' proA+B+Δ (lacIZY) zzf: tn10 (TetR) lacIQ1PN25-tetR luxCDE/endA1 recA1 galE15 galK16 nupG rpsL (StrR) ΔlacIYAaraD 139 Δ (ara, leu) 7697mcrA Δ (mrr-hsdRMS-mcrBC) proBA: 116 araE201 ΔrpoZ Δflu ΔcspgcCDG ΔC-.
It should be noted that the host bacterium in the present invention is not limited to E.coli S1030, and may be replaced by any E.coli carrying F factor, and may be obtained by a conventional molecular biology technique, which is well known in the art, and may be obtained by a person skilled in the art according to a conventional method. As will be appreciated by those skilled in the art, the present invention satisfies the full disclosure without providing for preservation of the seed. The reporter gene is also not limited to luxAB and may be replaced by any other fluorescent protein gene. The expression vector is electrotransformed or chemotransformed into S1030 competent cells, positive clones are screened by ampicillin and spectinomycin, and engineering strains of different ZraS mutants are obtained; the competent preparation and transformation methods by E.coli are a fundamental skill in the art, and can be performed by those skilled in the art by routine procedures or related laboratory manuals.
Example 3
Sensitive detection of ZraS mutant engineering strain on metallic lead ions
The mutant engineering bacteria S1030 (A214S), S1030 (S154T/A214S) and S1030 (Q313R/T415A) in example 2 were selected, and the wild type engineering bacteria S1030 (WT) was used as a control to detect metallic lead ions. The specific implementation process is as follows:
preparation of M9-HEPES Medium: as in example 1.
2. Bacterial resuscitation and activation
(1) Taking out the 3 mutants and the wild type engineering strain from a refrigerator at the temperature of minus 80 ℃, thawing the strains at the temperature of 4 ℃, picking a one-ring fungus solution, streaking the strain on LB agar medium added with 100 mug/mL of ampicillin and spectinomycin hydrochloride, and placing the strain in an incubator at the temperature of 37 ℃ for overnight culture;
(2) single colonies were picked in 2mL M9-HEPES medium (100. Mu.g/mL final concentrations of ampicillin and spectinomycin hydrochloride) and shake-cultured overnight at 37℃and 200 rpm;
(3) adding seed solution into 5mL of M9-HEPES culture medium at 1% inoculum size, shaking at 37deg.C and 200rpm, measuring OD600 every 30min, and inoculating into fresh M9-HEPES culture medium with ampicillin and spectinomycin hydrochloride resistance at 20% inoculum size when OD600 rises to about 0.2;
(4) repeating the step (3) once;
3. detection of extracellular lead ions
(1) Fully mixing bacterial liquid cultured by M9-HEPES culture medium, sub-packaging in 96-well enzyme-labeled pore plates, adding 150 mu L of bacterial liquid into each well, adding a certain amount of lead ion or zinc ion standard liquid, and repeating the steps for 3 times until the final concentration of lead ion or zinc ion is 0 mu M, 1 mu M, 2 mu M, 5 mu M and 10 mu M respectively;
(2) the elisa plate was placed in a multifunctional microplate reader (BiotekS 1 LFA) and the program method and parameters were set as shown in table 5:
table 5 method and parameters for detecting extracellular lead ions by using multifunctional enzyme-labeled instrument
The detection principle and the detection process of heavy metal lead ions by using mutant membrane proteins are shown in figure 13, when lead ions with a certain concentration exist outside cells, membrane proteins ZraS recognize ion signals, regulatory proteins ZraR are activated to cause phosphorylation of the regulatory proteins ZraR, phosphorylated ZraP regulatory proteins are combined with recognition sites on the upstream of ZraP promoters, the promoters zrPp are activated, the expression of reporter genes luxAB is induced, and simultaneously, the expression quantity of regulatory proteins ZraR on the downstream of the reporter genes is increased, so that the promoters zrPp are further activated, the signal of the reporter genes is stronger, and the induction sensitivity of the system to the lead ions is enhanced.
(3) And (3) data processing: unit optical signal intensity (Unit photon count, UPC) =pc/OD 600 Wherein PC (photon counts) is photon number, OD of the bacterial liquid 600 The absorbance of the bacterial liquid. And comparing the difference between the concentrations of the detected lead ions and zinc ions of different mutants and wild types by taking the ion concentration or time as an abscissa and the unit optical signal intensity as an ordinate.
(1) Detection of lead ions: as shown in fig. 9, the 3 mutants had an increased range of regulation of lead ions compared to the wild type, and the detection sensitivity was significantly improved. The lead ion is in the concentration range of 0-10 mu M, and the optical signal intensity of 3 mutants is obviously increased after 2h of culture and is positively correlated with the ion concentration. As shown in FIG. 10, which illustrates the linear relationship between the light signal intensity and the ion concentration of the wild type and the three mutants after culturing for 3h and 6h, the wild type has no obvious light signal response at 3h and 6h, and the three mutants have quadratic linear relationship with the ion concentration, wherein the response capacity of the mutants S1030 (A214S) and S1030 (S154T/A214S) to lead ions is equivalent and stronger than that of the mutants S1030 (Q313R/T415A).
