CN104946681A - Microbiological method for detecting heavy-metal cadmium in water body - Google Patents

Abstract

The invention relates to a microbiological method for detecting heavy-metal cadmium, particularly a construction method of an Escherichia coli engineering strain for detecting cadmium and a method for detecting cadmium in water by using the Escherichia coli engineering strain. The construction method of the engineering strain comprises the following steps: knocking out Escherichia coli cadmium-resistant zntA and zntR genes by using a Red recombination system, and substituting a pmerR-MerR(M)-pmerR-gfpmut2 report gene to the zntR coding gene position by using a gene knock-in technique. The lowest detection range of the engineering strain for cadmium conforms to Sewage Comprehensive Discharge Standard-GB8978-1996. The engineering strain overcomes the defects of high fluorescent background level, poor detection signal and the like in the plasmid-vector-based engineering strain. The engineering strain provided by the invention can be complementary with AAS (atomic absorption spectroscopy) and other physicochemical analysis processes to analyze the biological availability and total amount of the cadmium, thereby providing references for objective evaluation on biological toxicity of the cadmium.

Description

A kind of micro-biological process detecting heavy metal in water cadmium

Technical field

The present invention relates to a kind of micro-biological process detecting heavy metal in water cadmium, be specifically related to the construction process of the colon bacillus utilizing genetic engineering techniques, and set up it in the method detecting heavy metal cadmium in water body environment.

Background technology

Heavy metal cadmium (Cadmium, Cd) is classified as the human carcinogen of the first kind by IARC (IARC); American National Toxicology Program (NTP) also confirms as human carcinogen cadmium.Heavy metal in water cadmium pollution shows as nuisance disease in China partial area, as Guangdong cadmium rice event, Longjiang, Guangxi cadmium pollution event etc., causes the great attention of people.Cadmium is highly stable in the environment, not easily be biodegradable, content in water body and soil increases year by year, then at some plants or animal cylinder accumulation, as the rice, shell seafood, animal liver kidney etc. of In Cadmium Polluted Area, and by food chain at people's body accumulation, be one of poisonous substance accumulated the most in vivo known at present.Cadmium is very large to human toxicity, all can produce toxicity, mainly put aside at kidney, cause the changes of function of urinary system kidney, lung, liver, testis, brain, bone and blood system, will be difficult to get rid of once enter human body.Cadmium can disturb calcium in bone, if take in Trace Cadmium for a long time, bone is seriously softened, and bone is broken into pieces, and causes itai-itai disease, and it also can cause the dirty functional disorder of stomach, and disturbs the enzyme system of zinc in human body and organism, causes vascular hypertension to rise.Along with developing rapidly of industry, cadmium and compound thereof are widely used in the industries such as plating.Cadmium, to the pollution of environment, is mainly caused by industrial wastewater discharge that (China's " integrated wastewater discharge standard "---GB8978-1996 specifies that the maximal emission of cadmium in industrial wastewater is 0.1mgL ^-1).Therefore, the detection of heavy metal cadmium in environment water is seemed particularly necessary.

Current monitoring and detection heavy metal contaminants mainly contain two kinds of methods: (1) physico chemical analysis, as inductively coupled plasma atomic emission spectrum (ICP-AES), inductivity coupled plasma mass spectrometry (ICP-MS) etc., its advantage possesses high detection sensitivity and high specific, but also come with some shortcomings, as plant and instrument is expensive, complicated operation, sense cycle is long, the most important is, traditional physico-chemical process mainly measures environment total metals, can not detect the bioavailability of heavy metal; (2) based on the microbial method of biological organism, it is generally divided into based on plasmid with based on two kinds, genome conformity type.Plasmid integration type has that cost is low, high specificity, the advantage such as quick, easy to operate, but there is plasmid copy number heterogeneity after sub-culturing bacteria based on the engineering strain of plasmid vector, excessive number or loss and cause that detection signal is unstable, autofluorescent background value is high and the repeated defect such as bad of independent experiment.

Namely the ultimate principle of Measurement for Biotechnique is the biological molecular reaction mechanism to particular chemicals of Land use models.The element that needs three kinds are required for Measurement for Biotechnique: for specific or there is the inductor block of chemical substance of similar quality; The promotor controlled by inductor block; By the reporter gene that this promotor controls.When designing Measurement for Biotechnique, existing in a large number in the environment because bacterium has, grow the advantages such as quick, low cost and easy care and be subject to the general parent of researchist and look at.The specific pollutants utilizing bioassay method to come in testing environment in research in the past has caused great concern, so far have developed a series of engineering strain for specific organism and mineral compound and products thereof, as: heavy metal, first benzene and its derivative etc.

At present the Measurement for Biotechnique that cadmium ion measures also is researched and developed to some extent, but the concrete tolerance mechanism of colon bacillus to cadmium is not clear yet at present, colon bacillus has meticulous regulator control system and makes cadmium ion in cell remain on lower level, but we have found the responsible P-Type ATPase gene of MP zntA and Function protein gene zntR thereof that are pumped by cadmium ion of encoding in colon bacillus.Because the promotor of zntA gene is to cadmium and non-fully is special, it can also by zinc, the non-specific startup of plumbous plasma, so can not directly utilize the promotor of zntA gene to mediate specific reaction.According to another Kaisa M.Hakkila etc. at document Cd-specific mutants of mercury-sensing regulatory protein MerR, generated by directed evolution, Appl Environ Microbiol.2011Sep; 77 (17): 6215-24. reports, mercury ion Function protein MerR mutant in gram-negative bacteria after rite-directed mutagenesis, can with cadmium ion specific combination thus specific promoter starts downstream gene expression, it is confirmed to the specificity of cadmium ion.Therefore we are using the encoding gene of the MerR mutant gene MerR (M) after introduce rite-directed mutagenesis in colon bacillus engineering bacteria as cadmium ion specific reaction albumen.We attempt Function protein gene M erR (M), the promotor pmerR of allos and reporter gene gfpmut2 being merged the measuring element of composition containing double-promoter, are incorporated in colon bacillus genome.Fluorescence protein gene is conventional reporter gene, this invents us and is used for replacing promotor downstream gene as reporter gene by gfpmut2 fluorescin, so after the cadmium ion in environment is combined with regulatory gene, promotor starts the expression of downstream fluorescence protein gene, thus by fluorescent protein fluorescence intensity reaction concentration of cadmium ions.This mechanism provides a good detecting pattern, but cadmium is a kind of common hypertoxicity environmental pollution heavy metal, and most of bacterium has tolerance and resistance to cadmium, therefore its susceptibility is not high.And these researchs are based on plasmid as expression vector, but it is high to there is background values based on the biological testing element of plasmid vector, plasmid copy number heterogeneity after sub-culturing bacteria, excessive number or loss, the defect such as cause detection signal unstable.Its as detect Host Strains of cadmium can cause the instability of detection and lowest detectable limit higher, these are unfavorable for the detection of pollutent very much, new method must be adopted to improve the susceptibility of biological detection method detection cadmium ion, effectively to solve the bottleneck problem run in current environment monitoring.Riether K etc. at document Assessment of heavy metal bioavailability using Escherichia coli zntAp::lux and copAp::lux-based biosensors.Applied Microbiology and Biotechnology, 2002; The colon bacillus biosensor of 57 (5-6): the pZNT-lux based on plasmid built in 712-6., its construction strategy is that colon bacillus zntA promotor and luxCDABE gene are carried out gene fusion, is connected on pUCD615 plasmid vector.It by cadmium, lead, mercury, zinc induced luminescence, therefore, can lack specificity simultaneously, and is on wild colon bacillus basis, build the model animals detecting cadmium ion, there is above-mentioned defect.At patent " a kind of Microbial cell-based biosensors detecting cadmium bioavailability " CN publication number: in 102911906A, colon bacillus E.coli carries recombinant plasmid as host cell.Recombinant plasmid is the pUC18 plasmid of promoter sequence containing streptococcus aureus Staphylococcus aureu p1258 plasmid cadmium resistance operon cad, cadmium Resistance system regulatory gene cadC, luciferase gene luc and T7 terminator tandem sequence, the microbiological sensor based on plasmid equally, exist above-mentioned based on the number of drawbacks of plasmid as expression vector, and streptococcus aureus Staphylococcus aureu p1258 plasmid cadmium resistance operon cad, can be induced by lead, cadmium, zinc simultaneously, lack the characteristic of specific detection cadmium.

Colon bacillus engineering strain of the present invention can overcome that these are not enough, building a kind of E bacteria strain of special, responsive and rapid detection cadmium, laying the foundation for detecting Cadmium In The Water Body.Through retrieving the engineering strain of this research and establishment at home and abroad all without bibliographical information.

Summary of the invention

The object of the invention is the colon bacillus engineering strain be incorporated into by gene knockout and a kind of measuring element of gene knock-in technique construction on genome, thus set up a kind of micro-biological process of special, stable and rapid detection Heavy Metals in Waters cadmium, the present invention specifically build and fluoroscopic examination schematic diagram as shown in Figure 1.

