CN115141787B

CN115141787B - Submarine microbial sensor and preparation method and application thereof

Info

Publication number: CN115141787B
Application number: CN202210639825.2A
Authority: CN
Inventors: 杨建明; 李美洁; 吕书喆; 汤若昊; 王兆宝
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2023-08-15
Anticipated expiration: 2042-06-08
Also published as: CN115141787A

Abstract

The invention discloses a submarine microbial sensor and a preparation method and application thereof. The submarine microbial sensor comprises a sensing element containing sensing protein Cad sensing metal cadmium Cd (II) and an electric signal element protein mtrCAB, and the lactic dehydrogenase 1dh gene is knocked out, so that releasable electrons in cells are increased, and the over-expressed porin gene OprF enhances a bacterial adhesion electrode and increases membrane permeability. The submarine microorganism sensor constructed by the invention can sense low-concentration metal cadmium Cd (II) ions to generate strong electric signals, thereby realizing the real-time detection of the low-concentration metal cadmium ions. Compared with other detection methods, the submarine microorganism sensor is simpler and more convenient to detect metal cadmium ions and easier to collect signals, can be used for detecting the metal cadmium ions released by oxidation-reduction reaction of submarine shells in the ocean, and has wide application prospects.

Description

Submarine microbial sensor and preparation method and application thereof

Technical Field

The invention belongs to the technical fields of genetic engineering and molecular biology, and particularly relates to a submarine microbial sensor and a preparation method and application thereof.

Background

In the maritime combat action, submarines are called "deep sea strainers" because of their advanced performance, superior concealment, and long-lasting cruising ability. Each country is under scrutiny for submarine technology to effectively spread the maritime forces. In addition, how to efficiently and rapidly monitor the fight submarines of the enemy, thereby realizing the expelling and capturing of the enemy submarines and having important strategic significance for the marine national defense safety of China. The current traditional submarine detection method mainly comprises the following steps: sonar detection, infrared detection, electromagnetic detection and other methods, wherein the sonar detection is the most main method used in various countries at present. However, the submarine detection is also prone to exposing its own position when using sonar detection.

Under the condition that the submarine is submerged in the ocean for a long time, some parts are easily corroded by seawater, and then a certain amount of metal ions are released. The biological induction technology is to modify bacterial strain by genetic engineering means, so that the microorganism can generate detected change under the condition of inducing specific compounds, thereby achieving the purpose of detecting the specific compounds. The purpose of real-time detection of the submarine can be achieved by inducing metal ions released by the submarine and constructing a microbial sensor. The sensing element includes a promoter, ribosome binding site, terminator, transcription regulatory protein and the like responsible for gene transcription, and can specifically sense a metal ion to cause a downstream reporter element to generate a signal. Commonly used reporter elements include Green Fluorescent Protein (GFP), yellow Fluorescent Protein (YFP) and Red Fluorescent Protein (RFP), which generate detectable sensor signals such as green fluorescence, yellow fluorescence and red fluorescence, respectively. But this signal output method cannot cope with the complex environment in the ocean and numerous disturbance factors.

Therefore, there is a need to optimize reporting elements and hosts to obtain a submarine microbial sensor that facilitates real-time detection in seawater.

Disclosure of Invention

The invention provides a submarine microbial sensor and a preparation method and application thereof. The submarine biosensor contains a sensing element for sensing metal ions Cd (II) and an electric signal element protein mtrCAB, and the lactate dehydrogenase ldh gene of host escherichia coli is knocked out, and a porin gene OprF is overexpressed, so that the intracellular current signal intensity is increased. The submarine microorganism sensor provided by the invention can realize real-time fluorescence detection of metal cadmium ions in the ocean, so that the submarine can be further monitored in real time.

In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:

the invention provides a submarine biosensor which comprises an electric signal element protein gene, a sensing element and a porin gene and simultaneously knocks out an escherichia coli lactic dehydrogenase gene.

Furthermore, the electric signal element protein gene is an mtrCAB gene, and the nucleotide sequence of the electric signal element protein gene is shown as SEQ ID NO. 1; the sensing element is cad gene, and the nucleotide sequence of the sensing element is shown as SEQ ID NO. 3.

Furthermore, the porin gene is an oprF gene, and the nucleotide sequence of the porin gene is shown as SEQ ID NO. 6; the escherichia coli lactate dehydrogenase gene is an ldh gene, and the nucleotide sequence of the escherichia coli lactate dehydrogenase gene is shown as SEQ ID NO. 4.

Further, the submarine biosensor has the function of sensing metal cadmium ions Cd (II).

The invention also provides a preparation method of the submarine biological sensor, which comprises the following steps:

(1) Amplifying and purifying and recovering an electric signal element protein mtrCAB gene by using PCR, carrying out double enzyme digestion with a plasmid pACYCDuet-1 by using EcoR I and Hind III, carrying out seamless connection on an mtrCAB fragment and a plasmid enzyme slice fragment, transforming an escherichia coli competent cell by a connection product, and screening positive clones on an LB solid plate containing antibiotics to obtain a recombinant plasmid p-mtrCAB;

(2) Amplifying a sensing element Cad gene, purifying and recovering, performing single enzyme digestion with a recombinant plasmid p-mtrCAB by using EcoR I, performing seamless connection between a plasmid enzyme digestion fragment and Cad, transforming a connection product into escherichia coli competent cells, and screening positive clones on an LB solid plate containing antibiotics to obtain the recombinant plasmid p-Cad-mtrCAB;

(3) Amplifying an upstream fragment ldh-up and a downstream fragment ldh-down of an ldh gene in an escherichia coli genome, amplifying an porin gene oprF, performing overlap PCR on the three fragments to obtain an ldh-up-oprF-ldh-down fragment, and connecting the fragments to a HindIII site of a suicide plasmid pRE112 by seamless cloning to obtain pRE 112-delta ldh-oprF plasmid;

(4) pRE 112-Deltaldh-oprF was transformed into donor E.coli χ7213, host E.coliBL21 (DE 3) as recipient bacteria; activation of donor and acceptor bacteria, ddH ₂ O washing, re-suspending the bacterial cells by 1% DAP, coating on a non-resistance plate, and jointing; ddH ₂ O washing, re-suspending, coating on a chloramphenicol plate, and performing colony PCR verification; culturing positive clones in non-resistance LB, coating on a 10% sucrose plate for screening, and screening positive clones on a sucrose plate and a chloramphenicol plate to finally obtain recombinant escherichia coli E.coli BL21 (DE 3) delta ldh: oprF;

(5) E.coll BL21 (DE 3) delta ldh is converted by recombinant plasmid p-Cad-mtrCAB, wherein the competent cells of oprF are positive clones screened on LB solid plates containing antibiotics, and engineering strains ECCad are obtained, namely the submarine microbial sensor.