(2) Detection of zinc ions: as shown in FIG. 11, the zinc ions were in the concentration range of 0 to 10. Mu.M, the wild type and the three mutants had no obvious response to zinc, and the optical signals of the three mutants showed a gradual decrease trend with the increase of the culture time. It shows that when the ion concentration is in the range of 0-10 mu M, the three mutants can realize specific and sensitive detection of lead ions.
In conclusion, the invention constructs the mutant membrane protein ZraS of the escherichia coli bi-component regulation system, the response sensitivity of the mutant to metal lead ions is obviously improved compared with that of a wild type system, the regulation range of lead is enlarged, and the detection limit is reduced to 1 mu M; meanwhile, when the ion concentration is in the range of 0-10 mu M, the specific and sensitive detection of heavy metal lead ions can be realized.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Guangzhou advanced technology research institute
<120> E.coli membrane protein ZraS mutant, gene encoding the mutant, recombinant vector, preparation method and application thereof
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3852
<212> DNA
<213> Escherichia coli Membrane protein zraS wild type (zraS-WT)
<400> 1
ctgtgttaca gcgcagggta agcgctgata aaagatggca tgatttctgc tgtcagaaag 60
ggatgagcag gcaaagaaga agatgcgttt tatgcaacgt tctaaagact ccttagctaa 120
atggttaagc gcgatcctcc ccgtggtcat tgttgggctg gtgggattgt ttgcggtaac 180
tgtgattcgt gattatgggc gggcaagcga ggcagaccgc caggcattac tggaaaaagg 240
taatgtgctt atccgcgctc tggagtcggg aagccgcgta gggatgggga tgcgaatgca 300
ccatgtacag caacaggcgc ttctggaaga gatggcggga cagccgggag tgttgtggtt 360
cgcagtcacc gatgcgcagg gcatcattat tcttcatagc gaccccgata aggtcgggcg 420
tgcgctctat tcgccggatg aaatgcagaa attaaagcca gaggaaaact cccgctggcg 480
gctgcttggg aaaacggaaa ctacgcctgc acttgaggtc tatcgtttgt tccagccaat 540
gtcagcgccc tggcggcatg gaatgcacaa tatgccgcgc tgtaacggca aagctgtgcc 600
acaagtagat gcacaacagg ctatttttat cgccgttgat gccagtgatc tggttgcaac 660
ccagagtggg gaaaagcgca ataccctgat tatcctcttc gccctggcga cggtcttgct 720
ggcaagcgta ttgtcattct tctggtatcg ccgctatctg cgctcgcgcc agcttctaca 780
agatgaaatg aagcgcaaag agaagctggt ggcgctgggg catcttgcgg caggcgttgc 840
ccacgaaatc cgtaacccac tttcctcgat taaaggactg gcgaaatact ttgccgagcg 900
cgcgcctgca gggggagaag cgcatcaact ggcgcaggtg atggcgaaag aggccgaccg 960
tttaaaccgc gtggtaagcg agttgctgga actggttaag ccaacgcatc tggctttgca 1020
ggcggtggat ctcaacacgc tgattaacca ctcattacag ctggtaagtc aggatgcaaa 1080
cagccgggag atccagttac gctttaccgc caacgacaca ttaccggaaa ttcaggccga 1140
cccggacagg ctgactcagg tcctgttgaa tctctatctc aatgctattc aggcgattgg 1200
tcagcatggc gtgattagcg tgacggccag cgaaagcggc gcgggcgtga aaatcagcgt 1260
taccgacagc ggtaagggaa ttgcggcaga tcagcttgat gccatcttca ctccgtactt 1320
caccactaaa gccgaaggca ccggattggg gctggcggtc gtgcataata ttgttgaaca 1380
acacggtggt acaattcagg tcgcaagcca ggagggaaaa ggctcaacgt tcaccctctg 1440
gcttccggtc aatattacgc gtaaggaccc acaaggatga gaattcactt aattaacggc 1500
actcctcagc aaatataatg accctcttga taacccaaga gggcattttt taatgcccat 1560
ggcgtttatt tgccgactac cttggtgatc tcgcctttca cgtagtggac aaattcttcc 1620
aactgatctg cgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct 1680
tcaagtatga cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca 1740
tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact 1800
