CN112725373B

CN112725373B - Construction method for amplifying cadmium ion whole-cell biosensor circuit

Info

Publication number: CN112725373B
Application number: CN202011407100.8A
Authority: CN
Inventors: 贾晓强; 刘腾; 马玉冰
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2023-02-07
Anticipated expiration: 2040-12-04
Also published as: CN112725373A

Abstract

The invention relates to a construction method for amplifying a cadmium ion whole-cell biosensor circuit; the research firstly constructs the cadmium whole cell biosensor p2T7RNAPMut-68 by adding a T7RNAPMut (the 40 th amino acid is mutated into a stop codon) amplification module. The sensitivity and specificity of the biosensor are improved by amplifying the circuit through the T7RNAPMut (the 40 th amino acid is mutated into a stop codon) amplification module, so that the defect of the traditional cadmium ion biosensor in detection is overcome. The plasmids pCDFDuet-2 and pGN68 are used as vectors, and the cadmium ion whole-cell biosensor is obtained by utilizing cadmium specific binding protein CadR, reporter elements mCherry, cadO operon and a T7RNApMut amplification module. The invention provides a construction method of a whole-cell biosensor sensitive to cadmium ions, and the construction method has wide adaptability.

Description

Construction method for amplifying cadmium ion whole-cell biosensor circuit

Technical Field

The invention relates to a construction method for amplifying a cadmium ion whole-cell biosensor circuit; a method for carrying out circuit amplification on a cadmium ion whole-cell biosensor by utilizing T7RNAPMut (the 40 th amino acid is mutated into a stop codon) is used for carrying out quantitative analysis on cadmium ions in a water sample.

Background

By 2020, the global population has increased to 75 billion, corresponding to the rapid advance of industrialization. The rapid industrialization can continuously improve the life quality of people and cause serious heavy metal pollution. A series of health problems are caused by the toxic, non-degradable nature of heavy metals, and the tendency to accumulate in the human body through the food chain. In 2019, 7, 23 and 23 days, cadmium and cadmium compounds are listed in the book of poisonous and harmful water pollutants (first batch). Cadmium pollution is more and more paid attention by people, so that the establishment of a high-efficiency and rapid detection means for heavy metal cadmium ions becomes more important.

Heavy metals are highly toxic, difficult to degrade and prone to accumulate in the human body through the food chain, thus easily causing a series of health problems. Cadmium intake can lead to bone softening and osteoporosis. In addition, cadmium, as an endocrine disruptor, can have an effect on the human reproductive system. Cadmium, as a mutagen, despite its weak mutagenicity, is teratogenic and genotoxic and interferes with major DNA repair pathways. The target organs of cadmium carcinogenesis are pancreas, pituitary, liver, adrenal gland, prostate and hematopoietic lineage.

The biological recognition element of a whole cell biosensor is a microbial cell that responds to input from the environment and produces a measurable output. Currently, with the development of sequencing technology, we obtain a lot of sequence information about the environmental metagenome, from which we can select appropriate elements and modules. Gene synthesis and genome editing techniques have greatly facilitated the construction of custom biological systems for the detection of specific molecules or the synthesis of biochemicals. In recent years, synthetic biology has enabled the design of a variety of whole cell biosensors based on a modular concept. For a heavy metal ion whole cell biosensor, the detection module is composed of elements such as transcription factors (activators/repressors) capable of specifically binding to metal ions or sensory kinases of a two-component system, and regulatory elements (such as promoters, operators, and ribosome binding sites). The reporter module is composed of a reporter gene regulated by the expression of a promoter/operator, and usually the reporter gene is composed of a gene encoding an enzyme (such as beta-galactosidase and luciferase) capable of catalyzing the formation of a measurable product or a protein (such as green fluorescent protein EGFP, red fluorescent protein mCherry and other fluorescent proteins) capable of being measured. The whole-cell biosensor essentially makes a specific response to harmful heavy metals based on the metal response transcription or the two-component signal transduction of cells, so that the whole-cell biosensor has the advantages of sensitivity, simplicity, convenience, rapidness, strong selectivity, strong anti-interference capability and the like, and has wide application potential in environmental monitoring. At present, poor specificity and sensitivity are bottleneck problems which limit the application of heavy metal microbial sensors.