The invention also provides application of the submarine biosensor in real-time detection of metal Cd (II) ions.

Further, the concentration of Cd (II) ions which can be detected by the submarine biosensor is not lower than 0.1 mu mol/L.

The invention also provides application of the submarine biosensor in preparing a detection device for detecting the submarine in the ocean in real time.

Further, the construction method of the detection device comprises the following steps: placing a sealing ring between the anode chamber and the cathode chamber, clamping and assembling the anode chamber and the cathode chamber, and fixing anode materials in the anode chamber and the cathode chamber respectively by leads; inoculating an activated submarine microbial sensor in the anode chamber, and applying voltage to promote the formation of a biological film on the anode material; electrolyte is loaded in the cathode chamber.

Furthermore, the detection device detects Cd (II) ions released by the submarine in the ocean in real time, and converts the Cd (II) ions into a changed electric signal to be output, so that the purpose of detecting the submarine in the ocean in real time is achieved.

Further, the concentration of Cd (II) ions which can be sensed by the detection device is not lower than 0.1 mu mol/L.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention connects the metal ion sensing element with the extracellular electronic transmission path mtrCAD, and utilizes porin to improve membrane permeability, improve bacterial adhesion electrode and increase membrane permeability, and knock out lactic acid synthesis path, increase intracellular releasable electrons, amplify current signal, thereby generating current signal easy to be detected. The submarine microorganism sensor constructed by the invention can detect trace metal ions and generate instantaneous electric signal output, so that the detection of the metal ions released by the submarine in the ocean is realized, and the method is convenient and simple to operate, high in safety and high in sensitivity.

Drawings

FIG. 1 is a plasmid map of the constructed vector p-Cad-mtrCAB.

FIG. 2 shows the results of ECCad-induced electrogenesis detection of the constructed engineering strain

Detailed Description

The invention will be further illustrated with reference to specific examples, but the invention is not limited to the examples.

The specific techniques or conditions are not identified in the examples and are performed according to techniques described in the literature in this field or according to product specifications. The reagents or apparatus used were conventional products available commercially without the manufacturer's attention.

Example 1: construction of vector p-Cad-mtrCAB

1. Construction of p-mtrCAB expression vector

The PCR is carried out by taking S.oneidensis genome as a template, primer mtrCAB-F and primer mtrCAB-R, and the mtrCAB fragment is amplified, and the PCR amplification system is as follows:

the PCR procedure was: 3min at 95 ℃;30× (95 ℃ 15s,55 ℃ 15s,72 ℃ 4 min); 72 ℃ for 5min:16 ℃ infinity.

The primer sequences are shown below:

mtrCAB-F：5’-CAAGGAGAAAAAAATGATGAACGCACAAAAATC-3’(SEQ ID NO.8)；

mtrCAB-R：5’-GCCGCAAGCTTTTAGAGTTTGTAACTCATGCTC-3’(SEQ ID NO.9)。

the pACYCDuet-1 plasmid was digested simultaneously with restriction enzyme 1EcoR I (TaKaRa, cat. 1611) and restriction enzyme 2 Hind III (TaKaRa, cat. 1615), and the digestion system was:

the enzyme digestion system is incubated for 1h at 37 ℃ for gel recovery and purification.

The mtrCAB fragment was cloned into pACYCDuet-1 plasmid using a seamless clone as follows:

the ligation system was incubated at 50℃for 30min. The ligation product was transformed into E.coli DH 5. Alpha. Competent, spread on LB solid plates containing 34mg/L chloramphenicol, PCR screened positive clones, recombinant plasmid p-mtrCAB was extracted from positive clones, and identified by restriction enzyme digestion and sequencing. The nucleotide sequence of mtrCAB is shown as SEQ ID NO.1, and the amino acid sequence of mtrCAB is shown as SEQ ID NO. 2.

2. Construction of p-Cad-mtrCAB expression vector

The sequence of the synthesized sensing element Cad is shown as SEQ ID NO.3, and a Polymerase Chain Reaction (PCR) is carried out by using a primer Cad-F and a primer Cad-R to amplify Cad fragments, wherein the PCR amplification system is shown as follows:

the PCR procedure was: 3min at 95 ℃;30 cycles X (95 ℃ C. 15s,58 ℃ C. 15s,72 ℃ C. 4 min); 72 ℃ for 5min;16 ℃ infinity.

The primer sequences are shown below:

Cad-F：5’-CCGAATTCGTGTATTTTTTAATAAATTATTTTAC-3’(SEQ ID NO.10)；

Cad-R：5’-CATGAATTCTCAGACATTGACCTTCACTTCT-3’(SEQ ID NO.11)。

the PCR product was subjected to gel recovery and purification using gel recovery and purification kit (Vazyme, cat. DC 301-01).

The p-mtrCAB plasmid was digested with restriction enzyme 1EcoR I (TaKaRa, cat. No. 1611), the system for the digestion was:

The Cad fragment was cloned on the p-mtrbab plasmid using seamless cloning, the system of which is as follows:

the ligation system was incubated at 50℃for 30min. The ligation product was transformed into E.coli DH 5. Alpha. Competent, spread on LB solid plates containing 34mg/L chloramphenicol, PCR screened positive clones, recombinant plasmid p-Cad-mtrCAB (FIG. 1) was extracted from positive clones, and identified by restriction enzyme digestion and sequencing.