One of summary of the invention: a kind of colon bacillus engineering strain detecting Heavy Metals in Waters cadmium is provided, colon bacillus of the present invention (Escherichia coli) WMC-009, on September 29th, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, it is referred to as CGMCC No.9747 (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is colon bacillus (Escherichia coli) WMC-009, deposit number is CGMCC No.9747.

1. the construction process of E.coli WMC-009CGMCC No.9747 of the present invention is as follows:

ZntA and zntR gene in 1.1 gene knockout wild-type colon bacillus genomes

Adopt Red recombination system, knock out two cadmium ion " efflux pump " gene: zntA in wild-type colon bacillus genome, sequence is as shown in SEQ ID NO.1 and zntR, and sequence, as shown in SEQ ID NO.2, builds colon bacillus dual-gene knock-out bacterial strain Δ zntA Δ zntR;

1.2 build cadmium metal measuring element pmerR-MerR (M)-pmerR-gfpmut2 fusion gene

By allos cadmium ion regulatory gene MerR (M), sequence is as shown in SEQ ID NO.5, promotor pmerR, sequence is as shown in SEQ ID NO.4 and fluorescent reporter gene gfpmut2, sequence is as shown in SEQ ID NO.3, by intersecting round pcr, build the measuring element of the heavy metal cadmium ion specific reaction containing double-promoter: pmerR-MerR (M)-pmerR-gfpmut2 fusion gene;

1.3 adopt gene knock-in technique construction to detect the colon bacillus engineering strain of heavy metal cadmium

Adopt gene knock-in technology will replace zntR genes encoding frame position in Δ zntA Δ zntR strain gene group containing enhanced green fluorescence protein reporter gene measuring element pmerR-MerR (M)-pmerR-gfpmut2.Concrete grammar is: by intersecting round pcr, formed containing fusion gene pmerR-MerR (M)-pmerR-gfpmut2 and zntR gene both sides homology arm, fusion fragment zntR _no-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntR _ci-Co, this DNA merges clip size and is about 2.3kb, and zntR gene N holds homology arm gene zntR-N, sequence is as shown in SEQ ID NO.26 and zntR gene C end homology arm gene zntR-C, sequence, as shown in SEQ ID NO.27, is then connected to pKOV condition plasmid replication, builds pKOV-zntR _no-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntR _ci-Cotargeting vector, and be transformed into by this targeting vector in Δ zntA Δ zntR bacterial strain, by the selection of temperature and sucrose, selects the bacterial strain that twice homologous recombination successfully occurs, and further by bacterium colony PCR and order-checking evaluation and screening object bacterial strain.

2. E bacteria strain of the present invention can the principle of detection by quantitative Heavy Metals in Waters cadmium as follows:

E bacteria strain detection by quantitative Cadmium In The Water Body ion of the present invention adopts pmerR promotor directly to start gfpmut2 gene expression pattern.In this builds, when not adding exogenous cadmium ion, MerR (M) protein binding that in E.coli WMC-009 genome, MerR (M) genes encoding produces, in pmerR promoter sequence district, stops GFPmut2 to express; When exogenous cadmium ion enters after in E mycetocyte, Cd ²⁺with MerR (M) protein binding, MerR (M) albumen is come off from promotor, activate the expression of pmerR-MerR (M)-pmerR-gfpmut2 fusion gene in E.coli WMC-009 genome, thus produce GFPmut2 albumen, and the amount of GFPmut2 albumen produced or its fluorescence intensity be dose-dependence with concentration of cadmium ions, so pass through the object that mensuration fluorescence intensity can reach detection by quantitative concentration of cadmium ions.

Summary of the invention two: the E bacteria strain set up constructed by a kind of the present invention detects the micro-biological process of Cadmium In The Water Body.

The application implementation scheme that E bacteria strain of the present invention detects Heavy Metals in Waters cadmium is as follows:

1. the preparation of environment water body example mensuration:

Get 10-50ml environment water body example to be measured, the centrifugal 5min of 12,000g, gets supernatant, and regulate the pH value of Supernatant samples to be 6-8 with 0.5M dilute hydrochloric acid or sodium hydroxide solution, then use the aseptic syringe needle frit of 0.22 μm, post-filtration samples is used for follow-up mensuration.

The preparation of 2.LB substratum:

LB media components concentration is 1% (W/V) Tryptone, 0.5% (W/V) Yeast Extract, 1% (W/V) NaCl, 80% (V/V) deionized water, be cooled to room temperature after autoclave sterilization, add Homogeneous phase mixing after the aseptic MOPS damping fluid of the 400mmol/L of 10% (V/V) volume pH 7.2.

3. the formulation of typical curve:

The cadmium single element standard substance pure water of aseptic MilliQ level is diluted to suitable concentration gradient.

3.1 get 5-10 μ l from the glycerol stock E.coli WMC-009 bacterial strain preserved accesses fresh 5ml LB substratum, 37 DEG C, 250rpm incubated overnight;

The 3.2 E.coli WMC-009 getting 5-10 μ l step 3.1 bacterium that spends the night joins in LB substratum, and 37 DEG C, 250rpm, isothermal vibration is cultured to OD ₆₀₀value is at 0.02-0.6, OD ₆₀₀be optimum value when being worth 0.3, get 90-900 μ l bacterium liquid divide be filled to 96 hole clear bottom black microwell plates or 15 × 150mm specification detector tube in, in each detect aperture or detector tube, add 10-100 μ l cadmium single element standard substance simultaneously, separately add equal-volume aseptic MilliQ level pure water as negative control, often kind of water body example or standard substance do 3 parallel holes or 3 parallel pipes, 37 DEG C, 250rpm oscillation incubation 2-6h, best incubation time is 5h;

3.3 by the bacterium liquid of step 3.2 after induction, 4,000rpm, horizontal centrifugal 10min, abandons supernatant, and the thalline of results is resuspended in 1ml DB damping fluid, make bacteria suspension, wherein DB damping fluid contains 500mMNaCl, 20mM Tris-HCl, 20 μ g/ml paraxin regulate pH to 8.0;

3.4 by the bacteria suspension of step 3.3 dilution 0-20 doubly, make the OD of the full cell suspending liquid of E.coli WMC-009 ₆₀₀≤ 0.3, mixing, measures its diluent OD respectively ₆₀₀value and fluorescent value, excitation wavelength is 481nm, and emission wavelength is 510nm;

3.5 data processing:

Relative fluorescence (Relative fluorescence unit, RFU): RFU _m=F _m/ OD _600M, F _mfor hatching the fluorescent value of E.coli WMC-009 bacteria suspension, OD with cadmium single element standard substance _600Mfor hatching the absorbancy of E.coli WMC-009 bacteria suspension with cadmium single element standard substance; RFU _w=F _w/ OD _600W, F _wfor hatching the background fluorescence activity of E.coli WMC-009 bacteria suspension, OD with MilliQ level pure water _600Wfor hatching the absorbancy of E.coli WMC-009 bacteria suspension with MilliQ level pure water;

Absolute fluorescence value (Absolutee fluorescence unit, AFU): AFU=RFU _m-RFU _w, RFU _mfor hatching the relative fluorescence of E.coli WMC-009 bacteria suspension, RFU with cadmium single element standard substance _wfor hatching the relative background fluorescence activity of E.coli WMC-009 bacteria suspension with MilliQ level pure water.

3.6 with the absolute fluorescence value of cadmium single element standard substance induction for ordinate zou, cadmium single element standard concentration value is X-coordinate, drawing standard curve, and the acquisition standard equation that carries out curve fitting.

4. the mensuration of cadmium concentration in environment water:

4.1 get 5-10 μ l from the glycerol stock E.coli WMC-009 bacterial strain preserved accesses fresh LB, 37 DEG C, 250rpm incubated overnight;

The 4.2 E.coli WMC-009 getting 5-10 μ l step 4.1 bacterium that spends the night joins in LB substratum, and 37 DEG C, 250rpm, isothermal vibration is cultured to OD ₆₀₀value is at 0.02-0.6, OD ₆₀₀be optimum value when being worth 0.3, get 90-900 μ l bacterium liquid divide be filled to 96 hole clear bottom black microwell plates or 15 × 150mm specification detector tube in, in each detect aperture or detector tube, add the 10-100 μ l water body example of step 1 simultaneously, separately add equal-volume aseptic MilliQ level pure water as negative control, often kind of a water body example does 3 parallel holes or 3 parallel pipes, 37 DEG C, 250rpm oscillation incubation 2-6h, best incubation time is 5h;

4.3 by the bacterium liquid of step 4.2 after induction, 4,000rpm, horizontal centrifugal 10min, abandons supernatant, and the thalline of results is resuspended in 1ml DB damping fluid, make bacteria suspension, wherein DB damping fluid contains 500mMNaCl, 20mM Tris-HCl, 20 μ g/ml paraxin regulate pH to 8.0;

4.4 by the bacteria suspension of step 4.3 dilution 0-20 doubly, make the OD of the full cell suspending liquid of E.coli WMC-009 ₆₀₀≤ 0.3, mixing, measures its OD respectively ₆₀₀value and diluent fluorescent value, excitation wavelength is 481nm, and emission wavelength is 510nm;

4.6 data processing: the typical curve and the equation thereof that then make the acquisition of green fluorescence intensity typical curve according to the cadmium ion standardized solution induction with batch concentration known, the circumstances not known water body example fluorescence intensity level recorded is substituted in the equation of step 3.6, calculates the concentration of cadmium in circumstances not known water body example.