Disclosure of Invention

In order to solve the problems of the prior art; the research can optimize the gene circuit of the whole-cell biosensor by coupling with an amplifier, amplify a fluorescence response signal and further obtain the biosensor with high sensitivity and high specificity.

The research firstly constructs the cadmium whole cell biosensor p2T7RNAPMut-68 by adding a T7RNAPMut (the 40 th amino acid is mutated into a stop codon) amplification module. The sensitivity and specificity of the biosensor are improved by amplifying the circuit through the T7RNAPMut (the 40 th amino acid is mutated into a stop codon) amplification module, so that the defect of the traditional cadmium ion biosensor in detection is overcome.

The cadmium ion whole-cell biosensor is obtained by taking plasmids pCDFDuet-2 and pGN68 as vectors and utilizing cadmium specific binding protein CadR, reporter elements mCherry, cadO operon and a T7RNApMut amplification module.

The whole cell is a recombinant bacterium which is transformed with cadmium ions, comprises a detection element and a report element and has life metabolic activity. The sensor P2T7 RNAcumut-68 comprises a cadmium-specific protein-regulated promoter P _cad SEQ ID No.1, cadmium specific binding protein CadR gene SEQ ID No.2 and T7RNAP 40 th amino acid from Glutamic Acid (GAG) mutation to Terminator (TAG) T7RNAmut SEQ ID No.4 as core element composed of detection plasmid;

a T7 promoter P _T7 And the reporter plasmid consists of SEQ ID No.5, cadO operon SEQ ID No.6 and red fluorescent protein mCherry gene SEQ ID No.3 as core elements.

The host bacterium used in the invention is pseudomonas putida P.putida KT2440, and the performance of the sensor for detecting cadmium ions is obtained by detecting the growth curve, time-fluorescence response, concentration-fluorescence response and specificity of the chassis cells.

The technical scheme of the invention is as follows:

a method for constructing a cadmium ion whole-cell biosensor and optimizing and amplifying a circuit thereof; the detection performance of the cadmium ion whole-cell biosensor is optimized by adding a T7RNAPMut (40 th amino acid is mutated into a stop codon) amplification module.

The sensor construction method; the method comprises the following steps:

(1) Construction of a cadmium ion whole-cell biosensor sheet p2T7 RNAmut-68: is a cadmium-specific protein regulated promoter P _cad SEQ ID No.1, detection plasmid consisting of T7RNAmutSEQ ID No.4 in which amino acid at position 40 of cadmium specific binding protein CadR gene SEQ ID No.2 and T7RNAP is mutated from Glutamic Acid (GAG) to Terminator (TAG) and a T7 promoter P _T7 And the reporter plasmid consists of SEQ ID No.5, cadO operon SEQ ID No.6 and red fluorescent protein mCherry gene SEQ ID No.3 as core elements.

(2) Pseudomonas putida KT2440 is used as a host, and recombinant plasmids are transferred into the host to obtain cadmium ion whole-cell biosensor chassis cells and cadmium ion whole-cell biosensor chassis cells amplified by a circuit.

The performance of the cadmium ion whole-cell biosensor is detected, and the detection limit, sensitivity and specificity of the sensor to cadmium ions are researched through a chassis cell growth curve, time-fluorescence response, cadmium ion concentration-fluorescence response and specificity experiments.

The performance detection method for the cadmium ion whole-cell biosensor comprises the following steps:

(1) And (3) culturing test tube seed liquid: individual colonies of P.putida KT2440 containing the sensor plasmid were shake-cultured overnight at 220rpm at 30 ℃ in LB medium containing antibiotics. Then diluted to OD with LB medium ₆₀₀ 1, inoculating the strain into an LB liquid culture medium without resistance according to the proportion of 1 percent; absorbance OD of bacterial liquid ₆₀₀ Adding cadmium ion solution when the concentration is 0.6-0.8.