Example 2: construction of the host E.coli BL21 (DE 3) Δldh:: oprF

(1) Using escherichia coli genome as a template, amplifying an upstream fragment ldh-up and a downstream fragment ldh-down of an ldh gene, amplifying an porin gene oprF, performing overlap PCR on the three fragments to obtain an ldh-up-oprF-ldh-down fragment, and connecting the fragments to a Sac I site of a suicide plasmid pRE112 through seamless cloning to obtain pRE 112-delta ldh-oprF plasmid; wherein the nucleotide sequence of the ldh gene is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO. 5; the nucleotide sequence of the porin gene oprF is shown as SEQ ID NO.6, and the amino acid sequence is shown as SEQ ID NO. 7.

The primer sequences are shown below:

ldh-up-F：5’-AGCCAAGTAATTTCCACTCCTTGTGGTGGC-3’(SEQ ID NO.12)：

ldh-up-R：5’-GCATGCGATATCGAGCTCACTGGTCGCGCAGAACATCTTTC-3’(SEQ ID NO.13)；

ldh-down-F：5’-ATGAATTCCCGGGAGAGCTCCGTTATGATGGCGTCGCTAG-3’(SEQ ID NO.14)；

ldh-down-R：5’-TCAGTTTCATAAATTACGGATGGCAGAGTA-3’(SEQ ID NO.15)；

oprF-F：5’-TCCGTAATTTATGAAACTGAAGAACACCTTAG-3’(SEQ ID NO.16)：

oprF-R：5’-CAAGGAGTGGAAATTACTTGGCTTCAGCTTCTACT-3’(SEQ ID NO.17)。

(2) Transforming the constructed pRE 112-delta ldh-oprF into donor bacteria E.coli χ7213 and using a host E.coli BL21 (DE 3) as acceptor bacteria; activating donor bacteria and acceptor bacteria, transferring the activated bacteria into 10ml LB liquid medium at 37deg.C, 180rpm for about 2.5 hr to OD ₆₀₀ The value was 0.5.

(3) 1.5mL of each bacterial liquid is taken in a centrifuge tube, centrifuged at 10000rpm for 1min, the supernatant is discarded, the supernatant is centrifuged once for a short time, the residual LB of the supernatant is thoroughly removed, and 1mL of ddH is used ₂ O suspended precipitate, 10000rpm, centrifugate for 1min, discard supernatant, and reuse 1mL ddH ₂ O suspended precipitate, 10000rpm, centrifuge for 1min, discard supernatant to remove antibiotics.

(4) With 500. Mu.L of ddH ₂ After O re-suspending the bacterial pellet, 100. Mu.L of each was mixed uniformly, centrifuged at 10000rpm for 1min, the supernatant was discarded, and 50. Mu.L of 1% DAP was used to re-suspend the bacterial pellet, which was spread on a non-resistant LB plate, and cultured at 37℃for 12h. ddH ₂ O washing and resuspension, diluting the resuspension bacterial liquid 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 Multiple, each multiple was applied to a chloramphenicol plate at 30 μl, and colony PCR validation; positive clones were cultured in non-resistant LB, plated on 10% sucrose plates for screening,positive clones are screened on a sucrose plate and a chloramphenicol plate, and finally recombinant E.coli BL21 (DE 3) delta ldh: oprF is obtained.

Example 3: construction of microorganism sensor of submarine

The p-Cad-mtrCAB recombinant plasmid was transformed E..coli BL21 (DE 3) Δldh:: oprF competent cells were plated on LB solid plates containing 34mg/L chloramphenicol, and positive clones were obtained by PCR screening, thereby obtaining an engineering strain ECCad containing the vector p-Cad-mtrCAB.

Example 4: application of submarine biological sensor in detecting metal cadmium ions

(1) Construction of detection device

The activated ecad was inoculated into an anode chamber containing LB medium at 1% inoculum size, and a voltage of 0.3V was applied to promote biofilm formation on the carbon fiber felt anode. And a sealing ring is arranged between the anode chamber and the cathode chamber, and then the anode chamber and the cathode chamber are clamped and assembled, so that the solution is ensured not to leak outwards. The carbon felt is connected by wire and is respectively positioned in anode chamber and cathode chamber, in which the position in anode chamber is parallel to water inlet, in cathode chamber is perpendicular to bottom, the anode chamber is loaded with LB culture medium, and the cathode chamber is loaded with K ₃ [Fe(CN) ₆ ]、K ₂ HPO ₄ And KH ₂ PO ₄ An electrolyte is disposed. The electrochemical workstation is used for outputting detection of the electric signal.

(2) Detection with electrical signal as output

And (3) introducing a buffer solution with the concentration content of metal ions Cd (II) of 5 mu mol/L into the constructed device, detecting the output change of an electric signal by using an electrochemical workstation, and detecting once every 1 hour for 12 hours.

As a result, as shown in FIG. 2, in the presence of 5. Mu. Mol/L metal ion Cd (II), there was a significant difference from the absence of metal ion, and the detection voltage reached a maximum of 0.252V for 7 hours, and the current density was reduced to a plateau value after all metal ions were removed.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

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<120> a submarine microbial sensor, and preparation method and application thereof

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Met Lys Asn Cys Leu Lys Met Lys Asn Leu Leu Pro Ala Leu Thr Ile