acatttcgct catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt 1860
agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct 1920
accaaggcaa cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc 1980
gtggctggct cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt 2040
tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct 2100
acagcgcgga gaatctcgct ctctccaggg gaagccgaag tttccaaaag gtcgttgatc 2160
aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca 2220
ctgtgtggct tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc 2280
ggttcgagat ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg 2340
atcaccgctt ccctcatact cttccttttt caatattatt gaagcattta tcagggttat 2400
tgtctcatga gcggatacat atttgaatgt atttagaaaa ataggccaaa taggccgttt 2460
gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 2520
cggtggtttg tttgccggat caagagctac cacctctttt tccgaaggta actggcttca 2580
gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 2640
agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 2700
ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 2760
cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 2820
acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 2880
gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 2940
ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 3000
agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 3060
cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 3120
tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 3180
gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc 3240
ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta 3300
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 3360
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3420
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3480
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 3540
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 3600
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 3660
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 3720
acgagagagg atgctcacga tacgggttac tgatgatgaa cctgcagaag aggacatccg 3780
gtcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttgct gaggagactt 3840
agggaccggt ac 3852
<210> 2
<211> 465
<212> PRT
<213> Escherichia coli Membrane protein zraS wild type (zraS-WT)
<400> 2
Met Arg Phe Met Gln Arg Ser Lys Asp Ser Leu Ala Lys Trp Leu Ser
1 5 10 15
Ala Ile Leu Pro Val Val Ile Val Gly Leu Val Gly Leu Phe Ala Val
20 25 30
Thr Val Ile Arg Asp Tyr Gly Arg Ala Ser Glu Ala Asp Arg Gln Ala
35 40 45
Leu Leu Glu Lys Gly Asn Val Leu Ile Arg Ala Leu Glu Ser Gly Ser
50 55 60
Arg Val Gly Met Gly Met Arg Met His His Val Gln Gln Gln Ala Leu
65 70 75 80
Leu Glu Glu Met Ala Gly Gln Pro Gly Val Leu Trp Phe Ala Val Thr
85 90 95
Asp Ala Gln Gly Ile Ile Ile Leu His Ser Asp Pro Asp Lys Val Gly
100 105 110
Arg Ala Leu Tyr Ser Pro Asp Glu Met Gln Lys Leu Lys Pro Glu Glu
115 120 125
Asn Ser Arg Trp Arg Leu Leu Gly Lys Thr Glu Thr Thr Pro Ala Leu
130 135 140
Glu Val Tyr Arg Leu Phe Gln Pro Met Ser Ala Pro Trp Arg His Gly
145 150 155 160
Met His Asn Met Pro Arg Cys Asn Gly Lys Ala Val Pro Gln Val Asp
165 170 175
Ala Gln Gln Ala Ile Phe Ile Ala Val Asp Ala Ser Asp Leu Val Ala
180 185 190
Thr Gln Ser Gly Glu Lys Arg Asn Thr Leu Ile Ile Leu Phe Ala Leu
195 200 205
Ala Thr Val Leu Leu Ala Ser Val Leu Ser Phe Phe Trp Tyr Arg Arg
210 215 220