Engineering strain E.coli WMC-009 of the present invention, has following innovative point and technical superiority:

1. autofluorescent background value is low: the background values existed relative to the engineering strain of plasmid vector is high, and measuring element of the present invention is incorporated in genome, and merges double-promoter, obviously reduces background values, has superiority;

2. detection signal is stablized: relative to plasmid copy number heterogeneity after the sub-culturing bacteria that the engineering strain of plasmid vector exists, excessive number or loss, and causing the defect of detection signal instability, the present invention has detection signal and stablizes, and independent experiment is reproducible, the characteristic of credible result;

3. fluorescent signal is strong: E bacteria strain of the present invention adopts enhanced green fluorescence protein GFPmut2 fluoresce as detection signal, and it is stronger than the fluorescence of wild-type GFP more than 100 times, improves E bacteria strain to Cd ²⁺the sensitivity detected.And using gfpmut2 as reporter gene, possess without the need to the participation of substrate and cofactor, easy to detect, naked eyes just observable, highly sensitive, fluorescent stabilization, toxicological harmless, versatility, be easy to carrier construction and the features such as viable cell timing position observation can be carried out;

4. do not need specialized instrument and equipment, professional operator and Yi in basic unit and rig-site utilization and cheap, be convenient to promote: relative to traditional detection method as atomic absorption spectrophotometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) etc., the present invention is easy and simple to handle, sample pretreatment is simple, stdn testing process is easy and simple to handle, detection time is short, can produce in batches, cheap and be easy to promotion and implementation;

Any external source antibiotics resistance gene is not imported, not entail dangers to human health or to environment in 5.E.coli WMC-009 bacterial strain;

6. realizing biological effectiveness analyzing and testing: heavy metal cadmium is converted to biological effectiveness analyzing and testing from macroanalysis by the present invention, more can reflect physiological-toxicity, is the inexorable trend of Analysis of Heavy Metal detection method research.Bioavailability of heavy metals refers to and can be absorbed by organism and cause untoward reaction or affect the heavy metal of that a part of form of organism existence, is the important indicator weighing heavy metal virulence size.Heavy metal exists with variform in the environment, as residual form heavy metal almost can not be utilized by organism, what the physico-chemical analysis methods such as traditional AAS and ICP-MS and various chromatograph-mas spectrometers detected is total metals in sample, and microorganism cells is as a Living system, there is metabolism and the resistance mechanism of heavy metal, can the heavy metal of selective absorbing different shape, can the biological effectiveness of heavy metal and toxic effect in effective evaluation environment.In addition, microorganism is absolutely necessary to preservation of fertility and water body self-purification ability, and the biological effectiveness therefore studying heavy metal is also very important to environmental microorganism.

Accompanying drawing explanation

Fig. 1. structure of the present invention and fluoroscopic examination schematic diagram

E bacteria strain detection by quantitative Cadmium In The Water Body ion of the present invention adopts pmerR promotor directly to start gfpmut2 gene expression pattern.In this builds, when not adding exogenous cadmium ion, MerR (M) protein binding that in E.coli WMC-009 genome, MerR (M) genes encoding produces, in pmerR promoter sequence district, stops GFPmut2 to express; When exogenous cadmium ion enters after in E mycetocyte, Cd ²⁺with MerR (M) protein binding, MerR (M) albumen is come off from promotor, activate the expression of pmerR-MerR (M)-pmerR-gfpmut2 fusion gene in E.coli WMC-009 genome, thus produce GFPmut2 albumen, and the amount of GFPmut2 albumen produced or its fluorescence intensity be dose-dependence with concentration of cadmium ions, so pass through the object that mensuration fluorescence intensity can reach detection by quantitative concentration of cadmium ions.

Fig. 2. single-gene knocks out schema

The homology arm of H1/H2:zntA/zntR gene both sides; P1/P2:cat gene both sides homology arm; GeneA:zntA/zntR upstream region of gene gene; GeneB:zntA/zntR gene; GeneC:zntA/zntR downstream of gene gene; Gene cat: chloramphenicol resistance gene.

Fig. 3. gene knock-in schema

A:zntR upstream region of gene 500bp gene zntR-N; B:zntR gene; C:zntR downstream of gene 500bp gene zntR-C; D:pmerR-MerR (M)-pmerR-gfpmut2 measuring element.

Fig. 4 .cat gene PCR electrophorogram

M:DL 5,000DNA Marker; 1/2:cat gene (1200bp)

Fig. 5 .zntA single-gene knocks out PCR electrophorogram

M:DL 5,000DNA Marker; 1: wild-type zntA gene (2499bp); 2: wild-type (zntA::cat) (1249bp) containing cat gene; The mutant bacteria (498bp) of 3: successful knockout zntA.

Fig. 6 .zntR single-gene knocks out PCR electrophorogram

M:DL 5,000DNA Marker; 1: wild-type zntR gene (726bp); 2: wild-type (zntR::cat) (1249bp) containing cat gene; The mutant bacteria (408bp) of 3: successful knockout zntR

Fig. 7. Δ zntA-Δ zntR bacterial strain PCR identifies electrophorogram

M:DL 5,000DNA Marker; The Δ zntA (498bp) of 1: Δ zntA-Δ zntR bacterial strain; The Δ zntR (408bp) of 2: Δ zntA-Δ zntR bacterial strain.

Fig. 8 .pmerR-MerR (M), pmerR-gfpmut2, pmerR-MerR (M)-pmerR-gfpmut2 gene identification electrophorogram

M：DL 5,000DNA Marker；1：pmerR-MerR(M)(506bp)；2：pmerR-gfpmut2(788bp)；3：pmerR-MerR(M)-pmerR-gfpmut2(1294bp)。

Fig. 9 .pmerR-MerR (M)-pmerR-gfpmut2-pMD19-T is transformed into wild-type E dientification of bacteria electrophorogram

M：DL 5,000DNA Marker；1：zntA(2499bp)；2：zntR(426bp)；3：pmerR-MerR(M)-pmerR-gfpmut2(1294bp)。

Figure 10 .pmerR-MerR (M)-pmerR-gfpmut2-pMD19-T/ Δ zntA-Δ zntR identifies electrophorogram

M：DL 5,000DNA Marker；1：pmerR-MerR(M)-pmerR-gfpmut2(1294bp)；2：zntA(2499bp)；3：zntR(426bp)。

Embodiment

The preparation of embodiment 1. mutant bacteria

One, primer information and synthesis

Gene knockout primer: the primer sequence that gene knockout is provided according to http://ecogene.org/, Primer5.0 and DNAMAN software, design homologous recombination primer, in table 1,5 ' holds the homology arm into cat gene both sides, in table 1 without underscore part, 3 ' hold for for the chloramphenicol resistance gene that increases in table 1 underscore part.Upstream homology arm primer zntA-Nf, sequence is as shown in SEQ ID NO.6 and zntR-Nf, and sequence is as shown in SEQ ID NO.8; Downstream homology arm primer zntA-Cr, sequence is as shown in SEQ ID NO.7 and zntR-Cr, and sequence is as shown in SEQ ID NO.9.

Gene knockout primers designed: the outside design of crossing over gene to be knocked out for a pair utilizing Harvard Molecular Technology Group & Lipper Center for Computational Genetics website http://arep.med.harvard.edu/labgc/adnan/projects/EcoliKO-primer s/EcoliKOprimers.htm to provide knocks out primers designed, in table 2.Qualification zntA gene knockout design of primers: upstream primer zntA-H1P1, sequence, as shown in SEQ ID NO.10, is positioned at zntA upstream region of gene 150bp, downstream primer zntA-H2P2, and sequence, as shown in SEQ ID NO.11, is positioned at zntA downstream of gene 150bp; Qualification zntR gene knockout design of primers: upstream primer zntR-H1P1, sequence, as shown in SEQ ID NO.12, is positioned at zntR upstream region of gene 150bp, downstream primer zntR-H2P2, sequence, as shown in SEQ ID NO.13, is positioned at zntR downstream of gene 150bp, and single-gene knocks out schema and sees accompanying drawing 2.

Primer is synthesized by invitrogene company.Dissolved by primer with aseptic deionized water, being made into concentration is 10 μm of ol/L.