1 5 10 15

Thr Met Ala Met Ser Ala Val Met Ala Leu Val Val Thr Pro Asn Ala

20 25 30

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

35 40 45

Thr Leu Asp Lys Lys Phe Ala Glu Gly Asn Tyr Ser Pro Lys Gly Ala

50 55 60

Asp Ser Cys Leu Met Cys His Lys Lys Ser Glu Lys Val Met Asp Leu

65 70 75 80

Phe Lys Gly Val His Gly Ala Ile Asp Ser Ser Lys Ser Pro Met Ala

85 90 95

Gly Leu Gln Cys Glu Ala Cys His Gly Pro Leu Gly Gln His Asn Lys

100 105 110

Gly Gly Asn Glu Pro Met Ile Thr Phe Gly Lys Gln Ser Thr Leu Ser

115 120 125

Ala Asp Lys Gln Asn Ser Val Cys Met Ser Cys His Gln Asp Asp Lys

130 135 140

Arg Met Ser Trp Asn Gly Gly His His Asp Asn Ala Asp Val Ala Cys

145 150 155 160

Ala Ser Cys His Gln Val His Val Ala Lys Asp Pro Val Leu Ser Lys

165 170 175

Asn Thr Glu Met Glu Val Cys Thr Ser Cys His Thr Lys Gln Lys Ala

180 185 190

Asp Met Asn Lys Arg Ser Ser His Pro Leu Lys Trp Ala Gln Met Thr

195 200 205

Cys Ser Asp Cys His Asn Pro His Gly Ser Met Thr Asp Ser Asp Leu

210 215 220

Asn Lys Pro Ser Val Asn Asp Thr Cys Tyr Ser Cys His Ala Glu Lys

225 230 235 240

Arg Gly Pro Lys Leu Trp Glu His Ala Pro Val Thr Glu Asn Cys Val

245 250 255

Thr Cys His Asn Pro His Gly Ser Val Asn Asp Gly Met Leu Lys Thr

260 265 270

Arg Ala Pro Gln Leu Cys Gln Gln Cys His Ala Ser Asp Gly His Ala

275 280 285

Ser Asn Ala Tyr Leu Gly Asn Thr Gly Leu Gly Ser Asn Val Gly Asp

290 295 300

Asn Ala Phe Thr Gly Gly Arg Ser Cys Leu Asn Cys His Ser Gln Val

305 310 315 320

His Gly Ser Asn His Pro Ser Gly Lys Leu Leu Gln Arg Met Met Asn

325 330 335

Ala Gln Lys Ser Lys Ile Ala Leu Leu Leu Ala Ala Ser Ala Val Thr

340 345 350

Met Ala Leu Thr Gly Cys Gly Gly Ser Asp Gly Asn Asn Gly Asn Asp

355 360 365

Gly Ser Asp Gly Gly Glu Pro Ala Gly Ser Ile Gln Thr Leu Asn Leu

370 375 380

Asp Ile Thr Lys Val Ser Tyr Glu Asn Gly Ala Pro Met Val Thr Val

385 390 395 400

Phe Ala Thr Asn Glu Ala Asp Met Pro Val Ile Gly Leu Ala Asn Leu

405 410 415

Glu Ile Lys Lys Ala Leu Gln Leu Ile Pro Glu Gly Ala Thr Gly Pro

420 425 430

Gly Asn Ser Ala Asn Trp Gln Gly Leu Gly Ser Ser Lys Ser Tyr Val

435 440 445

Asp Asn Lys Asn Gly Ser Tyr Thr Phe Lys Phe Asp Ala Phe Asp Ser

450 455 460

Asn Lys Val Phe Asn Ala Gln Leu Thr Gln Arg Phe Asn Val Val Ser

465 470 475 480

Ala Ala Gly Lys Leu Ala Asp Gly Thr Thr Val Pro Val Ala Glu Met

485 490 495

Val Glu Asp Phe Asp Gly Gln Gly Asn Ala Pro Gln Tyr Thr Lys Asn

500 505 510

Ile Val Ser His Glu Val Cys Ala Ser Cys His Val Glu Gly Glu Lys

515 520 525

Ile Tyr His Gln Ala Thr Glu Val Glu Thr Cys Ile Ser Cys His Thr

530 535 540

Gln Glu Phe Ala Asp Gly Arg Gly Lys Pro His Val Ala Phe Ser His

545 550 555 560

Leu Ile His Asn Val His Asn Ala Asn Lys Ala Trp Gly Lys Asp Asn

565 570 575

Lys Ile Pro Thr Val Ala Gln Asn Ile Val Gln Asp Asn Cys Gln Val

580 585 590

Cys His Val Glu Ser Asp Met Leu Thr Glu Ala Lys Asn Trp Ser Arg

595 600 605

Ile Pro Thr Met Glu Val Cys Ser Ser Cys His Val Asp Ile Asp Phe

610 615 620

Ala Ala Gly Lys Gly His Ser Gln Gln Leu Asp Asn Ser Asn Cys Ile

625 630 635 640

Ala Cys His Asn Ser Asp Trp Thr Ala Glu Leu His Thr Ala Lys Thr

645 650 655

Thr Ala Thr Lys Asn Leu Ile Asn Gln Tyr Gly Ile Glu Thr Thr Ser

660 665 670

Thr Ile Asn Thr Glu Thr Lys Ala Ala Thr Ile Ser Val Gln Val Val

675 680 685

Asp Ala Asn Gly Thr Ala Val Asp Leu Lys Thr Ile Leu Pro Lys Val

690 695 700

Gln Arg Leu Glu Ile Ile Thr Asn Val Gly Pro Asn Asn Ala Thr Leu

705 710 715 720

Gly Tyr Ser Gly Lys Asp Ser Ile Phe Ala Ile Lys Asn Gly Ala Leu

725 730 735

Asp Pro Lys Ala Thr Ile Asn Asp Ala Gly Lys Leu Val Tyr Thr Thr

740 745 750

Thr Lys Asp Leu Lys Leu Gly Gln Asn Gly Ala Asp Ser Asp Thr Ala

755 760 765

Phe Ser Phe Val Gly Trp Ser Met Cys Ser Ser Glu Gly Lys Phe Val

770 775 780

Asp Cys Ala Asp Pro Ala Phe Asp Gly Val Asp Val Thr Lys Tyr Thr