(2) Growth curve of cadmium ion biosensor: to the tubes were added Cd to final concentrations of 0, 0.1, 1, 10, 100, 200, 300, 400, 500, 600. Mu.M ²⁺ Shaking culture was carried out at 30 ℃ and 220 rpm. Without addition of Cd ²⁺ Sampling every 1 hour before; addition of Cd ²⁺ Thereafter, samples were taken every 2 hours, and the absorbance OD was measured ₆₀₀ 。

(3) Time-fluorescence response of cadmium ion biosensor:to the tube was added Cd to final concentrations of 0, 0.01, 0.02, 0.04, 0.05, 0.1, 1, 10. Mu.M ²⁺ Culturing at 30 deg.C and 220rpm by shaking, collecting 200 μ L of the strain solution every 2 hr in 96-well plate, and measuring relative fluorescence value (RFU) and absorbance OD of the sample with microplate reader ₆₀₀ 。

(4) Concentration-fluorescence response of cadmium ion biosensor: to the tube was added Cd to final concentrations of 0, 0.01, 0.02, 0.04, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100. Mu.M ²⁺ Culturing at 30 deg.C and 220rpm, shaking for 8 hr, collecting 200 μ L bacterial solution, placing in 96-well plate, and measuring relative fluorescence value (RFU) and absorbance OD of sample with microplate reader ₆₀₀ 。

(5) Specificity experiment of cadmium ion biosensor: to the tube, cd was added to the final concentrations of 0.01, 0.1, 1, 10. Mu.M ²⁺ 、Pd ²⁺ 、Zn ²⁺ 、Cu ²⁺ 、As ³⁺ Culturing at 30 deg.C and 220rpm, shaking for 8 hr, collecting 200 μ L bacterial solution, placing in 96-well plate, and measuring relative fluorescence value (RFU) and absorbance OD of sample with microplate reader ₆₀₀ 。

(6) Fluorescence detection of cadmium ion whole cell biosensor: putting 200 mul of bacterial liquid into a clean enzyme label plate, and measuring the absorbance and the corresponding fluorescence signal intensity under the wavelength of 600nm in an enzyme label instrument; the red fluorescence measurement conditions were: the excitation wavelength is 580nm, and the excitation wavelength is 610nm.

(7) Data processing: calculating a relative fluorescence value (FIR) using the formula FIR = AFU/BFU, wherein FIR sample detected fluorescence value (AFU) is defined as the relative fluorescence value (RFU) divided by the sample absorbance; the control detected fluorescence (BFU) was defined as the Relative Fluorescence (RFU) detected in p.putida KT2440 cells (negative control) divided by the control absorbance. Control detected fluorescence (BFU) was set to 1.0 and the other samples were normalized to control detected fluorescence (BFU).

The invention has the advantages that:

the constructed whole-cell biosensor capable of detecting the concentration of cadmium ions and the whole-cell biosensor amplified by a circuit take P.putida KT2440 as host bacteria, and the performance of each sensor for detecting the cadmium ions is explored by detecting four performances of a growth curve, time-fluorescence response, concentration-fluorescence response, specificity and the like of a chassis cell.

The invention utilizes cadmium binding protein CadR and specific promoter P thereof _cad As a detection element, a red fluorescent protein gene mcherry is used as a report element, and then a T7RNAPMut amplification module is added to construct a biosensor chassis cell p2T7RNAPMut-68 capable of detecting cadmium ions, so that the construction method of the whole-cell biosensor sensitive to the cadmium ions is provided, and the sensor has better sensitivity and specificity to the cadmium ions.

The cadmium ion biosensor takes pseudomonas putida P.putida KT2440 as a host cell, has high sensitivity and specificity for cadmium ion detection, is not interfered by other metal ions, and has wide adaptability.

Drawings

FIG. 1 is an enlarged design of a cadmium ion whole cell biosensor circuit.

FIG. 2 is a graph of the growth of sensor p2T7RNAPMut-68.

FIG. 3 is a graph of time-fluorescence response of sensor p2T7RNAPMut-68.

FIG. 4 is a graph of sensor p2T7RNAPMut-68 concentration versus fluorescence response. The fitting curve comprises a cadmium ion concentration-fluorescence response fitting curve of the cadmium ion biosensor.

FIG. 5 is the specificity of sensor p2T7RNAPMut-68.