Table 1. gene knockout primer

Table 2. gene knockout primers designed

Two, the preparation of single mutation bacterium Δ zntA

2.1 plasmid pKD46 are converted into colon bacillus

With inoculating needle, from the Bacterial Plate of colon bacillus, picking 1 single colony inoculation is in 3ml LB liquid nutrient medium, and 37 DEG C, 250rpm shaking culture is spent the night and by cold CaCl ₂pKD46 Plastid transformation, for competent cell, enters in E bacterium competence cell by legal system, and then even coated plate is in LB-Amp ⁺flat board, is inverted dull and stereotyped, 30 DEG C of overnight incubation after bacterium liquid fully absorbs.Flat board grows bacterium colony and illustrate that pKD46 has proceeded in colon bacillus.

The cat gene PCR amplification in 2.2 band FRT sites

Get and spend the night bacterium liquid 1ml in the EP pipe of 1.5ml, 8,000rpm containing pKD3 plasmid, centrifugal 3min, abandons supernatant, thalline 1ml MilliQ H ₂o is resuspended wash twice after, the centrifugal 3min of boiling water boiling 10min, 12,000rpm, as template, mixing.Utilize primer zntA-Nf and zntA-Cr, prepare PCR reaction system by table 3.

Table 3

PCR reaction conditions is as follows: 94 DEG C of 1min; 88 DEG C of 4min; 94 DEG C of 10sec, 66 DEG C of 3min, 35 circulations; 72 DEG C 10min4 DEG C preservation.Get 5 μ l PCR primer with 1.0% Agarose gel electrophoresis qualification.PCR primer PCR primer purification kit carries out purifying, finally adds appropriate ddH ₂it is for subsequent use that O dissolves wash-out.As Fig. 4 display, increase and obtained the cat gene of about 1kb.

The 2.3 colon bacillus electricity containing pKD46 turn the preparation of competent cell

2.3.1 seed liquor preparation: LB-Amp from step 2.1 ⁺in the bacterium colony that flat board grows, get a single colony inoculation in 3ml LB-Amp ⁺liquid nutrient medium, 30 DEG C, 250rpm shaking culture is spent the night.

2.3.2 the bacterium 50 μ l that spends the night of step 2.3.1 is got in 10ml TB-Amp ⁺in test tube, about enlarged culturing 1.5h, add 20%L-Arab 5 μ l, final concentration is 0.02%, induction 1.5h;

2.3.3 take out step 2.3.2 bacterium liquid and put ice bath 15min, bacterium liquid is transferred in the 15ml centrifuge tube of precooling, 4 DEG C, the centrifugal 15min of 4,000rpm, abandons supernatant, add 10% glycerine 10ml of precooling, 4 DEG C, 4, the centrifugal 15min of 000rpm, abandon supernatant, repeating step 2.3.3 twice, then, add 40 μ l10% glycerine resuspended, the colon bacillus electricity namely prepared containing pKD46 turns competent cell.

2.4 electricity transform cat gene

Get 40 μ l competent cells prepared by 5 μ l cat gene PCR product after purification and step 2.3.3 prepared by step 2.2 to mix, be transferred to and shock by electricity in cup, operate on ice.Open electroporation, be adjusted to Manual, optimum configurations is: voltage: 2.5kV, electric capacity: 25 μ F, resistance: 200 Ω, electric shock cup: 2mm.Electric shock cup is put into electroporated instrument, clicks pusle key, after hearing buzzer, in electric shock cup, add rapidly 1ml TB resuspended, be transferred to EP pipe, 37 DEG C, 150rpm shaking culture 1h carries out resistance recovery.Then by bacterium liquid in 5,000rpm, 25 DEG C of centrifugal 5min, get the resuspended precipitation of 200 μ l supernatant, are applied to TB-CM ⁺flat board, 37 DEG C of overnight incubation, there is bacterium colony in flat board, illustrate and transform successfully.

The PCR qualification of 2.5cat gene

A single colony inoculation is got in 3ml TB-Cm from step 2.4 transformation plate ⁺in test tube, 37 DEG C, until bacterium liquid, 250rpm shaking culture 4h, occurs that getting 1ml bacterium liquid after obvious muddiness does PCR qualification.By bacterium liquid in the EP pipe of 1.5ml, 8,000rpm, centrifugal 3min, abandons supernatant, thalline 1ml MilliQ H ₂o is resuspended wash twice after, the centrifugal 3min of boiling water boiling 10min, 12,000rpm, as template, mixing.Utilize primer zntA-H1P1 and zntA-H2P2, prepare PCR reaction system by table 4.

Table 4

PCR reaction conditions is as follows: PCR reaction conditions is as follows: 95 DEG C of 5min; 95 DEG C of 40sec, 66 DEG C of 2min, 72 DEG C of 2min, 25 circulations; 4 DEG C of preservations.Get PCR primer 5 μ l with 1.0% Agarose gel electrophoresis qualification.As shown in Figure 5, there is bright band at about 1,500bp in result, proves that zntA gene is replaced by cat gene.

The conversion of 2.6pCP20

PCP20 Plastid transformation is entered by cold CaCl ₂the standby step 2.5 of legal system identifies the correct competent cell containing cat gene, and the even coated plate of bacterium liquid is in LB-Amp ⁺-Cm ⁺on plate, be inverted dull and stereotyped after bacterium liquid fully absorbs, 30 DEG C of overnight incubation.

The elimination of 2.7cat resistant gene

A single colony inoculation is got in 3ml LB liquid nutrient medium from step 2.6 Bacterial Transformation flat board, 42 DEG C, 250rpm about shaking culture 4h with inoculating needle; Getting collarium four ride is inoculated on LB flat board, 37 DEG C of overnight incubation.With inoculating needle respectively from picking 3 single bacterium colonies the Bacterial Plate of 37 DEG C of overnight incubation, now cat gene is removed, and pCP20 plasmid is lost, and dibbling is in LB-Cm respectively ⁺with on LB blank plate, be placed in 37 DEG C and cultivate about 6h; At LB-Cm ⁺flat board does not grow and the bacterium of LB grow on plates, and cat gene is successfully removed.Further by PCR qualification, prove that cat gene is removed completely.After picking colony directly adds and boils 10min in 100 μ l water, centrifugal as template, prepare PCR system by primers designed by table 4 and carry out pcr amplification.As shown in Figure 5, gene stripe size conforms to theoretical value result, illustrates and is successfully completed knocking out of znt4 gene, obtains zntA single mutation bacterium Δ zntA.

The purifying of 2.8 Δ znt4 single mutation bacterium

Picking list bacterium colony on Δ zntA tetra-ride flat board from step 2.7, dips a little bacterium four ride with transfering loop dull and stereotyped in new LB, 37 DEG C of overnight incubation.Remaining bacterium colony is chosen in 100 μ l sterilized waters, centrifugal after boiling 10min, goes supernatant to carry out PCR qualification as template primers designed by step in 2.5, obtains the single mutation bacterium of purifying after qualification is correct.

Three, prepared by two mutant bacteria Δ zntA Δ zntR

Δ zntA single mutation bacterium basis knocks out zntR gene, and prepare dual-gene mutant bacteria, method is with the preparation of single mutation bacterium, and concrete steps are as follows:

3.1PCR amplification is containing the chloramphenicol resistance gene in zntR upstream and downstream 50bp homology arm straps FRT site

The colon bacillus of getting containing plasmid pKD3 spends the night bacterium liquid 1ml in the EP pipe of 1.5ml, 12,000rpm, and centrifugal 1min, abandons supernatant, the aseptic MilliQ H of thalline 1ml ₂o is resuspended wash twice after, the centrifugal 3min of boiling water boiling 10min, 12,000rpm, prepares PCR reaction tubes according to table 5, mixing.

Table 5

PCR reaction conditions is as follows: 95 DEG C of 3min; 95 DEG C of 40sec, 48 DEG C of 40sec, 72 DEG C of 2min, 35 circulations; 72 DEG C of 10min, 4 DEG C of 30min.Product is identified, as accompanying drawing 4 with the Agarose gel electrophoresis of 1.0%.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H ₂it is for subsequent use that O dissolves wash-out.

The chloramphenicol resistance gene of amplification proceeds in the bacterial strain of Red recombinase by 3.2

The colon bacillus of pKD46 is had to be inoculated in 3ml LB-Amp conversion ⁺in liquid nutrient medium, 30 DEG C of overnight incubation, next day 1: 100 is seeded to containing 50ml LB-Amp ⁺in liquid nutrient medium, 30 DEG C are cultured to OD ₆₀₀when=0.1, add 20%L-pectinose at 1: 500, is induced to OD ₆₀₀prepare electricity during ≈ 0.5 and turn competence, 40 μ l/ often manage.The fragment of amplification in step 3.1 is got 5 μ l and adds competence bacterial strain, transform with Bio-Rad electric shock instrument electricity, electric shock condition: 200 Ω, 25 μ F, shock voltage 2.5kV, electric shock time 5ms, adds rapidly 1ml LB after electric shock, 250rpm, cultivates 1.5h, coats LB-Cm afterwards for 37 DEG C ⁺flat board, next day observes.