785 790 795 800

Gly Met Lys Ala Asp Leu Ala Phe Ala Thr Leu Ser Gly Lys Ala Pro

805 810 815

Ser Thr Arg His Val Asp Ser Val Asn Met Thr Ala Cys Ala Asn Cys

820 825 830

His Thr Ala Glu Phe Glu Ile His Lys Gly Lys Gln His Ala Gly Phe

835 840 845

Val Met Thr Glu Gln Leu Ser His Thr Gln Asp Ala Asn Gly Lys Ala

850 855 860

Ile Val Gly Leu Asp Ala Cys Val Thr Cys His Thr Pro Asp Gly Thr

865 870 875 880

Tyr Ser Phe Ala Asn Arg Gly Ala Leu Glu Leu Lys Leu His Lys Lys

885 890 895

His Val Glu Asp Ala Tyr Gly Leu Ile Gly Gly Asn Cys Ala Ser Cys

900 905 910

His Ser Asp Phe Asn Leu Glu Ser Phe Lys Lys Lys Gly Ala Leu Asn

915 920 925

Thr Ala Ala Ala Ala Asp Lys Thr Gly Leu Tyr Ser Thr Pro Ile Thr

930 935 940

Ala Thr Cys Thr Thr Cys His Thr Val Gly Ser Gln Tyr Met Val His

945 950 955 960

Thr Lys Glu Thr Leu Glu Ser Phe Gly Ala Val Val Asp Gly Thr Lys

965 970 975

Asp Asp Ala Thr Ser Ala Ala Gln Ser Glu Thr Cys Phe Tyr Cys His

980 985 990

Thr Pro Thr Val Ala Asp His Thr Lys Val Lys Met Met Lys Phe Lys

995 1000 1005

Leu Asn Leu Ile Thr Leu Ala Leu Leu Ala Asn Thr Gly Leu Ala Val

1010 1015 1020

Ala Ala Asp Gly Tyr Gly Leu Ala Asn Ala Asn Thr Glu Lys Val Lys

1025 1030 1035 1040

Leu Ser Ala Trp Ser Cys Lys Gly Cys Val Val Glu Thr Gly Thr Ser

1045 1050 1055

Gly Thr Val Gly Val Gly Val Gly Tyr Asn Ser Glu Glu Asp Ile Arg

1060 1065 1070

Ser Ala Asn Ala Phe Gly Thr Ser Asn Glu Val Ala Gly Lys Phe Asp

1075 1080 1085

Ala Asp Leu Asn Phe Lys Gly Glu Lys Gly Tyr Arg Ala Ser Val Asp

1090 1095 1100

Ala Tyr Gln Leu Gly Met Asp Gly Gly Arg Leu Asp Val Asn Ala Gly

1105 1110 1115 1120

Lys Gln Gly Gln Tyr Asn Val Asn Val Asn Tyr Arg Gln Ile Ala Thr

1125 1130 1135

Tyr Asp Ser Asn Ser Ala Leu Ser Pro Tyr Ala Gly Ile Gly Gly Asn

1140 1145 1150

Asn Leu Thr Leu Pro Asp Asn Trp Ile Thr Ala Gly Ser Ser Asn Gln

1155 1160 1165

Met Pro Leu Leu Met Asp Ser Leu Asn Ala Leu Glu Leu Ser Leu Lys

1170 1175 1180

Arg Glu Arg Thr Gly Leu Gly Phe Glu Tyr Gln Gly Glu Ser Leu Trp

1185 1190 1195 1200

Ser Thr Tyr Val Asn Tyr Met Arg Glu Glu Lys Thr Gly Leu Lys Gln

1205 1210 1215

Ala Ser Gly Ser Phe Phe Asn Gln Ser Met Met Leu Ala Glu Pro Val

1220 1225 1230

Asp Tyr Thr Thr Asp Thr Ile Glu Ala Gly Val Lys Leu Lys Gly Asp

1235 1240 1245

Arg Trp Phe Thr Ala Leu Ser Tyr Asn Gly Ser Ile Phe Lys Asn Glu

1250 1255 1260

Tyr Asn Gln Leu Asp Phe Glu Asn Ala Phe Asn Pro Thr Phe Gly Ala

1265 1270 1275 1280

Gln Thr Gln Gly Thr Met Ala Leu Asp Pro Asp Asn Gln Ser His Thr

1285 1290 1295

Val Ser Leu Met Gly Gln Tyr Asn Asp Gly Ser Asn Ala Leu Ser Gly

1300 1305 1310

Arg Ile Leu Thr Gly Gln Met Ser Gln Asp Gln Ala Leu Val Thr Asp

1315 1320 1325

Asn Tyr Arg Tyr Ala Asn Gln Leu Asn Thr Asp Ala Val Asp Ala Lys

1330 1335 1340

Val Asp Leu Leu Gly Met Asn Leu Lys Val Val Ser Lys Val Ser Asn

1345 1350 1355 1360

Asp Leu Arg Leu Thr Gly Ser Tyr Asp Tyr Tyr Asp Arg Asp Asn Asn

1365 1370 1375

Thr Gln Val Glu Glu Trp Thr Gln Ile Ser Ile Asn Asn Val Asn Gly

1380 1385 1390

Lys Val Ala Tyr Asn Thr Pro Tyr Asp Asn Arg Thr Gln Arg Phe Lys

1395 1400 1405

Val Ala Ala Asp Tyr Arg Ile Thr Arg Asp Ile Lys Leu Asp Gly Gly

1410 1415 1420

Tyr Asp Phe Lys Arg Asp Gln Arg Asp Tyr Gln Asp Arg Glu Thr Thr

1425 1430 1435 1440

Asp Glu Asn Thr Val Trp Ala Arg Leu Arg Val Asn Ser Phe Asp Thr

1445 1450 1455

Trp Asp Met Trp Val Lys Gly Ser Tyr Gly Asn Arg Asp Gly Ser Gln

1460 1465 1470

Tyr Gln Ala Ser Glu Trp Thr Ser Ser Glu Thr Asn Ser Leu Leu Arg

1475 1480 1485

Lys Tyr Asn Leu Ala Asp Arg Asp Arg Thr Gln Val Glu Ala Arg Ile

1490 1495 1500

Thr His Ser Pro Leu Glu Ser Leu Thr Ile Asp Val Gly Ala Arg Tyr

1505 1510 1515 1520

Ala Leu Asp Asp Tyr Thr Asp Thr Val Ile Gly Leu Thr Glu Ser Lys

1525 1530 1535

Asp Thr Ser Tyr Asp Ala Asn Ile Ser Tyr Met Ile Thr Ala Asp Leu