Tyr Leu Arg Ser Arg Gln Leu Leu Gln Asp Glu Met Lys Arg Lys Glu
225 230 235 240
Lys Leu Val Ala Leu Gly His Leu Ala Ala Gly Val Ala His Glu Ile
245 250 255
Arg Asn Pro Leu Ser Ser Ile Lys Gly Leu Ala Lys Tyr Phe Ala Glu
260 265 270
Arg Ala Pro Ala Gly Gly Glu Ala His Gln Leu Ala Gln Val Met Ala
275 280 285
Lys Glu Ala Asp Arg Leu Asn Arg Val Val Ser Glu Leu Leu Glu Leu
290 295 300
Val Lys Pro Thr His Leu Ala Leu Gln Ala Val Asp Leu Asn Thr Leu
305 310 315 320
Ile Asn His Ser Leu Gln Leu Val Ser Gln Asp Ala Asn Ser Arg Glu
325 330 335
Ile Gln Leu Arg Phe Thr Ala Asn Asp Thr Leu Pro Glu Ile Gln Ala
340 345 350
Asp Pro Asp Arg Leu Thr Gln Val Leu Leu Asn Leu Tyr Leu Asn Ala
355 360 365
Ile Gln Ala Ile Gly Gln His Gly Val Ile Ser Val Thr Ala Ser Glu
370 375 380
Ser Gly Ala Gly Val Lys Ile Ser Val Thr Asp Ser Gly Lys Gly Ile
385 390 395 400
Ala Ala Asp Gln Leu Asp Ala Ile Phe Thr Pro Tyr Phe Thr Thr Lys
405 410 415
Ala Glu Gly Thr Gly Leu Gly Leu Ala Val Val His Asn Ile Val Glu
420 425 430
Gln His Gly Gly Thr Ile Gln Val Ala Ser Gln Glu Gly Lys Gly Ser
435 440 445
Thr Phe Thr Leu Trp Leu Pro Val Asn Ile Thr Arg Lys Asp Pro Gln
450 455 460
Gly
465
<210> 3
<211> 1398
<212> DNA
<213> Escherichia coli Membrane protein zraS wild type (zraS-WT)
<400> 3
atgcgtttta tgcaacgttc taaagactcc ttagctaaat ggttaagcgc gatcctcccc 60
gtggtcattg ttgggctggt gggattgttt gcggtaactg tgattcgtga ttatgggcgg 120
gcaagcgagg cagaccgcca ggcattactg gaaaaaggta atgtgcttat ccgcgctctg 180
gagtcgggaa gccgcgtagg gatggggatg cgaatgcacc atgtacagca acaggcgctt 240
ctggaagaga tggcgggaca gccgggagtg ttgtggttcg cagtcaccga tgcgcagggc 300
atcattattc ttcatagcga ccccgataag gtcgggcgtg cgctctattc gccggatgaa 360
atgcagaaat taaagccaga ggaaaactcc cgctggcggc tgcttgggaa aacggaaact 420
acgcctgcac ttgaggtcta tcgtttgttc cagccaatgt cagcgccctg gcggcatgga 480
atgcacaata tgccgcgctg taacggcaaa gctgtgccac aagtagatgc acaacaggct 540
atttttatcg ccgttgatgc cagtgatctg gttgcaaccc agagtgggga aaagcgcaat 600
accctgatta tcctcttcgc cctggcgacg gtcttgctgg caagcgtatt gtcattcttc 660
tggtatcgcc gctatctgcg ctcgcgccag cttctacaag atgaaatgaa gcgcaaagag 720
aagctggtgg cgctggggca tcttgcggca ggcgttgccc acgaaatccg taacccactt 780
tcctcgatta aaggactggc gaaatacttt gccgagcgcg cgcctgcagg gggagaagcg 840
catcaactgg cgcaggtgat ggcgaaagag gccgaccgtt taaaccgcgt ggtaagcgag 900
ttgctggaac tggttaagcc aacgcatctg gctttgcagg cggtggatct caacacgctg 960
attaaccact cattacagct ggtaagtcag gatgcaaaca gccgggagat ccagttacgc 1020
tttaccgcca acgacacatt accggaaatt caggccgacc cggacaggct gactcaggtc 1080
ctgttgaatc tctatctcaa tgctattcag gcgattggtc agcatggcgt gattagcgtg 1140
acggccagcg aaagcggcgc gggcgtgaaa atcagcgtta ccgacagcgg taagggaatt 1200
gcggcagatc agcttgatgc catcttcact ccgtacttca ccactaaagc cgaaggcacc 1260
ggattggggc tggcggtcgt gcataatatt gttgaacaac acggtggtac aattcaggtc 1320
gcaagccagg agggaaaagg ctcaacgttc accctctggc ttccggtcaa tattacgcgt 1380
aaggacccac aaggatga 1398
<210> 4
<211> 1398
<212> DNA
<213> E.coli Membrane protein mutant zraS (zrafput-S154T/A214S)
<400> 4
atgcgtttta tgcaacgttc taaagactcc ttagctaaat ggttaagcgc gatcctcccc 60
gtggtcattg ttgggctggt gggattgttt gcggtaactg tgattcgtga ttatgggcgg 120
gcaagcgagg cagaccgcca ggcattactg gaaaaaggta atgtgcttat ccgcgctctg 180
gagtcgggaa gccgcgtagg gatggggatg cgaatgcacc atgtacagca acaggcgctt 240
ctggaagaga tggcgggaca gccgggagtg ttgtggttcg cagtcaccga tgcgcagggc 300
atcattattc ttcatagcga ccccgataag gtcgggcgtg cgctctattc gccggatgaa 360