3.3 select chlorampenicol resistant transformant

The bacterium colony in picking step 3.2, paraxin flat board grown, is inoculated in 5ml LB-Cm ⁺in liquid nutrient medium, 250rpm, 37 DEG C are cultured to macroscopic muddiness.Get 1ml in the EP pipe of 1.5ml, 12,000rpm, centrifugal 1min, abandons supernatant, the aseptic MilliQ H of thalline 1ml ₂o is resuspended wash twice after, the centrifugal 3min of boiling water boiling 10min, 12,000rpm, prepares PCR reaction system according to table 6, mixing.

Table 6

PCR reaction conditions is as follows: 95 DEG C of 3min; 95 DEG C of 40sec, 52 DEG C of 40sec, 72 DEG C of 2min, 25 circulations; 72 DEG C of 10min, 4 DEG C of 30min.Product is identified, as accompanying drawing 6 with the Agarose gel electrophoresis of 1.0%.

3.4FLP recombinase eliminates chloramphenicol resistance gene

Proceeded to by pCP20 in the bacterium colony of the step 3.3 chlorampenicol resistant positive, conversion temp to 42 DEG C after 30 DEG C of cultivation 10h, 250rpm shaking culture is spent the night.The transfering loop libation at an ancient wedding ceremony is got the bacterium liquid that spends the night and is coated non-resistant LB flat board, 37 DEG C of overnight incubation.The bacterium colony that nonreactive of spending the night flat board grows, is taken up in order of priority lattice to line new chlorampenicol resistant and non-resistant LB is dull and stereotyped, 37 DEG C of overnight incubation.Choose the single bacterium colony increasing bacterium of chlorampenicol resistant without growth and non-resistant LB plated growth, prepare PCR system by primers designed by table 6 and carry out pcr amplification.As shown in Figure 6, then bacterial strain is again with 2 kinds of primers designed qualifications, and in the band of pcr amplification, two is about 500bp for PCR qualification result, and illustrate and complete the two mutant bacteria preparation of Δ zntA-Δ zntR, result is as accompanying drawing 7.

Embodiment 2. merges the structure of reporter gene pmerR-MerR (M)-pmerR-gfpmut2

One, primer information and synthesis

Goal gene pmerR and MerR (M) sequence announced according to GenBank and known gfpmut2 gene order, use Primer5.0 software design primer, in table 7, part primer is held at the 5 ' end or 3 ' of gene and is introduced restriction enzyme site, the primer Pmer-MerR-m-F of design amplification pmerR-MerR (M), sequence is as shown in SEQ ID NO.20 and Pmer-MerR-m-R, sequence is as shown in SEQ ID NO.21, the primer Pmer-GFPmut2-F sequence of amplification pmerR-gfpmut2 gene as shown in SEQ ID NO.22 and GFPmut2-R sequence as shown in SEQ ID NO.23, and final fusion fragment is connected to the primers designed M13F of plasmid vector pMD19-T, sequence is as shown in SEQ ID NO.24 and M13R, sequence is as shown in SEQ ID NO.25.Specifying information is in table 7, and primer is synthesized by invitrogene company.Use aseptic ddH ₂primer dissolves by O, is made into the storage liquid that concentration is 10 μm of ol/L ,-20 DEG C of preservations.

Table 7. gene fusion primer details table

Two, pmerR-MerR (M)-pmerR-gfpmut2 gene fusion and be connected with pMD 19-T carrier

2.1 amplification pmerR-MerR (M) genes

Get and spend the night bacterium liquid 1ml in the EP pipe of 1.5ml, 8,000rpm containing plasmid merR-MerR (M)-pmerR-gfpmut2-pMD19-T (Clonetech), centrifugal 3min, abandons supernatant, thalline 1ml MilliQ H ₂o is resuspended wash twice after, the centrifugal 3min of boiling water boiling 10min, 12,000rpm, gets 24 μ l supernatants as template.According to the form below is made into system, does PCR qualification.

Table 8

PCR reaction conditions is as follows: 95 DEG C, 5min; 95 DEG C, 40sec, 50 DEG C, 40sec, 72 DEG C, 1.5min, 35 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.Result as shown in Figure 8.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H ₂it is for subsequent use that O dissolves wash-out.

2.2PCR amplification merR-gfpmut2 gene

Get and spend the night bacterium liquid 1ml in the EP pipe of 1.5ml, 8,000rpm containing plasmid merR-MerR (M)-pmerR-gfpmut2-pMD19-T, centrifugal 3min, abandons supernatant, thalline 1ml MilliQ H ₂o is resuspended wash twice after, the centrifugal 3min of boiling water boiling 10min, 12,000rpm, gets 24 μ l supernatants as template.According to the form below is made into system, does PCR qualification.

Table 9

PCR reaction conditions is as follows: 95 DEG C, 5min; 95 DEG C, 40sec, 50 DEG C, 40sec, 72 DEG C, 1.5min, 35 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.Result as shown in Figure 8.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H ₂it is for subsequent use that O dissolves wash-out.

The fusion of 2.3pmerR-MerR (M)-pmerR-gfpmut2 gene

By the PCR primer of step 2.1 and step 2.2, according to the form below 10 prepares system, and then according to the form below 11 adds system on table 10 basis again

Table 10

PCR reaction conditions is as follows: 95 DEG C, 3min; 95 DEG C, 40sec, 62 DEG C, 40sec ,-1 DEG C, 72 DEG C, 3min, 8 circulations; 72 DEG C, 2min, 4 DEG C of preservations.

Table 11

PCR reaction conditions is as follows: 95 DEG C, 3min; 95 DEG C, 40sec, 52 DEG C, 40sec, 72 DEG C, 2min, 35 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.Result as shown in Figure 8.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H ₂it is for subsequent use that O dissolves wash-out.

2.4PCR product adds A reaction and purifying

The PCR primer according to the form below of step 2.3 is made into reaction system:

Table 12

PCR reaction conditions is as follows: 72 DEG C, 1h, 4 DEG C of preservations.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H ₂it is for subsequent use that O dissolves wash-out.

2.5 object fragments are connected with pMD19-T simple carrier

The connection of carrier T and goal gene is carried out by the pMD19-T simpLe Vector test kit specification sheets of TaKaRa company.By the product of step 2.4 and pMD19-T simple Vector ligation system as follows:

Table 13

16 DEG C, connection is spent the night.

2.6 transform

By cold CaCl ₂step 2.5 is connected product conversion and enters in colon bacillus DH5 α competent cell by method.

2.7 order-checking

Select one through M13F/R primer, PCR identifies that correct mono-clonal bacterial strain send Shanghai Jierui Biology Engineering Co., Ltd to check order, the canonical reference sequence alignment that sequencing result and NCBI provide.

2.8 transform

2.8.1 the bacterial strain checking order correct through step 2.7 extracts plasmid, and the extraction of plasmid adopts the little upgrading grain of TaKaRa MiniBEST Plasmid Purification Kit Ver.3.0 test kit;

2.8.2 by cold CaCl ₂step 2.8.1 plasmid is transformed in Δ zntA-Δ zntR competent cell by method respectively, be labeled as in pmerR-MerR (M)-pmerR-gfpmut2-pMD19-T/ Δ zntA-Δ zntR and wild-type E bacterium competence cell, and transformed the exactness of bacterial classification by PCR qualification, result is as shown in accompanying drawing 9 and 10.

Embodiment 3. gene knock-in

One, primer information and synthesis

Use Primer5.0 software design primer, in table 14, the inner primer of design pmerR-MerR (M)-pmerR-gfpmut2 gene N-terminal and C-terminal, P ₂: zntR-cd-Ni, sequence is as shown in SEQ ID N0.15 and P ₅: zntR-cd-gfpmut2-Ci, sequence as shown in SEQ ID N0.16 and Outside primer, P ₁: zntR-No, sequence is as shown in SEQ ID N0.14 and P ₆: zntR-Co, sequence, as shown in SEQ ID N0.17, makes P ₂: zntR-cd-Ni and P ₃: pmerR-MerR (M)-pmerR-gfpmut2-F, sequence as shown in SEQ ID N0.18, P ₄: pmerR-MerR (M)-pmerR-gfpmut2-R, sequence as shown in SEQ ID N0.19, with P _s: there is complementary sequence between zmR-cd-gfpmut2-Ci, gene knockout two Outside primer are constant, P ₃with P ₄for the gene primer of pair for amplification pmerR-MerR (M)-pmerR-gfpmut2, P ₇: pkov-SalI, sequence is as shown in SEQ ID N0.28 and P ₈: pkov-NotI, sequence, as shown in SEQ ID N0.29, is the gene primer of pair for amplification pKOV.Specifying information is in table 14, and primer is synthesized by invitrogene company.Use aseptic ddH ₂primer dissolves by O, is made into the storage liquid that concentration is 10 μm of ol/L ,-20 DEG C of preservations.