1540 1545 1550

Leu Ala Thr Ala Phe Tyr Asn Tyr Gln Thr Ile Glu Ser Glu Gln Ala

1555 1560 1565

Gly Ser Ser Asn Tyr Ser Thr Pro Thr Trp Thr Gly Phe Ile Glu Asp

1570 1575 1580

Gln Val Asp Val Val Gly Ala Gly Ile Ser Tyr Asn Asn Leu Leu Glu

1585 1590 1595 1600

Asn Lys Leu Arg Leu Gly Leu Asp Tyr Thr Tyr Ser Asn Ser Asp Ser

1605 1610 1615

Asn Thr Gln Val Arg Gln Gly Ile Thr Gly Asp Tyr Gly Asp Tyr Phe

1620 1625 1630

Ala Lys Val His Asn Ile Asn Leu Tyr Ala Gln Tyr Gln Ala Thr Glu

1635 1640 1645

Lys Leu Ala Leu Arg Phe Asp Tyr Lys Ile Glu Asn Tyr Lys Asp Asn

1650 1655 1660

Asp Ala Ala Asn Asp Ile Ala Val Asp Gly Ile Trp Asn Val Val Gly

1665 1670 1675 1680

Phe Gly Ser Asn Ser His Asp Tyr Thr Ala Gln Met Leu Met Leu Ser

1685 1690 1695

Met Ser Tyr Lys Leu

1700

<210> 3

<211> 572

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gtgtattttt taataaatta ttttacttat tgaaatgtat tattttctaa tgtcataccc 60

tggtcaaaac cgttcgtttt tgagactaga attttatgcc ctacttactt cttttatttt 120

cattcaaata tttgcttgca tgatgagtcg aaaatggtta taatacactc aaataaatat 180

ttgaatgaag atgggatgat aatatgaaaa agaaagatac ttgtgaaatt ttttgttatg 240

acgaagaaaa ggttaatcga atacaagggg atttacaaac agttgatatt tctggtgtta 300

gccaaatttt aaaggctatt gccgatgaaa atagagcaaa aattacttac gctctgtgtc 360

aggatgaaga gttgtgtgtt tgtgatatag caaatatctt aggtgttacg atagcaaatg 420

catctcatca tttacgtacg ctttataagc aaggggtggt caactttaga aaagaaggaa 480

aactagcttt atattcttta ggtgatgaac atatcaggca gataatgatg atcgccctag 540

cacataagaa agaagtgaag gtcaatgtct ga 572

<210> 4

<211> 1716

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atgtcttcca tgacaacaac tgataataaa gcctttttga atgaacttgc tcgtctggtg 60

ggttcttcac acctgctcac cgatcccgca aaaacggccc gctatcgcaa gggcttccgt 120

tctggtcagg gcgacgcgct ggctgtcgtt ttccctggct cactactaga attgtggcgg 180

gtgctgaaag cctgcgtcac cgccgacaaa attattctga tgcaggccgc caatacaggc 240

ctgaccgaag gatcgacgcc aaacggtaac gattatgatc gcgatgtcgt tatcatcagc 300

accctgcgtc tcgacaagct gcacgttctt ggcaagggcg aacaggtgct ggcctatccg 360

ggcaccacgc tctattcgct ggaaaaagcc ctcaaaccgc tgggacgcga accgcactca 420

gtgattggat catcgtgtat aggcgcatcg gtcatcggcg gtatttgtaa caactccggc 480

ggctcgctgg tgcaacgtgg cccggcgtat accgaaatgt cgttattcgc gcgtataaat 540

gaagacggca aactgacgct ggtgaaccat ctggggattg atctgggcga aacgccggag 600

cagatcctta gcaagctgga tgatgatcgc atcaaagatg acgatgtgcg tcacgatggt 660

cgtcacgccc acgattatga ctatgtccac cgcgttcgtg atattgaagc cgacacgccc 720

gcacgttata acgccgatcc tgatcggtta tttgaatctt ctggttgcgc cgggaagctg 780

gcggtctttg cagtacgtct tgataccttc gaagcggaaa aaaatcagca ggtgttttat 840

atcggcacca accagccgga agtgctgacc gaaatccgcc gtcatattct ggctaacttc 900

gaaaatctgc cggttgccgg ggaatatatg caccgggata tctacgatat tgcggaaaaa 960

tacggcaaag acaccttcct gatgattgat aagttaggca ccgacaagat gccgttcttc 1020

tttaatctca agggacgcac cgatgcgatg ctggagaaag tgaaattctt ccgtccgcat 1080

tttactgacc gtgcgatgca aaaattcggt cacctgttcc ccagccattt accgccgcgc 1140

atgaaaaact ggcgcgataa atacgagcat catctgctgt taaaaatggc gggcgatggc 1200

gtgggcgaag ccaaatcgtg gctggtggat tatttcaaac aggccgaagg cgatttcttt 1260

gtctgtacgc cggaggaagg cagcaaagcg tttttacacc gtttcgccgc tgcgggcgca 1320

gcaattcgtt atcaggcggt gcattccgat gaagtcgaag acattctggc gttggatatc 1380

gctctgcggc gtaacgacac cgagtggtat gagcatttac cgccggagat cgacagccag 1440

ctggtgcaca agctctatta cggccatttt atgtgctatg tcttccatca ggattacata 1500

gtgaaaaaag gcgtggatgt gcatgcgtta aaagaacaga tgctggaact gctacagcag 1560

cgcggcgcgc agtaccctgc cgagcataac gtcggtcatt tgtataaagc accggagacg 1620

ttgcagaagt tctatcgcga gaacgatccg accaacagca tgaatccggg gatcggtaaa 1680

accagtaaac ggaaaaactg gcaggaagtg gagtaa 1716

<210> 5

<211> 571

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Met Ser Ser Met Thr Thr Thr Asp Asn Lys Ala Phe Leu Asn Glu Leu

