CN114921396A

CN114921396A - Electricity-producing geobacillus and construction method and application thereof

Info

Publication number: CN114921396A
Application number: CN202210606200.6A
Authority: CN
Inventors: 蒋永光; 石良; 王志高
Original assignee: China University of Geosciences
Current assignee: China University of Geosciences
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-08-19
Anticipated expiration: 2042-05-31
Also published as: CN114921396B

Abstract

The invention provides a geobacillus for electricity generation and a construction method and application thereof. The invention obtains over-expression plasmid by Gibson seamless cloning of nano-wire protein coding gene and PAWP78, and then introduces the over-expression plasmid into strain PCA through electrotransformation to obtain the electric geobacter strain for over-expressing nano-wire protein, which comprises strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ. The strain of the invention carries out anaerobic fermentation in MFC for not less than 150 hours, the maximum output voltage reaches 469-577 mV,compared with a control strain PCA/PAWP78, the maximum power density is improved by 63 percent, and the maximum power density reaches 1392-1580 mW/m ² Compared with the control strain PCA/PAWP78, the strain is improved by 1.62-1.97 times. The nano-wire protein is verified to be capable of enhancing the extracellular electron transfer rate and improving the electricity output capability of the electrogenesis bacteria.

Description

Electricity-producing geobacillus and construction method and application thereof

Technical Field

The invention relates to the technical field of microbial fuel cells, in particular to a geobacillus for generating electricity, a construction method and application thereof.

Background

Microbial Fuel Cells (MFCs) are devices that convert chemical energy generated during the oxidation of organic substances into electrical energy by using the catalytic activity of microorganisms. The MFCs can be applied to the fields of biological power generation, sewage treatment, biological hydrogen production and the like, and a new method is provided for recycling energy and resources.

The electrogenic bacteria are bacteria with Extracellular Electron Transfer (EET) capability, and as the most important component in MFC, the strength of the electrogenic capability directly determines the performance of MFC.

Improving the EET efficiency of the electrogenic bacteria is one of the important ways to enhance the electricity output capability of the electrogenic bacteria. The current EET method for improving the electrogenesis bacteria mainly comprises the following steps: the expression of c-type cytochrome related to electron transfer and electron transfer mediator in cell membrane is promoted. Compared with the wild strain, the electric output of the modified engineering strain is obviously improved, but still at a lower level.

Disclosure of Invention

The invention aims to provide an electricity generating bacillus and a construction method and application thereof aiming at the defects in the prior art.

The first purpose of the invention is to provide an electrogenic geobacillus which is obtained by overexpression of a nano-conductive protein gene through Geobacter sulfurreduce PCA, wherein the nano-conductive protein gene comprises any one of gene PilA, gene OmcS, gene OmcT or gene OmcZ; the nucleotide sequence of the gene PilA is shown as SEQ ID NO. 1, the nucleotide sequence of the gene OmcS is shown as SEQ ID NO. 2, the nucleotide sequence of the gene OmcT is shown as SEQ ID NO. 3, and the nucleotide sequence of the gene OmcZ is shown as SEQ ID NO. 4.

The second object of the present invention is to provide a method for constructing the above-mentioned geobacillus for electrogenesis, which comprises the following steps

S1, amplifying the gene of the nanowire protein by taking the genome of the Geobacter sulfureatedcA as a template to obtain an amplified gene segment of the nanowire protein; the amplified nanowire protein gene segment comprises any one of an amplified gene PilA segment, an amplified gene OmcS segment, an amplified gene OmcT segment or an amplified gene OmcZ segment, wherein the nucleotide sequence of the gene PilA is shown as SEQ ID NO. 1, the nucleotide sequence of the gene OmcS is shown as SEQ ID NO. 2, the nucleotide sequence of the gene OmcT is shown as SEQ ID NO. 3, and the nucleotide sequence of the gene OmcZ is shown as SEQ ID NO. 4;