Table 14. gene knock-in primer details table

Note: underscore is restriction enzyme site

Two, pKOV-zntR _no-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntR _ci-Cofusion and the structure of reporting bacterial strain

The amplification of 2.1 both sides homology arms

Get and spend the night bacterium liquid 1ml in the EP pipe of 1.5ml, 8,000rpm containing pmerR-MerR (M)-pmerR-gfpmut2-pMD19-T plasmid, centrifugal 3min, abandons supernatant, thalline 1ml MilliQ H ₂o is resuspended wash twice after, the centrifugal 3min of boiling water boiling 10min, 12,000rpm, gets 24 μ l supernatants as template.According to the form below is made into system, does PCR qualification.

Table 15

PCR reaction conditions is as follows: 94 DEG C, 1min; 88 DEG C, 4min; 94 DEG C, 10sec, 66 DEG C, 3min, 25 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H ₂it is for subsequent use that O dissolves wash-out.

The amplification of 2.2pmerR-MerR (M)-pmerR-gfipmut2 gene

Get and spend the night bacterium liquid 1ml in the EP pipe of 1.5ml, 12,000rpm containing pmerR-MerR (M)-pmerR-gfpmut2 plasmid, centrifugal 5min, abandons supernatant, thalline 1ml MilliQ H ₂o is resuspended to be washed twice, the centrifugal 5min of boiling water boiling 10min, 12,000rpm, gets 24 μ l supernatants as template.According to the form below is made into system, does PCR qualification.

Table 16

PCR reaction conditions is as follows: 94 DEG C, 1min; 88 DEG C, 4min; 94 DEG C, 10sec, 66 DEG C, 3min, 25 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQH ₂it is for subsequent use that O dissolves wash-out.

2.3 side homology arms and pmerR-MerR (M)-pmerR-gfpmut2 gene fusion

PCR reaction system is as follows:

Table 17

PCR reaction conditions is as follows: 94 DEG C, 1min; 88 DEG C, 4min; 94 DEG C, 10sec, 66 DEG C, 3min, 25 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H ₂it is for subsequent use that O dissolves wash-out.

2.4 both sides homology arms and pmerR-MerR (M)-pmerR-gfpmut2 gene fusion build zntRNo-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntRCi-Co

PCR reaction system is as follows:

Table 18

PCR reaction conditions is as follows: 94 DEG C, 1min; 88 DEG C, 4min; 94 DEG C, 10sec, 66 DEG C, 3min, 25 circulations; 72 DEG C, 10min, 4 DEG C of preservations.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H ₂it is for subsequent use that O dissolves wash-out.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.Result shows, both sides homology arm and pmerR-MerR (M)-pmerR-gfpmut2 gene successful fusion.

The preparation of 2.5pKOV linear carrier fragment

Get the bacterium that spends the night containing pKOV plasmid of 15ml TB substratum growth, adopt TaKaRa MiniBEST Plasmid Purification Kit Ver.5.0 test kit extracting plasmid.

The single endonuclease digestion of pKOV plasmid

Table 19

37 DEG C, enzyme cuts 2h.

Endonuclease reaction after product heats 5min in 65 DEG C, the activity of inactivator, then is template amplification pKOV carrier segments with digestion products, and PCR reaction system is as follows:

Table 20

PCR reaction conditions is as follows: PCR reaction conditions is as follows: 94 DEG C, 5min; 94 DEG C, 40sec; 63 DEG C, 40sec, 72 DEG C, 6min, 30 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.PCR primer purification kit carries out purifying, finally adds appropriate aseptic MilliQ H2O dissolving wash-out for subsequent use.

2.6 targeting vector pKOV-zntR _no-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntR _ci-Costructure and preservation

2.6.1 survey the linearizing pKOV carrier concn that glue reclaims fragment zntRNo-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntRCi-Co and purifying, ligation system is as follows:

Table 21

16 DEG C, connection is spent the night, and wherein homology arm merges the mol ratio of fragment and pKOV carrier is 10: 1.

2.6.2 plasmid pKOV-zntRNo-Ni-pmerR-MerR (the M)-pmerR-gfpmut2-zntRCi-Co extracted in the correct E.coli DH5 α of colony identification is transformed into and twoly strikes in bacterium Δ zntA-Δ zntR competence, is coated with LB CM ⁺flat board, 30 DEG C of constant temperature culture, grow bacterium colony, illustrate and transform successfully.

2.6.3PCR qualification positive colony bacterial strain

With inoculating needle picking above-mentioned steps 2.6.2LB CM ⁺single bacterium colony of grow on plates a little respectively streak inoculation in a new aseptic LB CM ⁺on flat board, be inverted in 30 DEG C of constant incubators and cultivate 10h.Then, with inoculating needle this LB CM of picking respectively ⁺single bacterium colony of grow on plates is a little, is mixed in the EP pipe containing 50 μ l sterilized waters, after the centrifugal 3min of boiling water boiling 10min, 12,000rpm, gets supernatant 5 μ l and adds in 200 μ l PCR reaction tubess as PCR qualification template.Getting following system adds in this pipe, mixing.

Table 22

PCR reaction conditions is as follows: 94 DEG C, 1min; 88 DEG C, 4min; 94 DEG C of 10sec, 66 DEG C of 5min, 25 circulations; 72 DEG C of 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification.Electrophoresis showed PCR fragment length is out selected at the mono-clonal of about 2.3kb, carries out plasmid extraction below.

2.7 the first step homologous recombination, pKOV-zntR _no-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntR _ci-Cobe incorporated into the genome of colon bacillus,

Be taken at the 30 DEG C of pKOV-zntRNo-Ni-pmerR-MerR of hatching (M)-pmerR-gfpmut2-zntRCi-Co/ Δ zntA-Δ zntR spend the night bacterium liquid 10 μ l be inoculated in 42 DEG C of preheatings containing 5ml LB CM ⁺in the test tube of substratum, in 42 DEG C, shaking culture 2h in the constant incubator of 250rpm, then get respectively 42 DEG C hatch 2h after bacterium liquid 20 μ l sectional streak be inoculated in the LB CM of 3-4 42 DEG C of preheatings ⁺flat board on.After bacterium liquid absorbs completely, be inverted in overnight incubation in 42 DEG C of constant incubators.

With the LB CM that inoculating needle picking is hatched through 42 DEG C ⁺single bacterium colony of grow on plates a little respectively streak inoculation in the LB CM of new 42 DEG C of preheatings ⁺on flat board, be inverted in 42 DEG C of constant incubators and cultivate 10h.Then, a little with single bacterium colony of this LB CM grow on plates of inoculating needle difference picking, be mixed in the EP pipe containing 50 μ l sterilized waters, after the centrifugal 3min of boiling water boiling 10min, 12,000rpm, get supernatant 5 μ l and add in 200 μ lPCR reaction tubess as PCR qualification template.PCR reaction system is with table 22.

PCR reaction conditions is as follows: 94 DEG C, 1min; 88 DEG C, 4min; 94 DEG C, 10sec, 66 DEG C, 5min, 25 circulations; 72 DEG C, 10min, 4 DEG C of preservations.Get 5 μ l products with 1.0% Agarose gel electrophoresis qualification, PCR primer is two bands, and its brightness is suitable, about 1,000bp and the length of about 2,300bp, prove to there occurs the first step homologous recombination, segment and plasmid have been incorporated on genome.

2.8 second step homologous recombination, pKOV plasmid and gene element from and lose

Choose in 5mL that the successful bacterial strain of strain step 2.7 the first step homologous recombination is inoculated in 30 DEG C of preheatings in the bacterium liquid 50 μ l that 42 DEG C of overnight incubation the are cultivated test tube containing the LB nutrient solution of 5% sucrose, in 30 DEG C, shaking culture 2h in the constant incubator of 250rpm, then get respectively 30 DEG C hatch 2h after bacterium liquid carry out ten times of serial dilutions, choose 10 ^-2, 10 ^-3, 10 ^-4the each 0.1mL of bacterium liquid of three dilution gradients coats on the LB flat board containing 5% sucrose of 30 DEG C of preheatings respectively.After bacterium liquid absorbs completely, be inverted culture dish, 30 DEG C are spent the night, and with single bacterium colony of inoculating needle picking 30 DEG C, 5% sucrose LB grow on plates, streak inoculation is in a LB CM respectively ⁺on dull and stereotyped and 5% sucrose LB flat board, be inverted overnight incubation in 30 DEG C of constant incubators, carry out the negative screening of paraxin flat board, then picking is at 5% sucrose LB grow on plates, corresponding to CM ⁺single bacterium colony that/LB flat board does not grow is bacterium colony PCR and identifies, PCR reaction system with table 22, get 5 μ l products with 1.0% Agarose gel electrophoresis qualification, the bacterium colony tentative confirmation that PCR primer is about 2.3kb is the positive bacterium colony knocked in of producer.Divide pure bacterium colony, PCR identifies further and confirms gene knock-in success.And be placed in-80 DEG C of preservations, for subsequent use.