atgcagaaat taaagccaga ggaaaactcc cgctggcggc tgcttgggaa aacggaaact 420
acgcctgcac ttgaggtcta tcgtttgttc cagccaatga cagcgccctg gcggcatgga 480
atgcacaata tgccgcgctg taacggcaaa gctgtgccac aagtagatgc acaacaggct 540
atttttatcg ccgttgatgc cagtgatctg gttgcaaccc agagtgggga aaagcgcaat 600
accctgatta tcctcttcgc cctggcgacg gtcttgctgt caagcgtatt gtcattcttc 660
tggtatcgcc gctatctgcg ctcgcgccag cttctacaag atgaaatgaa gcgcaaagag 720
aagctggtgg cgctggggca tcttgcggca ggcgttgccc acgaaatccg taacccactt 780
tcctcgatta aaggactggc gaaatacttt gccgagcgcg cgcctgcagg gggagaagcg 840
catcaactgg cgcaggtgat ggcgaaagag gccgaccgtt taaaccgcgt ggtaagcgag 900
ttgctggaac tggttaagcc aacgcatctg gctttgcagg cggtggatct caacacgctg 960
attaaccact cattacagct ggtaagtcag gatgcaaaca gccgggagat ccagttacgc 1020
tttaccgcca acgacacatt accggaaatt caggccgacc cggacaggct gactcaggtc 1080
ctgttgaatc tctatctcaa tgctattcag gcgattggtc agcatggcgt gattagcgtg 1140
acggccagcg aaagcggcgc gggcgtgaaa atcagcgtta ccgacagcgg taagggaatt 1200
gcggcagatc agcttgatgc catcttcact ccgtacttca ccactaaagc cgaaggcacc 1260
ggattggggc tggcggtcgt gcataatatt gttgaacaac acggtggtac aattcaggtc 1320
gcaagccagg agggaaaagg ctcaacgttc accctctggc ttccggtcaa tattacgcgt 1380
aaggacccac aaggatga 1398
<210> 5
<211> 465
<212> PRT
<213> E.coli Membrane protein mutant zraS (zrafput-S154T/A214S)
<400> 5
Met Arg Phe Met Gln Arg Ser Lys Asp Ser Leu Ala Lys Trp Leu Ser
1 5 10 15
Ala Ile Leu Pro Val Val Ile Val Gly Leu Val Gly Leu Phe Ala Val
20 25 30
Thr Val Ile Arg Asp Tyr Gly Arg Ala Ser Glu Ala Asp Arg Gln Ala
35 40 45
Leu Leu Glu Lys Gly Asn Val Leu Ile Arg Ala Leu Glu Ser Gly Ser
50 55 60
Arg Val Gly Met Gly Met Arg Met His His Val Gln Gln Gln Ala Leu
65 70 75 80
Leu Glu Glu Met Ala Gly Gln Pro Gly Val Leu Trp Phe Ala Val Thr
85 90 95
Asp Ala Gln Gly Ile Ile Ile Leu His Ser Asp Pro Asp Lys Val Gly
100 105 110
Arg Ala Leu Tyr Ser Pro Asp Glu Met Gln Lys Leu Lys Pro Glu Glu
115 120 125
Asn Ser Arg Trp Arg Leu Leu Gly Lys Thr Glu Thr Thr Pro Ala Leu
130 135 140
Glu Val Tyr Arg Leu Phe Gln Pro Met Thr Ala Pro Trp Arg His Gly
145 150 155 160
Met His Asn Met Pro Arg Cys Asn Gly Lys Ala Val Pro Gln Val Asp
165 170 175
Ala Gln Gln Ala Ile Phe Ile Ala Val Asp Ala Ser Asp Leu Val Ala
180 185 190
Thr Gln Ser Gly Glu Lys Arg Asn Thr Leu Ile Ile Leu Phe Ala Leu
195 200 205
Ala Thr Val Leu Leu Ser Ser Val Leu Ser Phe Phe Trp Tyr Arg Arg
210 215 220
Tyr Leu Arg Ser Arg Gln Leu Leu Gln Asp Glu Met Lys Arg Lys Glu
225 230 235 240
Lys Leu Val Ala Leu Gly His Leu Ala Ala Gly Val Ala His Glu Ile
245 250 255
Arg Asn Pro Leu Ser Ser Ile Lys Gly Leu Ala Lys Tyr Phe Ala Glu
260 265 270
Arg Ala Pro Ala Gly Gly Glu Ala His Gln Leu Ala Gln Val Met Ala
275 280 285
Lys Glu Ala Asp Arg Leu Asn Arg Val Val Ser Glu Leu Leu Glu Leu
290 295 300
Val Lys Pro Thr His Leu Ala Leu Gln Ala Val Asp Leu Asn Thr Leu
305 310 315 320
Ile Asn His Ser Leu Gln Leu Val Ser Gln Asp Ala Asn Ser Arg Glu
325 330 335
Ile Gln Leu Arg Phe Thr Ala Asn Asp Thr Leu Pro Glu Ile Gln Ala
340 345 350
Asp Pro Asp Arg Leu Thr Gln Val Leu Leu Asn Leu Tyr Leu Asn Ala
355 360 365
Ile Gln Ala Ile Gly Gln His Gly Val Ile Ser Val Thr Ala Ser Glu
370 375 380
Ser Gly Ala Gly Val Lys Ile Ser Val Thr Asp Ser Gly Lys Gly Ile
385 390 395 400
Ala Ala Asp Gln Leu Asp Ala Ile Phe Thr Pro Tyr Phe Thr Thr Lys
405 410 415
Ala Glu Gly Thr Gly Leu Gly Leu Ala Val Val His Asn Ile Val Glu
420 425 430