1 5 10 15

Ala Arg Leu Val Gly Ser Ser His Leu Leu Thr Asp Pro Ala Lys Thr

20 25 30

Ala Arg Tyr Arg Lys Gly Phe Arg Ser Gly Gln Gly Asp Ala Leu Ala

35 40 45

Val Val Phe Pro Gly Ser Leu Leu Glu Leu Trp Arg Val Leu Lys Ala

50 55 60

Cys Val Thr Ala Asp Lys Ile Ile Leu Met Gln Ala Ala Asn Thr Gly

65 70 75 80

Leu Thr Glu Gly Ser Thr Pro Asn Gly Asn Asp Tyr Asp Arg Asp Val

85 90 95

Val Ile Ile Ser Thr Leu Arg Leu Asp Lys Leu His Val Leu Gly Lys

100 105 110

Gly Glu Gln Val Leu Ala Tyr Pro Gly Thr Thr Leu Tyr Ser Leu Glu

115 120 125

Lys Ala Leu Lys Pro Leu Gly Arg Glu Pro His Ser Val Ile Gly Ser

130 135 140

Ser Cys Ile Gly Ala Ser Val Ile Gly Gly Ile Cys Asn Asn Ser Gly

145 150 155 160

Gly Ser Leu Val Gln Arg Gly Pro Ala Tyr Thr Glu Met Ser Leu Phe

165 170 175

Ala Arg Ile Asn Glu Asp Gly Lys Leu Thr Leu Val Asn His Leu Gly

180 185 190

Ile Asp Leu Gly Glu Thr Pro Glu Gln Ile Leu Ser Lys Leu Asp Asp

195 200 205

Asp Arg Ile Lys Asp Asp Asp Val Arg His Asp Gly Arg His Ala His

210 215 220

Asp Tyr Asp Tyr Val His Arg Val Arg Asp Ile Glu Ala Asp Thr Pro

225 230 235 240

Ala Arg Tyr Asn Ala Asp Pro Asp Arg Leu Phe Glu Ser Ser Gly Cys

245 250 255

Ala Gly Lys Leu Ala Val Phe Ala Val Arg Leu Asp Thr Phe Glu Ala

260 265 270

Glu Lys Asn Gln Gln Val Phe Tyr Ile Gly Thr Asn Gln Pro Glu Val

275 280 285

Leu Thr Glu Ile Arg Arg His Ile Leu Ala Asn Phe Glu Asn Leu Pro

290 295 300

Val Ala Gly Glu Tyr Met His Arg Asp Ile Tyr Asp Ile Ala Glu Lys

305 310 315 320

Tyr Gly Lys Asp Thr Phe Leu Met Ile Asp Lys Leu Gly Thr Asp Lys

325 330 335

Met Pro Phe Phe Phe Asn Leu Lys Gly Arg Thr Asp Ala Met Leu Glu

340 345 350

Lys Val Lys Phe Phe Arg Pro His Phe Thr Asp Arg Ala Met Gln Lys

355 360 365

Phe Gly His Leu Phe Pro Ser His Leu Pro Pro Arg Met Lys Asn Trp

370 375 380

Arg Asp Lys Tyr Glu His His Leu Leu Leu Lys Met Ala Gly Asp Gly

385 390 395 400

Val Gly Glu Ala Lys Ser Trp Leu Val Asp Tyr Phe Lys Gln Ala Glu

405 410 415

Gly Asp Phe Phe Val Cys Thr Pro Glu Glu Gly Ser Lys Ala Phe Leu

420 425 430

His Arg Phe Ala Ala Ala Gly Ala Ala Ile Arg Tyr Gln Ala Val His

435 440 445

Ser Asp Glu Val Glu Asp Ile Leu Ala Leu Asp Ile Ala Leu Arg Arg

450 455 460

Asn Asp Thr Glu Trp Tyr Glu His Leu Pro Pro Glu Ile Asp Ser Gln

465 470 475 480

Leu Val His Lys Leu Tyr Tyr Gly His Phe Met Cys Tyr Val Phe His

485 490 495

Gln Asp Tyr Ile Val Lys Lys Gly Val Asp Val His Ala Leu Lys Glu

500 505 510

Gln Met Leu Glu Leu Leu Gln Gln Arg Gly Ala Gln Tyr Pro Ala Glu

515 520 525

His Asn Val Gly His Leu Tyr Lys Ala Pro Glu Thr Leu Gln Lys Phe

530 535 540

Tyr Arg Glu Asn Asp Pro Thr Asn Ser Met Asn Pro Gly Ile Gly Lys

545 550 555 560

Thr Ser Lys Arg Lys Asn Trp Gln Glu Val Glu

565 570

<210> 6

<211> 1053

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atgaaactga agaacacctt aggcgttgtc atcggctcgc tggttgccgc ttcggcaatg 60

aacgcctttg cccagggcca gaactcggta gagatcgaag ccttcggcaa gcgctacttc 120

accgacagcg ttcgcaacat gaagaacgcg gacctgtacg gcggctcgat cggttacttc 180

ctgaccgacg acgtcgagct ggcgctgtcc tacggtgagt accatgacgt tcgtggcacc 240

tacgaaaccg gcaacaagaa ggtccacggc aacctgacct ccctggacgc catctaccac 300

ttcggtaccc cgggcgtagg tctgcgtccg tacgtgtcgg ctggtctggc tcaccagaac 360

atcaccaaca tcaacagcga cagccaaggc cgtcagcaga tgaccatggc caacatcggc 420

gctggtctga agtactactt caccgagaac ttcttcgcca aggccagcct cgacggccag 480

tacggtctgg agaagcgtga caacggtcac cagggcgagt ggatggctgg cctgggcgtc 540

ggcttcaact tcggtggttc gaaagccgct ccggctccgg aaccggttgc cgacgtttgc 600

tccgactccg acaacgacgg cgtttgcgac aacgtcgaca agtgcccgga taccccggcc 660

aacgtcaccg ttgacgccaa cggctgcccg gctgtcgccg aagtcgtacg cgtacagctg 720

gacgtgaagt tcgacttcga caagtccaag gtcaaagaga acagctacgc tgacatcaag 780

aacctggctg acttcatgaa gcagtacccg tccacttcca ccaccgttga aggtcacacc 840

gactccgtcg gcaccgacgc ttacaaccag aagctgtccg agcgtcgtgc caacgccgtt 900

cgtgacgtac tggtcaacga gtacggtgta gaaggtggtc gcgtgaacgc tgttggttac 960

ggcgagtccc gcccggttgc cgacaacgcc accgctgaag gccgcgctat caaccgtcgc 1020

gttgaagccg aagtagaagc tgaagccaag taa 1053

<210> 7

<211> 350

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Met Lys Leu Lys Asn Thr Leu Gly Val Val Ile Gly Ser Leu Val Ala