Embodiment 4. engineering strain detects the formulation of Heavy Metals in Waters cadmium concentration flow process

The preparation that 4.1 environment water body examples measure:

Get 10-50ml environment water body example to be measured, the centrifugal 5min of 12,000g, gets supernatant, and regulate the pH value of Supernatant samples to be 6-8 with 0.5M dilute hydrochloric acid or sodium hydroxide solution, then use the aseptic syringe needle frit of 0.22 μm, post-filtration samples is used for follow-up mensuration.

The preparation of 4.2LB substratum:

LB media components concentration is 1% (W/V) Tryptone, 0.5% (W/V) Yeast Extract, 1% (W/V) NaCl, 80% (V/V) deionized water, be cooled to room temperature after autoclave sterilization, add aseptic MOPS damping fluid (400mmol/L) Homogeneous phase mixing afterwards of 10% (V/V) volume pH 7.2.

The formulation of 4.3 typical curves:

The cadmium single element standard substance pure water of aseptic MilliQ level is diluted to suitable concentration gradient.

4.3.1 from the glycerol stock E.coli WMC-009 bacterial strain preserved, get 5-10 μ l and access fresh 5ml LB substratum, 37 DEG C, 250rpm incubated overnight;

4.3.2 the E.coli WMC-009 the getting 5-10 μ l step 4.3.1 bacterium that spends the night joins in LB substratum, and 37 DEG C, 250rpm, isothermal vibration is cultured to OD ₆₀₀value is at 0.02-0.6, OD ₆₀₀be optimum value when being worth 0.3, get 90-900 μ l bacterium liquid divide be filled to 96 hole clear bottom black microwell plates or 15 × 150mm specification detector tube in, in each detect aperture or detector tube, add 10-100 μ l cadmium single element standard substance simultaneously, separately add equal-volume aseptic MilliQ level pure water as negative control, often kind of water body example or standard substance do 3 parallel holes or 3 parallel pipes, 37 DEG C, 250rpm oscillation incubation 2-6h, best incubation time is 5h;

4.3.3 by the bacterium liquid of step 4.3.2 after induction, 4,000rpm, horizontal centrifugal 10min, abandons supernatant, and the thalline of results is resuspended in 1ml DB damping fluid, make bacteria suspension, wherein DB damping fluid contains 500mMNaCl, 20mM Tris-HCl, 20 μ g/ml paraxin regulate pH to 8.0;

4.3.4 by the bacteria suspension of step 4.3.3 dilution 0-20 doubly, the OD of the full cell suspending liquid of E.coli WMC-009 is made ₆₀₀≤ 0.3, mixing, measures its diluent OD respectively ₆₀₀value and fluorescent value, excitation wavelength is 481nm, and emission wavelength is 510nm;

4.3.5 data processing:

Relative fluorescence (Relative fluorescence unit, RFU): RFU _m=F _m/ OD _600M, F _mfor hatching the fluorescent value of E.coli WMC-009 bacteria suspension, OD with cadmium single element standard substance _600Mfor hatching the absorbancy of E.coli WMC-009 bacteria suspension with cadmium single element standard substance; RFU _w=F _w/ OD _600W, F _wfor hatching the background fluorescence activity of E.coli WMC-009 bacteria suspension, OD with MilliQ level pure water _600Wfor hatching the absorbancy of E.coli WMC-009 bacteria suspension with MilliQ level pure water;

Absolute fluorescence value (Absolutee fluorescence unit, AFU): AFU=RFU _m-RFU _w, RFU _mfor hatching the relative fluorescence of E.coli WMC-009 bacteria suspension, RFU with cadmium single element standard substance _wfor hatching the relative background fluorescence activity of E.coli WMC-009 bacteria suspension with MilliQ level pure water.

4.3.6 with the absolute fluorescence value of cadmium single element standard substance induction for ordinate zou, cadmium single element standard concentration value is X-coordinate, drawing standard curve, and the acquisition standard equation that carries out curve fitting.

The mensuration of cadmium concentration in 4.4 environment waters:

4.4.1 from the glycerol stock E.coli WMC-009 bacterial strain preserved, get 5-10 μ l access fresh LB, 37 DEG C, 250rpm incubated overnight;

4.4.2 the E.coli WMC-009 the getting 5-10 μ l step 4.4.1 bacterium that spends the night joins in LB substratum, and 37 DEG C, 250rpm, isothermal vibration is cultured to OD ₆₀₀value is at 0.02-0.6, OD ₆₀₀be optimum value when being worth 0.3, get 90-900 μ l bacterium liquid divide be filled to 96 hole clear bottom black microwell plates or 15 × 150mm specification detector tube in, in each detect aperture or detector tube, add the 10-100 μ l water body example of step 1 simultaneously, separately add equal-volume aseptic MilliQ level pure water as negative control, often kind of a water body example does 3 parallel holes or 3 parallel pipes, 37 DEG C, 250rpm oscillation incubation 2-6h, best incubation time is 5h;

4.4.3 by the bacterium liquid of step 4.4.2 after induction, 4,000rpm, horizontal centrifugal 10min, abandons supernatant, and the thalline of results is resuspended in 1ml DB damping fluid, make bacteria suspension, wherein DB damping fluid contains 500mM NaCl, 20mM Tris-HCl, 20 μ g/ml paraxin regulate pH to 8.0;

4.4.4 by the bacteria suspension of step 4.4.3 dilution 0-20 doubly, the OD of the full cell suspending liquid of E.coli WMC-009 is made ₆₀₀≤ 0.3, mixing, measures its OD respectively ₆₀₀value and diluent fluorescent value, excitation wavelength is 481nm, and emission wavelength is 510nm;

4.4.5 data processing: the typical curve and the equation thereof that then make the acquisition of green fluorescence intensity typical curve according to the cadmium ion standardized solution induction with batch concentration known, the circumstances not known water body example fluorescence intensity level recorded is substituted in the equation of step 4.3.6, calculates the concentration of cadmium in circumstances not known water body example.

Embodiment 5. the present invention detects Methodological evaluation and the application example of Heavy Metals in Waters cadmium concentration

One, engineering strain E.coli WMC-009 is to the recovery experiment of cadmium ion in strong acid and strong base simulated samples

Draw the E.coli WMC-009 calculated bacterium seed liquor of spending the night with liquid-transfering gun to join in the fresh LB liquid nutrient medium of 90ml, make starter bacteria liquid OD ₆₀₀value is about 0.02, and 37 DEG C, 250rpm is induced to OD=0.02-0.6, best when OD is 0.3, and packing, 0.9mL/ manages, and adds the sample of 100 μ l, six kinds of different pH value range at 0.42-13.84 respectively, and then adds equal-volume Cd respectively ²⁺solution to each pipe bacterium liquid makes its final concentration all consistent, separately adds the aseptic MilliQ H of equal-volume ₂o is as blank.Often kind of a pH value sample makees 3 parallel pipes.By above bacterium liquid in 37 DEG C, after 250rpm shaking culture 2-6h, best incubation time is 5h, 4,000rpm, horizontal centrifugal 10min, abandon supernatant, the thalline of results is resuspended in 1ml Desalting buffer, then gets the resuspended bacterium liquid 20 of 50 μ l and is doubly diluted in 0.95mL Desalting buffer, mixing, measures its diluent fluorescent value and OD respectively ₆₀₀value.Experimental result is the mean value ± SD value of three independent experiments, n=3.

Experimental result: E.coli WMC-009 to the rate of recovery of cadmium ion in strong acid and strong base simulated samples all between 80%-120%.The result measured by the method is close with the result adopting atomic absorption spectrometry (AAS) to measure.Illustrate E.coli WMC-009 possess objective detection Cadmium In The Water Body content and not by the impact of strong acid and strong base in water body.

Two, engineering strain E.coli WMC-009 is to the recovery experiment of cadmium ion in the paper mill effluent sample of disease in Lucheng District of Wenzhou City

A paper mill effluent sample is gathered, through the sterile filter of 0.22 μm from disease in Lucheng District of Wenzhou City.Draw the E.coli WMC-009 calculated bacterium seed liquor of spending the night with liquid-transfering gun to join in the fresh LB liquid nutrient medium of 90ml, make starter bacteria liquid OD ₆₀₀value is about 0.02, and 37 DEG C, 250rpm is induced to OD=0.02-0.6, best OD=0.3, and packing (0.9mL/ pipe), adds the Cd of equal-volume different concns ²⁺solution, to each pipe bacterium liquid, makes its final concentration 10 ^-9-10 ^-3between M/I, and then add the lake water extremely often pipe bacterium liquid after 100 μ l filtrations respectively, often kind of concentration makees 3 parallel pipes.By above bacterium liquid in 37 DEG C, 250rpm shaking culture 2-6h, best incubation time is 5h, 4,000rpm, horizontal centrifugal 10min, abandon supernatant, the thalline of results is resuspended in 1ml Desalting buffer, then gets the resuspended bacterium liquid 20 of 50 μ l and is doubly diluted in 0.95ml Desalting buffer, mixing, measures its diluent fluorescent value and OD respectively ₆₀₀value.Experimental result is the mean value ± SD value of three independent experiments, n=3.