step S2, using the PAWP78 plasmid as a template, and using primers to perform PCR amplification on a linearized vector to obtain a linearized vector fragment PAWP 78;

step S3, cloning the amplified nano-lead protein gene segment obtained in the step S1 to the linearized vector segment PAWP78 obtained in the step S2 by a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP 78-nano-lead protein gene, wherein the vector PAWP 78-nano-lead protein gene comprises any one of a vector PAWP78-PilA, a vector PAWP78-OmcS, a vector PAWP78-OmcT or a vector PAWP 78-OmcZ;

step S4, transforming the vector PAWP 78-nanowire protein gene obtained in the step S3 into E.coli DH5 alpha, and obtaining an over-expression plasmid PAWP 78-nanowire protein gene through sequencing verification, wherein the over-expression plasmid PAWP 78-nanowire protein gene comprises any one of an over-expression plasmid PAWP78-PilA, an over-expression plasmid PAWP78-OmcS, an over-expression plasmid PAWP78-OmcT or an over-expression plasmid PAWP 78-OmcZ;

and step S5, electrically transforming the overexpression plasmid obtained in the step S4 into Geobacter sulfuridunduens PCA for culture to obtain the electrogenesis geobacillus, wherein the electrogenesis geobacillus comprises any one of the strain PCA/PilA, the strain PCA/OmcS, the strain PCA/OmcT or the strain PCA/OmcZ.

Further, in step S1, the primers used for amplifying the upstream fragment of the gene PilA are PilA-1 and PilA-2, the nucleotide sequence of the primer PilA-1 is shown as SEQ ID NO. 5, and the nucleotide sequence of the primer PilA-2 is shown as SEQ ID NO. 6; the primers used for amplifying the downstream segment of the gene PilA are PilA-3 and PilA-4, the nucleotide sequence of the primer PilA-1 is shown as SEQ ID NO. 7, and the nucleotide sequence of the primer PilA-2 is shown as SEQ ID NO. 8.

Further, in step S1, the primers used for amplifying the gene OmcS are OmcS-1 and OmcS-2, the nucleotide sequence of the primer OmcS-1 is shown in SEQ ID NO. 9, and the nucleotide sequence of the primer OmcS-2 is shown in SEQ ID NO. 10.

Further, in step S1, the primers used for amplifying the upstream fragment of gene OmcT are OmcS-1 and OmcT-1, the nucleotide sequence of primer OmcS-1 is shown in SEQ ID NO. 9, and the nucleotide sequence of primer OmcT-1 is shown in SEQ ID NO. 11; the primers used for amplifying the downstream segment of the gene OmcT are OmcT-2 and OmcT-3, the nucleotide sequence of the primer OmcT-2 is shown as SEQ ID NO. 12, and the nucleotide sequence of the primer OmcT-3 is shown as SEQ ID NO. 13.

Further, in step S1, the primers used for amplifying the gene OmcZ are OmcZ-1 and OmcZ-2, the nucleotide sequence of the primer OmcZ-1 is shown in SEQ ID NO. 14, and the nucleotide sequence of the primer OmcZ-2 is shown in SEQ ID NO. 15.

Further, in step S2, the primers for carrying out PCR amplification on the linearized vector include PAWP78-1 and PAWP78-2, wherein the nucleotide sequence of the PAWP78-1 is shown in SEQ ID NO. 16, and the nucleotide sequence of the PAWP78-2 is shown in SEQ ID NO. 17.

Further, in step S5, the culture is performed anaerobically at 30 ℃ using NBAF medium.

It is a third object of the present invention to provide use of the above-mentioned geobacillus for electricity generation in a microbial fuel cell MFC.

Further, the time for the anaerobic fermentation of the electrogenic bacillus in the MFC is not less than 150 hours.