Gln His Gly Gly Thr Ile Gln Val Ala Ser Gln Glu Gly Lys Gly Ser
435 440 445
Thr Phe Thr Leu Trp Leu Pro Val Asn Ile Thr Arg Lys Asp Pro Gln
450 455 460
Gly
465
<210> 6
<211> 1398
<212> DNA
<213> E.coli Membrane protein mutant zraS (zrafput-A214S)
<400> 6
atgcgtttta tgcaacgttc taaagactcc ttagctaaat ggttaagcgc gatcctcccc 60
gtggtcattg ttgggctggt gggattgttt gcggtaactg tgattcgtga ttatgggcgg 120
gcaagcgagg cagaccgcca ggcattactg gaaaaaggta atgtgcttat ccgcgctctg 180
gagtcgggaa gccgcgtagg gatggggatg cgaatgcacc atgtacagca acaggcgctt 240
ctggaagaga tggcgggaca gccgggagtg ttgtggttcg cagtcaccga tgcgcagggc 300
atcattattc ttcatagcga ccccgataag gtcgggcgtg cgctctattc gccggatgaa 360
atgcagaaat taaagccaga ggaaaactcc cgctggcggc tgcttgggaa aacggaaact 420
acgcctgcac ttgaggtcta tcgtttgttc cagccaatgt cagcgccctg gcggcatgga 480
atgcacaata tgccgcgctg taacggcaaa gctgtgccac aagtagatgc acaacaggct 540
atttttatcg ccgttgatgc cagtgatctg gttgcaaccc agagtgggga aaagcgcaat 600
accctgatta tcctcttcgc cctggcgacg gtcttgctgt caagcgtatt gtcattcttc 660
tggtatcgcc gctatctgcg ctcgcgccag cttctacaag atgaaatgaa gcgcaaagag 720
aagctggtgg cgctggggca tcttgcggca ggcgttgccc acgaaatccg taacccactt 780
tcctcgatta aaggactggc gaaatacttt gccgagcgcg cgcctgcagg gggagaagcg 840
catcaactgg cgcaggtgat ggcgaaagag gccgaccgtt taaaccgcgt ggtaagcgag 900
ttgctggaac tggttaagcc aacgcatctg gctttgcagg cggtggatct caacacgctg 960
attaaccact cattacagct ggtaagtcag gatgcaaaca gccgggagat ccagttacgc 1020
tttaccgcca acgacacatt accggaaatt caggccgacc cggacaggct gactcaggtc 1080
ctgttgaatc tctatctcaa tgctattcag gcgattggtc agcatggcgt gattagcgtg 1140
acggccagcg aaagcggcgc gggcgtgaaa atcagcgtta ccgacagcgg taagggaatt 1200
gcggcagatc agcttgatgc catcttcact ccgtacttca ccactaaagc cgaaggcacc 1260
ggattggggc tggcggtcgt gcataatatt gttgaacaac acggtggtac aattcaggtc 1320
gcaagccagg agggaaaagg ctcaacgttc accctctggc ttccggtcaa tattacgcgt 1380
aaggacccac aaggatga 1398
<210> 7
<211> 465
<212> PRT
<213> E.coli Membrane protein mutant zraS (zrafput-A214S)
<400> 7
Met Arg Phe Met Gln Arg Ser Lys Asp Ser Leu Ala Lys Trp Leu Ser
1 5 10 15
Ala Ile Leu Pro Val Val Ile Val Gly Leu Val Gly Leu Phe Ala Val
20 25 30
Thr Val Ile Arg Asp Tyr Gly Arg Ala Ser Glu Ala Asp Arg Gln Ala
35 40 45
Leu Leu Glu Lys Gly Asn Val Leu Ile Arg Ala Leu Glu Ser Gly Ser
50 55 60
Arg Val Gly Met Gly Met Arg Met His His Val Gln Gln Gln Ala Leu
65 70 75 80
Leu Glu Glu Met Ala Gly Gln Pro Gly Val Leu Trp Phe Ala Val Thr
85 90 95
Asp Ala Gln Gly Ile Ile Ile Leu His Ser Asp Pro Asp Lys Val Gly
100 105 110
Arg Ala Leu Tyr Ser Pro Asp Glu Met Gln Lys Leu Lys Pro Glu Glu
115 120 125
Asn Ser Arg Trp Arg Leu Leu Gly Lys Thr Glu Thr Thr Pro Ala Leu
130 135 140
Glu Val Tyr Arg Leu Phe Gln Pro Met Ser Ala Pro Trp Arg His Gly
145 150 155 160
Met His Asn Met Pro Arg Cys Asn Gly Lys Ala Val Pro Gln Val Asp
165 170 175
Ala Gln Gln Ala Ile Phe Ile Ala Val Asp Ala Ser Asp Leu Val Ala
180 185 190
Thr Gln Ser Gly Glu Lys Arg Asn Thr Leu Ile Ile Leu Phe Ala Leu
195 200 205
Ala Thr Val Leu Leu Ser Ser Val Leu Ser Phe Phe Trp Tyr Arg Arg
210 215 220
Tyr Leu Arg Ser Arg Gln Leu Leu Gln Asp Glu Met Lys Arg Lys Glu
225 230 235 240
Lys Leu Val Ala Leu Gly His Leu Ala Ala Gly Val Ala His Glu Ile
245 250 255
Arg Asn Pro Leu Ser Ser Ile Lys Gly Leu Ala Lys Tyr Phe Ala Glu
260 265 270
Arg Ala Pro Ala Gly Gly Glu Ala His Gln Leu Ala Gln Val Met Ala
275 280 285
Lys Glu Ala Asp Arg Leu Asn Arg Val Val Ser Glu Leu Leu Glu Leu
290 295 300