Experimental result: E.coli WMC-009 to the rate of recovery of paper mill effluent cadmium ion all between 90%-120%.The result measured by the method is close with the result adopting atomic absorption spectrometry (AAS) to measure.Illustrate that E.coli WMC-009 possesses the application prospect of the content of the actual Cadmium In The Water Body of objective detection.

Claims (5)

1. the application of a strain colon bacillus WMC-009, is characterized in that described colon bacillus (Escherichia coli) WMC-009CGMCC No.9747 is applied to the detection of heavy metal in water cadmium.

2. the application of a strain colon bacillus WMC-009 according to claim 1, it is characterized in that described colon bacillus WMC-009 obtains by the following method: adopt Red recombination system to knock out zntA and the zntR gene of imparting wild-type colon bacillus cadmium resistance, obtain after then adopting gene knock-in technology that pmerR-MerR (M)-pmerR-gfpmut2 reporter gene is replaced zntR encoding gene position.

3. the application of a strain colon bacillus WMC-009 according to claim 1, is characterized in that the construction process of colon bacillus WMC-009 bacterial strain is as follows:

3.1 adopt Red recombination system to knock out zntA and the zntR gene of imparting wild-type colon bacillus cadmium resistance;

3.2 design zntR gene N-terminal and the inner primer of C-terminal and the primers of Outside primer and pmerR-MerR (M)-pmerR-gfpmut2 fusion gene, adopt and intersect round pcr and build containing the fusion gene fragment of fusion gene pmerR-MerR (M)-pmerR-gfpmut2 with the two ends homology arm of zntR gene;

3.3 will containing the fusion gene fragment zntR of fusion gene pmerR-MerR (M)-pmerR-gfpmut2 with the two ends homology arm of zntR gene _no-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntR _ci-Cobe connected on pKOV carrier, build and merge report carrier pKOV-zntR _no-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntR _ci-Co;

Described recombinant vectors pKOV-zntRNo-Ni-pmerR-MerR (M)-pmerR-gfpmut2-zntRCi-Co is transferred in the intestinal bacteria △ zntA △ zntR bacterial strain of step 3.1 by 3.4, by twice homologous recombination, pmerR-MerR (M)-pmerR-gfpmut2 reporter gene is replaced the zntR encoding gene position in step 3.1 intestinal bacteria △ zntA △ zntR strain gene group.

4. the application of a strain colon bacillus WMC-009 described in claim 1, is characterized in that the method detecting Heavy Metals in Waters cadmium is as follows:

The preparation that 4.1 environment water body examples measure:

Get 10-50ml environment water body example to be measured, the centrifugal 5min of 12,000g, gets supernatant, and regulate the pH value of Supernatant samples to be 6-8 with 0.5M dilute hydrochloric acid or sodium hydroxide solution, then use the aseptic syringe needle frit of 0.22 μm, post-filtration samples is used for follow-up mensuration.

The preparation of 4.2LB substratum:

LB media components concentration is 1% (W/V) Tryptone, 0.5% (W/V) Yeast Extract, 1% (W/V) NaCl, 80% (V/V) deionized water, be cooled to room temperature after autoclave sterilization, add Homogeneous phase mixing after the aseptic MOPS damping fluid of the 400mmol/L of 10% (V/V) volume pH 7.2.

The formulation of 4.3 typical curves:

The cadmium single element standard substance pure water of aseptic MilliQ level is diluted to suitable concentration gradient.

4.3.1 from the glycerol stock E.coli WMC-009 bacterial strain preserved, get 5-10 μ l and access fresh 5ml LB substratum, 37C, 250rpm incubated overnight;

4.3.2 the E.coli WMC-009 the getting 5-10 μ l step 4.3.1 bacterium that spends the night joins in LB substratum, and 37 DEG C, 250rpm, isothermal vibration is cultured to OD ₆₀₀value is at 0.02-0.6, OD ₆₀₀be optimum value when being worth 0.3, get 90-900 μ l bacterium liquid divide be filled to 96 hole clear bottom black microwell plates or 15 × 150mm specification detector tube in, in each detect aperture or detector tube, add 10-100 μ l cadmium single element standard substance simultaneously, separately add equal-volume aseptic MilliQ level pure water as negative control, often kind of water body example or standard substance do 3 parallel holes or 3 parallel pipes, 37 DEG C, 250rpm oscillation incubation 2-6h, best incubation time is 5h;

4.3.3 by the bacterium liquid of step 4.3.2 after induction, 4,000rpm, horizontal centrifugal 10min, abandons supernatant, and the thalline of results is resuspended in 1ml DB damping fluid, make bacteria suspension, wherein DB damping fluid contains 500mM NaCl, 20mM Tris-HCl, 20 μ g/ml paraxin regulate pH to 8.0;

4.3.4 by the bacteria suspension of step 4.3.3 dilution 0-20 doubly, the OD of the full cell suspending liquid of E.coli WMC-009 is made ₆₀₀≤ 0.3, mixing, measures its diluent OD respectively ₆₀₀value and fluorescent value, excitation wavelength is 481nm, and emission wavelength is 510n _x;

4.3.5 data processing:

Relative fluorescence (Relative fluorescence unit, RFU): RFU _m=F _m/ OD _600M, F _mfor hatching the fluorescent value of E.coli WMC-009 bacteria suspension, OD with cadmium single element standard substance _600Mfor hatching the absorbancy of E.coli WMC-009 bacteria suspension with cadmium single element standard substance; RFU _w=F _w/ OD _600W, F _wfor hatching the background fluorescence activity of E.coli WMC-009 bacteria suspension, OD with MilliQ level pure water _600Wfor hatching the absorbancy of E.coli WMC-009 bacteria suspension with MilliQ level pure water;

Absolute fluorescence value (Absolutee fluorescence unit, AFU): AFU=RFU _m-RFU _w, RFU _mfor hatching the relative fluorescence of E..coli WMC-009 bacteria suspension, RFU with cadmium single element standard substance _wfor hatching the relative background fluorescence activity of E.coli WMC-009 bacteria suspension with MilliQ level pure water.

4.3.6 with the absolute fluorescence value of cadmium single element standard substance induction for ordinate zou, cadmium single element standard concentration value is X-coordinate, drawing standard curve, and the acquisition standard equation that carries out curve fitting.

The mensuration of cadmium concentration in 4.4 environment waters:

4.4.1 from the glycerol stock E.coli WMC-009 bacterial strain preserved, get 5-10 μ l access fresh LB, 37 DEG C, 250rpm incubated overnight;

4.4.2 the E.coli WMC-009 the getting 5-10 μ l step 4.4.1 bacterium that spends the night joins in LB substratum, and 37 DEG C, 250rpm, isothermal vibration is cultured to OD ₆₀₀value is at 0.02-0.6, OD ₆₀₀be optimum value when being worth 0.3, get 90-900 μ l bacterium liquid divide be filled to 96 hole clear bottom black microwell plates or 15 × 150mm specification detector tube in, in each detect aperture or detector tube, add the 10-100 μ l water body example of step 1 simultaneously, separately add equal-volume aseptic MilliQ level pure water as negative control, often kind of a water body example does 3 parallel holes or 3 parallel pipes, 37 DEG C, 250rpm oscillation incubation 2-6h, best incubation time is 5h;

4.4.3 by the bacterium liquid of step 4.4.2 after induction, 4,000rpm, horizontal centrifugal 10min, abandons supernatant, and the thalline of results is resuspended in 1ml DB damping fluid, make bacteria suspension, wherein DB damping fluid contains 500mM NaCl, 20mM Tris-HCl, 20 μ g/ml paraxin regulate pH to 8.0;

4.4.4 by the bacteria suspension of step 4.4.3 dilution 0-20 doubly, the OD of the full cell suspending liquid of E.coli WMC-009 is made ₆₀₀≤ 0.3, mixing, measures its OD respectively ₆₀₀value and diluent fluorescent value, excitation wavelength is 481nm, and emission wavelength is 510nm;

4.4.5 data processing: the typical curve and the equation thereof that then make the acquisition of green fluorescence intensity typical curve according to the cadmium ion standardized solution induction with batch concentration known, the circumstances not known water body example fluorescence intensity level recorded is substituted in the equation of step 4.3.6, calculates the concentration of cadmium in circumstances not known water body example.

5. the application of a strain colon bacillus WMC-009 described in claim 1, it is characterized in that: in the LB substratum containing 40mM MOPS buffer composition, the pH value of E.coli WMC-009 bacterial strain to water body example has stronger immunity from interference, and can be applied to the detection of cadmium ion in environment water body example.Relative to atomic absorption spectrometry, E.coli WMC-009 bacterial strain is Cd in the water body example of 0.42 ~ 13.84 to pH value range ²⁺recovery of standard addition be 80% ~ 120%, to Cd in the water sample of paper mill, disease in Lucheng District of Wenzhou City ²⁺recovery of standard addition be 90%-120%.