Detailed Description

The invention is described in further detail below with reference to the following figures and specific examples:

the cadR gene SEQ ID No.1 and the promoter P in the genome of P.putida KT2440 are utilized _cad SEQ ID No.1 and the cadO operon SEQ ID No.6; escherichia coli (Escherichia coli) pGN68 carrying red fluorescent protein genes mcherry SEQ ID No.3, T7RNAPMutSEQ ID No.4 and promoter P _T7 SEQ ID No.5, wherein the red fluorescent protein gene mcherrySEQ ID No.3 is codon optimized by Pseudomonas putida. Construction of a cadmium ion whole-cell biosensor is realized in P.putida KT 2440.

The plasmids pCDFDuet-2 and pGN68 are used as expression vectors for constructing detection and fluorescent protein of the cadmium ion biosensor. Wherein, the plasmid pCDFDuet-2 is a commercial plasmid, and the sequence of the plasmid pGN68 is shown as SEQ ID No. 7.

The LB medium consisted of: 10g/L NaCl, 10g/L peptone and 5g/L yeast powder, the balance being distilled water, and sterilizing at 121 ℃ under 0.1MPa for 20min.

The present invention will be further described with reference to the following examples.

Example 1 construction of Whole cell biosensor p2T7RNAPMut-68 capable of detecting cadmium ion content

1. Construction of plasmids (see FIG. I)

Cadmium-specific promoter P obtained by PCR amplification _cad SEQ ID No.1 and the sequence of the cadR gene of the specific binding protein, SEQ ID No.2 and T7RNAPmutSEQ ID No.4 (partial amino acid sequences are shown in FIG. 2). In the fragment cadR-P _cad Xho I and EcoRI cleavage sites are added at both ends respectively, and Xho I and EcoRI cleavage sites are added at both ends of the fragment RBS-T7 RNAmumut respectively. T7 promoter P amplified by PCR _T7 SEQ ID No.5, cadO sequence SEQ ID No.6 and red fluorescent protein gene mcherry SEQ ID No.3. In fragment-P _T7 BamHI and EcoRI sites were added to the ends of cadO-RBS-mcherry-respectively. Performing enzyme digestion by using Fastdigest endonuclease, wherein the reaction system 1) comprises the following steps: mu.L 10 × FD buffer, 2.5. Mu.L XhoI, 2.5. Mu.L EcoRI, 30. Mu.L cadR-P plus enzymatic cleavage site _cad Fragments and 10. Mu.L of ultrapure water; the reaction system 2) is as follows: mu.L of 10 × FD buffer, 2.5. Mu.L of XhoI, 2.5. Mu.L of EcoRI, 30. Mu.L of the-RBS-T7 RNAPMut-fragment to which the enzyme cleavage site was added, and 10. Mu.L of ultrapure water. The reaction system 3) is: mu.L 10 × FD buffer, 2.5. Mu.L BamHI, 2.5. Mu.L EcoRI, 30. Mu.L of-P plus enzymatic cleavage site _T7 A cadO-RBS-mcherry-fragment and 10. Mu.L of ultrapure water. The reaction conditions are as follows: 37 ℃ for 2h. Adding 250 mu L of Bingding Buffer solution into 50 mu L of enzyme digestion product by using a PCR purification kit, uniformly mixing, adding into an adsorption column, standing for one minute, centrifuging for 1 minute at 10,000xg, and removing effluent. 650. Mu.L of Wash Buffer,10,000g was added and centrifuged for 1 min, and the effluent was discarded. Centrifuge at 10,000xg for 2 minutes to remove the residual Wash Buffer. Placing the adsorption column in a clean centrifuge tube, adding the adsorbent column at the center of the columnAdding 30 μ L of Elution Buffer (the Elution Buffer is preheated in a water bath kettle at 65 ℃ in advance), standing at room temperature for 1 minute, centrifuging at 10,000xg for 1 minute, and eluting the enzyme-digested-cadR-P _cad -, RBS-T7RNAPMut and P _T7 -cadO-RBS-mcherry. Digesting the obtained product-cadR-P _cad Ligation with RBS-T7 RNAPMut. The reaction system is as follows: mu.L of 10 × T4 DNA ligand buffer, 1. Mu. L T4 DNA ligand, 4. Mu.L of cleaved-cadR-P _cad Fragment and 4. Mu.L of cleaved-RBS-T7 RNAPMut-fragment. The reaction conditions are as follows: 22 ℃ for 10min. PCR amplification is carried out by taking the product after enzyme digestion connection as a template to obtain-cadR-P _cad -RBS-T7 RNAPMut-fragment, cleaved enzymatically using FastDiget endonuclease, in the reaction system: mu.L 10 × FD buffer, 5.0. Mu.L EcoRI, 30. Mu.L-cadR-P plus enzymatic cleavage site _cad -RBS-T7 rnapimut-fragment and 10 μ L ultrapure water; the pCDFDuet-2 plasmid was similarly digested with Fastdigest endonucleases EcoRI, the pGN68 plasmid was digested with Fastdigest endonucleases BamHI and EcoRI, and purified and recovered with a PCR purification kit.