1 5 10 15

Ala Ser Ala Met Asn Ala Phe Ala Gln Gly Gln Asn Ser Val Glu Ile

20 25 30

Glu Ala Phe Gly Lys Arg Tyr Phe Thr Asp Ser Val Arg Asn Met Lys

35 40 45

Asn Ala Asp Leu Tyr Gly Gly Ser Ile Gly Tyr Phe Leu Thr Asp Asp

50 55 60

Val Glu Leu Ala Leu Ser Tyr Gly Glu Tyr His Asp Val Arg Gly Thr

65 70 75 80

Tyr Glu Thr Gly Asn Lys Lys Val His Gly Asn Leu Thr Ser Leu Asp

85 90 95

Ala Ile Tyr His Phe Gly Thr Pro Gly Val Gly Leu Arg Pro Tyr Val

100 105 110

Ser Ala Gly Leu Ala His Gln Asn Ile Thr Asn Ile Asn Ser Asp Ser

115 120 125

Gln Gly Arg Gln Gln Met Thr Met Ala Asn Ile Gly Ala Gly Leu Lys

130 135 140

Tyr Tyr Phe Thr Glu Asn Phe Phe Ala Lys Ala Ser Leu Asp Gly Gln

145 150 155 160

Tyr Gly Leu Glu Lys Arg Asp Asn Gly His Gln Gly Glu Trp Met Ala

165 170 175

Gly Leu Gly Val Gly Phe Asn Phe Gly Gly Ser Lys Ala Ala Pro Ala

180 185 190

Pro Glu Pro Val Ala Asp Val Cys Ser Asp Ser Asp Asn Asp Gly Val

195 200 205

Cys Asp Asn Val Asp Lys Cys Pro Asp Thr Pro Ala Asn Val Thr Val

210 215 220

Asp Ala Asn Gly Cys Pro Ala Val Ala Glu Val Val Arg Val Gln Leu

225 230 235 240

Asp Val Lys Phe Asp Phe Asp Lys Ser Lys Val Lys Glu Asn Ser Tyr

245 250 255

Ala Asp Ile Lys Asn Leu Ala Asp Phe Met Lys Gln Tyr Pro Ser Thr

260 265 270

Ser Thr Thr Val Glu Gly His Thr Asp Ser Val Gly Thr Asp Ala Tyr

275 280 285

Asn Gln Lys Leu Ser Glu Arg Arg Ala Asn Ala Val Arg Asp Val Leu

290 295 300

Val Asn Glu Tyr Gly Val Glu Gly Gly Arg Val Asn Ala Val Gly Tyr

305 310 315 320

Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr Ala Glu Gly Arg Ala

325 330 335

Ile Asn Arg Arg Val Glu Ala Glu Val Glu Ala Glu Ala Lys

340 345 350

<210> 8

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

caaggagaaa aaaatgatga acgcacaaaa atc 33

<210> 9

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gccgcaagct tttagagttt gtaactcatg ctc 33

<210> 10

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ccgaattcgt gtatttttta ataaattatt ttac 34

<210> 11

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

catgaattct cagacattga ccttcacttc t 31

<210> 12

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

agccaagtaa tttccactcc ttgtggtggc 30

<210> 13

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gcatgcgata tcgagctcac tggtcgcgca gaacatcttt c 41

<210> 14

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

atgaattccc gggagagctc cgttatgatg gcgtcgctag 40

<210> 15

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tcagtttcat aaattacgga tggcagagta 30

<210> 16

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tccgtaattt atgaaactga agaacacctt ag 32

<210> 17

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

caaggagtgg aaattacttg gcttcagctt ctact 35

Claims

1. The submarine biosensor is characterized by comprising an electric signal element protein gene, a sensing element and a porin gene, and simultaneously knocking out an escherichia coli lactic dehydrogenase gene; the electric signal element protein gene is an mtrCAB gene, and the nucleotide sequence of the electric signal element protein gene is shown as SEQ ID NO. 1; the sensing element is cad gene, and the nucleotide sequence of the sensing element is shown as SEQ ID NO. 3; the porin gene is an oprF gene, and the nucleotide sequence of the porin gene is shown as SEQ ID NO. 6; the escherichia coli lactate dehydrogenase gene is an ldh gene, and the nucleotide sequence of the escherichia coli lactate dehydrogenase gene is shown as SEQ ID NO. 4; the submarine biosensor has the function of sensing metal cadmium ions Cd (II).

2. The method of manufacturing a submarine biosensor according to claim 1, comprising the steps of:

(1) Amplifying an electric signal element protein gene mtrCAB gene, and recovering and connecting with the double enzyme-digested plasmid fragments to obtain a recombinant plasmid p-mtrCAB; amplifying the Cad gene of the sensing element, and recovering and connecting the Cad gene with the single-restriction enzyme-digested recombinant plasmid p-mtrCAB to obtain the recombinant plasmid p-Cad-mtrCAB;

(2) Amplifying the upstream and downstream fragments of the ldh gene of the escherichia coli and the oprF gene of the porin gene, cloning the co-connected fragments on a suicide plasmid, and obtaining a recombinant plasmid transformed strain to obtain a recombinant strain;

(3) And (3) converting the recombinant plasmid p-Cad-mtrCAB in the step (1) into the recombinant strain in the step (2), screening positive clones, and obtaining the engineering strain which is the submarine microbial sensor finally.

3. Use of a submarine biosensor according to claim 1 for the real-time detection of metal Cd (ii) ions.

4. Use of a submarine biosensor according to claim 1 for the preparation of a detection device for real-time detection of submarines in the ocean.

5. The use according to claim 4, characterized in that the method of construction of the detection device is: placing a sealing ring between the anode chamber and the cathode chamber, clamping and assembling the anode chamber and the cathode chamber, and fixing anode materials in the anode chamber and the cathode chamber respectively by leads; inoculating an activated submarine microbial sensor in the anode chamber, and applying voltage to promote the formation of a biological film on the anode material; electrolyte is loaded in the cathode chamber.

6. The use according to claim 5, wherein the detection device detects the Cd (ii) ions released by the submarine in the ocean in real time and converts them into a variable electrical signal output, thereby achieving the purpose of detecting the submarine in the ocean in real time.

7. The use according to claim 6, wherein the concentration of Cd (ii) ions that can be sensed by the detection means is not less than 0.1 μmol/L.