The invention has the following beneficial effects:

the invention adopts a genetic engineering bacteria means to carry out over-expression on the nanowire protein gene to obtain the engineering strains of the geobacillus electrogenesis, the strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ, and the strain has obviously improved electrogenesis activity. The strain of the invention is subjected to anaerobic fermentation in MFC for not less than 150h, and the strains PCA/PilA and PCAThe maximum output voltages which can be reached by/OmcS, PCA/OmcT and PCA/OmcZ are 577mV, 533mV, 551mV and 469mV respectively, which are increased by 63%, 50%, 55% and 32% compared with the control strain PCA/PAWP78(355 mV); the maximum output power which can be realized by the strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ is 1580mW/m ² 、1502mW/m ² 、1481mW/m ² 1392mW/m ² Compared with the control strain PCA/PAWP78(532 mW/m) ² ) The improvement is 1.97 times, 1.82 times, 1.78 times and 1.62 times. The electric output capability of the electrogenesis geobacillus is obviously improved by the overexpression of the nanowire protein.

Drawings

FIG. 1 is a schematic diagram of the principle of Giboson seamless cloning according to the present invention;

FIG. 2 is a schematic diagram of the structure of an overexpression plasmid of the invention;

FIG. 3 is a comparative diagram of PCR verification of bacterial liquid of PCA engineering strain of thioredoxin transferred into plasmid;

FIG. 4 is a Western bolt comparison chart of the plasmid transferred thioredoxin PCA engineering strain;

FIG. 5 is a graph comparing the output voltage results of the strains PCA/PilA, PCA/OmcS, PCA/OmcT, PCA/OmcZ of the present invention with that of the control strain PCA/PAWP 78;

FIG. 6 is a graph comparing the results of electric power density produced by the strains PCA/PilA, PCA/OmcS, PCA/OmcT, PCA/OmcZ of the present invention with the control strain PCA/PAWP 78;

FIG. 7 is a graph comparing the results of MFC cyclic voltammetry for the strains PCA/PilA, PCA/OmcS, PCA/OmcT, PCA/OmcZ of the present invention with the control strain PCA/PAWP 78;

FIG. 8 is a graph comparing the results of AC impedance results of the strains PCA/PilA, PCA/OmcS, PCA/OmcT, PCA/OmcZ of the present invention with the control strain PCA/PAWP 78.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described below.

The original strain Geobacter sulfurreducens PCA used by the invention is a typical strain deposited by ATCC: ATCC 51573.

In the examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.

Description of the sources of the biological materials:

1. plasmid origin: it is commercially available.

Genomic template sources for Geobacter sulfurreducens PCA: using Kangji bacterial genome DNA kit, according to the instruction, taking 5mL of anaerobic cultured PCA bacterial liquid, obtaining 50uL of PCA genomic DNA.

3. Stock solution and culture medium preparation method

3.1 Core media stock: 7.6g of KCl and 4g of NH ₄ Cl and 1.38g NaH ₂ PO ₄ ·H ₂ O was added to a beaker having a volume of 1L, 1L of ultrapure water was added to the beaker, stirred well, and stored at 4 ℃.

3.2 Mg/Ca stock solution: adding 0.8g of CaCl ₂ ·2H2O、4g MgSO ₄ ·7H ₂ O was added to a beaker having a volume of 1L, 1L of ultrapure water was added to the beaker, stirred well, and stored at 4 ℃.

3.3 Trace Mineral stock solution: 950mL of ultrapure water was added to a 1L beaker, and 0.1g of MnCl was added ₂ 、0.5g FeSO ₄ ·7H ₂ O、0.17g CoCl ₂ ·6H ₂ O、0.1g ZnCl ₂ 、0.03g CuSO ₄ ·5H ₂ O、0.005g AlKSO ₄ ·12H ₂ O、0.005g H ₃ BO ₃ 、0.09g Na ₂ MoO ₄ 、0.05g NiCl ₂ 、0.02g Na ₂ WO ₄ ·2H ₂ O and 0.1g Na ₂ Se ₄ Adding into a beaker; stirring, adding ultrapure water to constant volume of 1L, and storing at 4 deg.C.