And (3) carrying out ligation reaction on the nucleotide and the plasmid after enzyme digestion. The reaction system is as follows: mu.L of 10 × T4 DNA Ligase buffer, 1. Mu. L T4 DNA Ligase, 6. Mu.L of the digested nucleotide fragment and 2. Mu.L of the digested plasmid. The reaction conditions are as follows: 22 ℃ for 10min. After enzyme digestion and connection, competent cells P.putida KT2440 are transformed, positive clone plasmid is screened by colony PCR, and sequencing verification is carried out.

2. Transformation of plasmid into Pseudomonas putida Chassis Strain P.putida KT2440

The detailed construction steps of the recombinant expression vector transformed into the chassis strain P.putida KT2440 are as follows:

1) The activated P.putida KT 2440. Mu.L was inoculated into 10ml of LB medium at 30 ℃ and 220rpm and cultured to OD ₆₀₀ When the concentration is 0.6, transferring the mixture into a 10ml centrifuge tube, centrifuging the mixture for 5min in a precooled 4 ℃ centrifuge at 4500rpm/min, removing supernatant, and collecting thalli;

2) Washing thallus with 5ml of precooled sterilized 0.1mol/L calcium chloride, centrifuging at 4500rpm/min for 5min in a precooled 4 ℃ centrifuge, removing supernatant, collecting thallus, and repeatedly washing twice;

3) Pouring the supernatant as far as possible, adding 50 mu L of 0.1mol/L calcium chloride and 50 mu L of 30% glycerol for resuspending the thalli, and preparing E.coli DH5 alpha competent cells;

4) mu.L of the plasmid constructed in example 1 was added to 100. Mu.L of the electroporation competent cells, and gently swirled to mix. After preventing on ice for half an hour, thermally shocking for 45s, rapidly carrying out ice bath for 2min, adding 1mL LB culture medium, recovering at 30 ℃ for 1h, coating on a kanamycin-resistant plate, and carrying out overnight culture;

5) And selecting positive transformants verified by colony PCR to be cultured in 5ml LB culture medium overnight to obtain the cadmium ion whole cell biosensor chassis cells.

Example 2 Performance testing of target Whole cell biosensor

1. Growth curves for target whole cell biosensors

1.1 growth curves of Chassis cells of target Whole-cell biosensor

Individual colonies of P.putida KT2440 containing the sensor plasmid were shake-cultured overnight at 220rpm at 30 ℃ in LB medium containing antibiotics. Then diluted to OD with LB medium ₆₀₀ 1, inoculating the strain into an LB liquid culture medium without resistance according to the proportion of 1 percent; absorbance OD of bacterial liquid ₆₀₀ Adding Cd to the tube at final concentration of 0.6-0.8, 0.1, 1, 10, 100, 200, 300, 400, 500, 600 μ M ²⁺ Shaking culture was carried out at 30 ℃ and 220 rpm. Without addition of Cd ²⁺ Sampling every 1 hour before; addition of Cd ²⁺ Thereafter, samples were taken every 2 hours, and the absorbance OD was measured ₆₀₀ . Wherein the absorbance is measured at a wavelength of 600 nm.

Data processing: calculating a relative fluorescence value (FIR) using the formula FIR = AFU/BFU, wherein the FIR sample detected fluorescence value (AFU) is defined as the relative fluorescence value (RFU) divided by the sample absorbance; the control detected fluorescence value (BFU) was defined as the relative fluorescence value (RFU) detected in p.pudida KT2440 cells (negative control) divided by the control absorbance. Control detected fluorescence (BFU) was set to 1.0 and the other samples were normalized to control detected fluorescence (BFU).