Val Lys Pro Thr His Leu Ala Leu Gln Ala Val Asp Leu Asn Thr Leu
305 310 315 320
Ile Asn His Ser Leu Gln Leu Val Ser Gln Asp Ala Asn Ser Arg Glu
325 330 335
Ile Gln Leu Arg Phe Thr Ala Asn Asp Thr Leu Pro Glu Ile Gln Ala
340 345 350
Asp Pro Asp Arg Leu Thr Gln Val Leu Leu Asn Leu Tyr Leu Asn Ala
355 360 365
Ile Gln Ala Ile Gly Gln His Gly Val Ile Ser Val Thr Ala Ser Glu
370 375 380
Ser Gly Ala Gly Val Lys Ile Ser Val Thr Asp Ser Gly Lys Gly Ile
385 390 395 400
Ala Ala Asp Gln Leu Asp Ala Ile Phe Thr Pro Tyr Phe Thr Thr Lys
405 410 415
Ala Glu Gly Thr Gly Leu Gly Leu Ala Val Val His Asn Ile Val Glu
420 425 430
Gln His Gly Gly Thr Ile Gln Val Ala Ser Gln Glu Gly Lys Gly Ser
435 440 445
Thr Phe Thr Leu Trp Leu Pro Val Asn Ile Thr Arg Lys Asp Pro Gln
450 455 460
Gly
465
<210> 8
<211> 1398
<212> DNA
<213> E.coli Membrane protein mutant zraS (zrafput-Q313R/T415A)
<400> 8
atgcgtttta tgcaacgttc taaagactcc ttagctaaat ggttaagcgc gatcctcccc 60
gtggtcattg ttgggctggt gggattgttt gcggtaactg tgattcgtga ttatgggcgg 120
gcaagcgagg cagaccgcca ggcattactg gaaaaaggta atgtgcttat ccgcgctctg 180
gagtcgggaa gccgcgtagg gatggggatg cgaatgcacc atgtacagca acaggcgctt 240
ctggaagaga tggcgggaca gccgggagtg ttgtggttcg cagtcaccga tgcgcagggc 300
atcattattc ttcatagcga ccccgataag gtcgggcgtg cgctctattc gccggatgaa 360
atgcagaaat taaagccaga ggaaaactcc cgctggcggc tgcttgggaa aacggaaact 420
acgcctgcac ttgaggtcta tcgtttgttc cagccaatgt cagcgccctg gcggcatgga 480
atgcacaata tgccgcgctg taacggcaaa gctgtgccac aagtagatgc acaacaggct 540
atttttatcg ccgttgatgc cagtgatctg gttgcaaccc agagtgggga aaagcgcaat 600
accctgatta tcctcttcgc cctggcgacg gtcttgctgg caagcgtatt gtcattcttc 660
tggtatcgcc gctatctgcg ctcgcgccag cttctacaag atgaaatgaa gcgcaaagag 720
aagctggtgg cgctggggca tcttgcggca ggcgttgccc acgaaatccg taacccactt 780
tcctcgatta aaggactggc gaaatacttt gccgagcgcg cgcctgcagg gggagaagcg 840
catcaactgg cgcaggtgat ggcgaaagag gccgaccgtt taaaccgcgt ggtaagcgag 900
ttgctggaac tggttaagcc aacgcatctg gctttgcggg cggtggatct caacacgctg 960
attaaccact cattacagct ggtaagtcag gatgcaaaca gccgggagat ccagttacgc 1020
tttaccgcca acgacacatt accggaaatt caggccgacc cggacaggct gactcaggtc 1080
ctgttgaatc tctatctcaa tgctattcag gcgattggtc agcatggcgt gattagcgtg 1140
acggccagcg aaagcggcgc gggcgtgaaa atcagcgtta ccgacagcgg taagggaatt 1200
gcggcagatc agcttgatgc catcttcact ccgtacttca ccgctaaagc cgaaggcacc 1260
ggattggggc tggcggtcgt gcataatatt gttgaacaac acggtggtac aattcaggtc 1320
gcaagccagg agggaaaagg ctcaacgttc accctctggc ttccggtcaa tattacgcgt 1380
aaggacccac aaggatga 1398
<210> 9
<211> 465
<212> PRT
<213> E.coli Membrane protein mutant zraS (zrafput-Q313R/T415A)
<400> 9
Met Arg Phe Met Gln Arg Ser Lys Asp Ser Leu Ala Lys Trp Leu Ser
1 5 10 15
Ala Ile Leu Pro Val Val Ile Val Gly Leu Val Gly Leu Phe Ala Val
20 25 30
Thr Val Ile Arg Asp Tyr Gly Arg Ala Ser Glu Ala Asp Arg Gln Ala
35 40 45
Leu Leu Glu Lys Gly Asn Val Leu Ile Arg Ala Leu Glu Ser Gly Ser
50 55 60
Arg Val Gly Met Gly Met Arg Met His His Val Gln Gln Gln Ala Leu
65 70 75 80
Leu Glu Glu Met Ala Gly Gln Pro Gly Val Leu Trp Phe Ala Val Thr
85 90 95
Asp Ala Gln Gly Ile Ile Ile Leu His Ser Asp Pro Asp Lys Val Gly
100 105 110
Arg Ala Leu Tyr Ser Pro Asp Glu Met Gln Lys Leu Lys Pro Glu Glu
115 120 125
Asn Ser Arg Trp Arg Leu Leu Gly Lys Thr Glu Thr Thr Pro Ala Leu
130 135 140
Glu Val Tyr Arg Leu Phe Gln Pro Met Ser Ala Pro Trp Arg His Gly
145 150 155 160