3.4 Vitamins stock: adding 950mL of ultrapure water into a 1L beaker, and adding 0.002g of biotin, 0.002g of folic acid, 0.01g of vitamin B6, 0.005g of riboflavin, 0.005g of vitamin B1, 0.005g of nicotinic acid, 0.005g of pantothenic acid, 0.0001g of vitamin B12, 0.005g of p-aminobenzoic acid and 0.005g of lipoic acid into the beaker; stirring, diluting to 1L, and storing at 4 deg.C in dark place.

3.5 NBFA liquid Medium: 850mL of ultrapure water was added to a 1L beaker; 4.64g (40mM) fumaric acid was added to the beaker; adjusting the pH value to 6.0-6.1 by NaOH (5M); adding 20mL of Core medium stock solution, 50mL of Mg/Ca stock solution and 10mL of Mineral stock solution into a beaker in sequence; 1.64g of anhydrous sodium acetate (20mM) was added to the beaker; adjusting pH to 6.8 with NaOH (5M), adding ultrapure water to constant volume of 1L, and adding 2g NaHCO ₃ (ii) a Packaging in anaerobic bottle, and adding CO ₂ /N ₂ (8:2) deoxidizing, sterilizing at 121 ℃ for 20min, and cooling to room temperature for use.

3.6 NBFA solid Medium: 850mL of ultrapure water was added to a 1L beaker; 4.64g (40mM) fumaric acid was added to the beaker; adjusting the pH value to 6.0-6.1 by NaOH (5M); adding 20mL of Core medium stock solution, 50mL of Mg/Ca stock solution and 10mL of Mineral stock solution into a beaker in sequence; 1.64g of anhydrous sodium acetate (20mM) was added to the beaker; adjusting pH to 6.8 with NaOH (5M), adding ultrapure water to constant volume of 1L, and adding 2g NaHCO ₃ (ii) a The culture medium is divided into 200mL to 250mL anaerobic bottles, 2g agar powder is added into each bottle, and CO is used ₂ /N ₂ (8:2), deoxidizing, sterilizing at 121 ℃, for 20min, cooling, storing at room temperature, dissolving in a high-pressure steam sterilization kettle at 121 ℃ for 5min before use, and pouring into a Coylab glove box.

3.7 EB buffer: 950mL of ultrapure water was added to a 1L beaker, and 0.24g of Hepes (1mM), 59.85g of Sucrose (175mM) and 0.095g of MgCl were added ₂ (1mM) was added to the beaker and the pH was adjusted to 7 with NaOH (1M).

Example 1

This example illustrates the construction of the geobacter aerogenes strain PCA/PilA. The specific process comprises the following steps:

step 1, amplifying a PilA gene by using a genome of Geobacter sulfureatedcans PCA as a template to obtain an amplified PilA gene fragment; the nucleotide sequence of the gene PilA is shown as SEQ ID NO. 1; the primers used for amplifying the upstream segment of the gene PilA are PilA-1 and PilA-2, the nucleotide sequence of the primer PilA-1 is shown as SEQ ID NO. 5, and the nucleotide sequence of the primer PilA-2 is shown as SEQ ID NO. 6; the primers used for amplifying the downstream segment of the gene PilA are PilA-3 and PilA-4, the nucleotide sequence of the primer PilA-1 is shown as SEQ ID NO. 7, and the nucleotide sequence of the primer PilA-2 is shown as SEQ ID NO. 8.

The reaction system of PCR is: mu.L of gold medal Mix (Hi-Fi DNA polymerase from Oncorhynchus organisms), 2. mu.L of forward primer (10. mu.M), 2. mu.L of reverse primer (10. mu.M), 1. mu.L of template DNA; the PCR reaction conditions are as follows:

the reaction system of Gibson is: 10 μ L of Gibson Mix (2X, NEB), 3uL of insert DNA, 2 μ L of vector fragment DNA, 5 μ L of deionized water. (the molar ratio of the DNA fragments was controlled at 1:1)

Reaction conditions for Gibson were: incubate at 50 ℃ for 30min, then store the sample at-20 ℃.