1.2 results of the experiment

The growth change curve of the cadmium ion whole cell biosensor p2T7RNAPMut-68 constructed in the invention is shown in figure 2 after cadmium ions with different concentrations are added.

When Cd is added ²⁺ When the concentration is less than 1 mu M, cd ²⁺ Growth of sensor p2T7RNAPMut-68 at concentrations less than 400. Mu.M and no Cd addition ²⁺ Substantially the same time, indicating the addition of 0-400. Mu.M Cd ²⁺ Has little influence on cell growth, and the sensor p2T7RNAPMut-68 increases the cell pair Cd ²⁺ The tolerance of (2).

2. Time-fluorescence response of target whole-cell biosensor

2.1 time-fluorescence response of target Whole cell biosensor

Individual colonies of P.putida KT2440 containing the sensor plasmid were shake-cultured overnight at 220rpm at 30 ℃ in LB medium containing antibiotics. Then diluted to OD with LB medium ₆₀₀ 1, inoculating the strain into an LB liquid culture medium without resistance according to the proportion of 1 percent; absorbance OD of bacterial liquid ₆₀₀ When the concentration is 0.6-0.8, cd is added into the test tube to final concentration of 0, 0.01, 0.02, 0.04, 0.05, 0.1, 1, 10 μ M ²⁺ Culturing at 30 deg.C and 220rpm by shaking, collecting 200 μ L bacterial liquid every 2 hr in 96-well plate, and measuring relative fluorescence value (RFU) and absorbance OD of sample with microplate reader ₆₀₀ 。

2.2 results of the experiment

The time-fluorescence response curve of the constructed cadmium ion whole-cell biosensor p2T7RNAPMut-68 after cadmium ions with different concentrations are added is shown in figure 3.

The fluorescence response of sensor p2T7RNAPMut-68 increased gradually with time. Adding Cd ²⁺ After 4h incubation, sensor p2T7RNAPMut-68The detection limit is reduced to 0.05 mu M from 1 mu M in 2 hours; adding Cd ²⁺ After 6h of incubation, the detection limit of the sensor p2T7RNAPMut-68 was reduced to 0.01. Mu.M (lower than the detection limit of 0.027. Mu.M for cadmium in WHO drinking water).

3. Concentration-fluorescence response of target whole-cell biosensor

3.1 concentration-fluorescence response of target Whole cell biosensor

Individual colonies of P.putida KT2440 containing the sensor plasmid were shake-cultured overnight at 220rpm at 30 ℃ in LB medium containing antibiotics. Then diluted to OD with LB medium ₆₀₀ 1, inoculating the strain into an LB liquid culture medium without resistance according to the proportion of 1 percent; absorbance OD of bacterial liquid ₆₀₀ When the concentration is 0.6-0.8, cd with final concentration of 0, 0.01, 0.02, 0.04, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 μ M is added into the test tube ²⁺ Culturing at 30 deg.C and 220rpm, shaking for 8 hr, collecting 200 μ L bacterial solution, placing in 96-well plate, and measuring relative fluorescence value (RFU) and absorbance OD of sample with microplate reader ₆₀₀ . The absorbance was measured at a wavelength of 600nm under the red fluorescence measurement conditions: the excitation wavelength is 580nm, and the excitation wavelength is 610nm.

Data processing: calculating a relative fluorescence value (FIR) using the formula FIR = AFU/BFU, wherein FIR sample detected fluorescence value (AFU) is defined as the relative fluorescence value (RFU) divided by the sample absorbance; the control detected fluorescence value (BFU) was defined as the relative fluorescence value (RFU) detected in p.pudida KT2440 cells (negative control) divided by the control absorbance. The control detection fluorescence value (BFU) was set to 1.0 and the other samples were normalized to the control detection fluorescence value (BFU).

3.2 results of the experiment

The concentration-fluorescence response curve of the constructed cadmium ion whole-cell biosensor p2T7RNAPMut-68 is shown in figure 4 after cadmium ions with different concentrations are added.

Sensor p2T7RNAPMut-68 Cd at 5. Mu.M ²⁺ The fluorescence response is best in the presence, a cadmium ion concentration-fluorescence response curve is subjected to linear fitting, the linear response range of the p2T7RNAPMut-68 is 0.01-0.5 mu M, and the sensitivity is 388.74.