Met His Asn Met Pro Arg Cys Asn Gly Lys Ala Val Pro Gln Val Asp
165 170 175
Ala Gln Gln Ala Ile Phe Ile Ala Val Asp Ala Ser Asp Leu Val Ala
180 185 190
Thr Gln Ser Gly Glu Lys Arg Asn Thr Leu Ile Ile Leu Phe Ala Leu
195 200 205
Ala Thr Val Leu Leu Ala Ser Val Leu Ser Phe Phe Trp Tyr Arg Arg
210 215 220
Tyr Leu Arg Ser Arg Gln Leu Leu Gln Asp Glu Met Lys Arg Lys Glu
225 230 235 240
Lys Leu Val Ala Leu Gly His Leu Ala Ala Gly Val Ala His Glu Ile
245 250 255
Arg Asn Pro Leu Ser Ser Ile Lys Gly Leu Ala Lys Tyr Phe Ala Glu
260 265 270
Arg Ala Pro Ala Gly Gly Glu Ala His Gln Leu Ala Gln Val Met Ala
275 280 285
Lys Glu Ala Asp Arg Leu Asn Arg Val Val Ser Glu Leu Leu Glu Leu
290 295 300
Val Lys Pro Thr His Leu Ala Leu Arg Ala Val Asp Leu Asn Thr Leu
305 310 315 320
Ile Asn His Ser Leu Gln Leu Val Ser Gln Asp Ala Asn Ser Arg Glu
325 330 335
Ile Gln Leu Arg Phe Thr Ala Asn Asp Thr Leu Pro Glu Ile Gln Ala
340 345 350
Asp Pro Asp Arg Leu Thr Gln Val Leu Leu Asn Leu Tyr Leu Asn Ala
355 360 365
Ile Gln Ala Ile Gly Gln His Gly Val Ile Ser Val Thr Ala Ser Glu
370 375 380
Ser Gly Ala Gly Val Lys Ile Ser Val Thr Asp Ser Gly Lys Gly Ile
385 390 395 400
Ala Ala Asp Gln Leu Asp Ala Ile Phe Thr Pro Tyr Phe Thr Ala Lys
405 410 415
Ala Glu Gly Thr Gly Leu Gly Leu Ala Val Val His Asn Ile Val Glu
420 425 430
Gln His Gly Gly Thr Ile Gln Val Ala Ser Gln Glu Gly Lys Gly Ser
435 440 445
Thr Phe Thr Leu Trp Leu Pro Val Asn Ile Thr Arg Lys Asp Pro Gln
450 455 460
Gly
465

Claims (7)

1. The escherichia coli bi-component regulation and control system membrane protein ZraS mutant is characterized in that: the mutant is one of S154T/A214S, A214S, Q313R/T415A; the amino acid sequence of the wild ZraS is shown as SEQ ID NO. 2.
2. A gene encoding the escherichia coli two-component regulatory system membrane protein ZraS mutant of claim 1.
3. An expression vector comprising the gene of claim 2.
4. The expression vector of claim 3, wherein: in the expression vectorZraSThe promoter of the gene is an endogenous promoterZraSp。
5. A recombinant plasmid comprising the gene of claim 2.
6. A recombinant cell comprising the recombinant plasmid of claim 5.
7. Use of the mutant according to claim 1 or the gene according to claim 2 or the expression vector according to claim 3 or 4 or the recombinant plasmid according to claim 5 or the recombinant cell according to claim 6 in the detection of low lead levels.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN107418964A (en) * 2016-05-24 2017-12-01 中国科学院深圳先进技术研究院 A kind of more the bacterium continuously-directional evolutionary systems and method of bacteriophage auxiliary
CN109943581A (en) * 2017-12-20 2019-06-28 深圳先进技术研究院 The continuously-directional evolutionary system and directed evolution method of a kind of plasmid and bacteriophage auxiliary

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AU2011348204B2 (en) * 2010-12-22 2017-03-02 President And Fellows Of Harvard College Continuous directed evolution

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN107418964A (en) * 2016-05-24 2017-12-01 中国科学院深圳先进技术研究院 A kind of more the bacterium continuously-directional evolutionary systems and method of bacteriophage auxiliary
CN109943581A (en) * 2017-12-20 2019-06-28 深圳先进技术研究院 The continuously-directional evolutionary system and directed evolution method of a kind of plasmid and bacteriophage auxiliary

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