4. Specificity of target Whole cell biosensor

4.1 specificity of target Whole cell biosensor

Individual colonies of P.putida KT2440 containing the sensor plasmid were shake-cultured overnight at 220rpm at 30 ℃ in LB medium containing antibiotics. Then diluted to OD with LB medium ₆₀₀ 1, inoculating the strain into an LB liquid culture medium without resistance according to the proportion of 1 percent; absorbance OD of bacterial liquid ₆₀₀ When the concentration is 0.6-0.8, adding Cd to the test tube at final concentration of 0.01, 0.1, 1, 10 μ M ²⁺ 、Pd ²⁺ 、Zn ²⁺ 、Cu ²⁺ 、As ³⁺ Culturing at 30 deg.C and 220rpm, shaking for 8 hr, collecting 200 μ L bacterial solution, placing in 96-well plate, and measuring relative fluorescence value (RFU) and absorbance OD of sample with microplate reader ₆₀₀ . The absorbance was measured at a wavelength of 600nm under the red fluorescence measurement conditions: the excitation wavelength is 580nm, and the excitation wavelength is 610nm.

4.2 results of the experiment

The specificity analysis of the constructed cadmium ion whole cell biosensor p2T7RNAPMut-68 is shown in FIG. 5 after cadmium ions with different concentrations are added.

Cd ²⁺ The fluorescence response of the sensor p2T7RNAPMut-68 is significantly higher than that of other heavy metal ions at concentrations of 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M and 10. Mu.M. p2T7 RNAcumut-68 in Cd ²⁺ The fluorescence response at a concentration of 0.1. Mu.M was 10. Mu.M Pd ²⁺ 、Zn ²⁺ 、Cu ²⁺ 、As ³⁺ 7.6-17.8 times of the case, the specificity of p2T7RNAPMut-68 is 7.6.

Wherein, zn ²⁺ The fluorescence response of the sensor is higher than that of other Zn when the concentration is 10 mu M ²⁺ Fluorescence response at concentration. This is due to cadmium responseThe CadR protein has 2 different types of functional sites, caused by the special structure of the transcription factor CadR protein: metal binding site I (S1 and S1 ') and metal binding site II (S2 and S2'). The metal binding site II (S2 and S2') is located in the middle of the molecule and is formed by 2 histidine residues (His 87 and His90 in the. Alpha.5 helix), 1 glutamic acid residue (Glu 62 in the. Alpha.4 helix) and a variable ligand from the His tail region, 3 histidine residues and 1 glutamic acid residue arranged in a tetrahedral configuration capable of interacting with Cd ²⁺ And Zn ²⁺ And (4) combining.

While the methods and techniques of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and/or modifications of the methods and techniques described herein may be made without departing from the spirit and scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.

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gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag 240

atgattgcac gcatcaacga ctggtttgag gaaatgaaag ctaagcgcgg caagcgcccg 300

acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag 360

accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420

atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480

cacttcaaga aatacgttga ggaacaactc aacaagcgcg tagggcgcgt ctacaagaaa 540

gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600

tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660

attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720

tctgagacta tcgaacccgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780

ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc 840

attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900

agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960

aacattgcgc aaaacaccgc atggaaaatc aacaagagag tcctagcggt cgccaacgta 1020

atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080

ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct 1140

gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200

atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg 1260

gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320

aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380

aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag 1440

ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact 1500

tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560

gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620

tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680

ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgtg 1740

attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag 1800

aacactggtg aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg 1860

ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg 1920

tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat 1980

tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg 2040

atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100

tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggagg gattcttcgc 2160

aagcgttgcg ctgtgcattg ggtaactccg gatggtttcc ctgtgtggca ggaatacaag 2220

aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280

attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct 2340

aactttgtac acagccaaga cggtagccac cttcgtaaga cggtagtgtg ggcacacgag 2400

aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac 2460

gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520

gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa 2580

atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc 2640

gcgttcgcgt aa 2652

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taatacgact cactatagg 19

<210> 6

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctatagtggc tacagggtgt tcacttggca acaggctcaa tttaaggatg 50

<210> 7

<211> 3062

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaattcatac gtatttaaat caggagtgga aatgagtaaa ggagaagaac ttttcactgg 60

agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag 120

tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac 180

tggaaaacta cctgttccat ggccaacact tgtcactact ttgacttatg gtgttcaatg 240

cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg ccatgcccga 300

aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtgc 360

tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag gtattgattt 420

taaagaagat ggaaacattc ttggacacaa attggaatac aactataact cacacaatgt 480

atacatcatg gcagacaaac aaaagaatgg aatcaaagtt aacttcaaaa ttagacacaa 540

cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc caattggcga 600

tggccctgtc cttttaccag acaaccatta cctgtccaca caatctgccc tttcgaaaga 660

tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac 720

acatggcatg gatgaactat acaaataata aggatccaac taaagattaa ctttataagg 780

aggaaaaaca tatgccttct ctagttgata attatcgaaa aataaatata gcaaataata 840

aatcaaacaa cgatttaacc aaaagagaaa aagaatgttt agcgtgggca tgcgaaggaa 900

aaagctcttg ggatatttca aaaatattag gctgcagtga gcgtactgtc actttccatt 960

taaccaatgt gcaaatgaaa ctcaatacaa caaaccgctg ccaaagtatt tctaaagcaa 1020

ttttaacagg agcaattgat tgcccatact ttaaaaatta ataagcggcc gcttaattaa 1080

ttaatctaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 1140

atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccgcc ctagacctag 1200

ggcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 1260

gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 1320

cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 1380

aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 1440

tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1500

ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 1560

ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1620

cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1680

ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1740

gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1800

atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1860

aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1920

aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1980

gaaaactcac gttaagggat tttggtcatg actagtgctt ggattctcac caataaaaaa 2040

cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg 2100

atctatcaac aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat 2160

cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca cagacggcat 2220

gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca 2280

tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga 2340

aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat 2400

aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga 2460

aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg 2520

tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa 2580

attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt 2640

gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat 2700

aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata 2760

tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa 2820

atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg 2880

aacctcttac gtgccgatca acgtctcatt ttcgccagat atcgacgtca gtcctttgat 2940

tctaataaat tggatttttg tcacactatt gtatcgctgg gaatacaatt acttaacata 3000

agcacctgta ggatcgtaca ggtttacgca agaaaatggt ttgttatagt cgaataaacg 3060

ca 3062

Claims

1. A construction method for amplifying a cadmium ion whole-cell biosensor circuit; it is characterized by that it uses plasmids pCDFDuet-2 and pGN68 as carrier, utilizes detection element CadR, report element mCherry, cadO operon and T7RNAPMut amplification module, and adopts the sequence of cadR-P _cad -RBS-T7RNAPmut+P _T7 Assembling gene circuits of the cadO-RBS-mcherry by PCR to obtain a cadmium ion whole cell biosensor p2T7 RNAcumut-68; the detection plasmid comprises a cadmium-specific protein regulated promoter P _cad SEQ ID No.1, cadmium specific binding protein CadR gene SEQ ID No.2 and T7RNAP 40 th amino acid from Glutamic Acid (GAG) mutation to Terminator (TAG) T7RNAmut SEQ ID No.4 as core element composed of detection plasmid; the reporter plasmid comprises a T7 promoter P _T7 The reporter plasmid consists of the core elements of SEQ ID No.5, cadO operon SEQ ID No.6 and red fluorescent protein mCherry gene SEQ ID No.3.

2. The method of claim 1, comprising the steps of:

(1) Regulation of promoter P by cadmium-specific proteins _cad SEQ ID No.1, cadmium specific binding protein CadR gene SEQ ID No.2 and T7RNAP mutation of amino acid at position 40 from Glutamic Acid (GAG) to Terminator (TAG)Detection plasmid composed of T7RNAmutSEQID No.4 as core element and T7 promoter P _T7 A report plasmid which is composed of SEQ ID No.5, cadO operon SEQ ID No.6 and red fluorescent protein mCherry gene SEQ ID No.3 as core elements;

3. The method as set forth in claim 1, wherein the performance of the cadmium ion whole cell biosensor is measured as P.

The putida KT2440 is a host bacterium, and the performance of the sensor for detecting cadmium ions is obtained by detecting the growth curve, time-fluorescence response, concentration-fluorescence response and specificity of the chassis cells.