And 2, carrying out PCR amplification on the linearized vector by using the PAWP78 plasmid as a template and using primers to obtain a linearized vector fragment PAWP78, wherein the primers comprise PAWP78-1 and PAWP78-2, the nucleotide sequence of the PAWP78-1 is shown in SEQ ID NO. 16, and the nucleotide sequence of the PAWP78-2 is shown in SEQ ID NO. 17.

Step 3, cloning the amplified nanowire protein gene segment obtained in the step 1 to the linearized vector segment PAWP78 obtained in the step 2 by a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP 78-PilA;

step S4, transforming the vector PAWP78-PilA obtained in the step 3 into E.coli DH5 alpha, and obtaining an expression plasmid PAWP78-PilA through sequencing verification;

and 5, electrically converting the over-expression plasmid PAWP78-PilA obtained in the step 4 into Geobacter sulfurducens PCA for culture to obtain the geobacillus electrogenesis strain PCA/PilA. The specific process is as follows: inoculating 1mL of frozen PCA wild strain into 10mL of NBFA liquid culture medium in a Coylab anaerobic glove box, inoculating 5mL of bacterial liquid into 50mL of NBFA liquid culture medium when the bacterial liquid OD600 is equal to 0.3 after the bacterial liquid is cultured at 30 ℃, and inoculating 10mL of bacterial liquid into 100mL of NBFA liquid culture medium when the bacterial liquid OD600 is equal to 0.3 after the bacterial liquid is cultured at 30 ℃, and culturing until the bacterial strain grows until the OD600 is equal to 0.3; placing the bacterial liquid and the high-speed centrifugal tube in a Coylab anaerobic glove box on ice, and centrifuging the bacterial liquid in the high-speed centrifugal tube at 4300 Xg and 4 ℃ for 10 min. Removing supernatant on ice, and resuspending the thalli by PCA EB buffer with the same volume as the supernatant; repeating the operation of the previous step on ice, cleaning the thalli twice, and then resuspending 100mL of centrifugally collected thalli by 200 mu L of PCA EB buffer to obtain competent cells; adding 1000ng of plasmid into 100 mu L of PCA competent cells, gently mixing uniformly, and then transferring the mixture into an electric shock cup; the electric rotating instrument is set to be 1.47KV for electric rotation; after electrotransfer, adding 1mL of NBFA liquid culture medium into an electric shock cup, sucking all liquid in the electric shock cup by using a syringe, adding the liquid into 10mL of NBFA liquid culture medium, and performing anaerobic resuscitation culture at 30 ℃ for 24 hours; concentrating the thallus in the last step to 100 mu L, then coating the thallus on an NBFA solid plate containing Kana (working concentration 200 mu g/mL), and carrying out anaerobic culture at 30 ℃; when a single bacterial colony grows on the NBFA solid plate, selecting the single bacterial colony in a Coylab anaerobic glove box to be cultured in an NBFA liquid culture medium containing Kana (working concentration 200 mug/mL) in an anaerobic manner at the temperature of 30 ℃; and (3) after red bacterial liquid grows out of the culture medium, centrifuging 500 mu L of bacterial liquid, removing supernatant, resuspending the bacteria by using 20 mu L of sterilized deionized water, and performing PCR verification by using the resuspended bacterial liquid as a template to obtain the bacterial strain PAWP78-PilA into which the plasmid is correctly introduced.

Example 2

This example illustrates the construction of the E.coli strain PCA/OmcS. The specific process comprises the following steps:

step 1, amplifying a PilA gene by taking a genome of Geobacter sulfurducencens PCA as a template to obtain an amplified OmcS gene fragment; the nucleotide sequence of the gene OmcS is shown as SEQ ID NO. 2; the primers used for amplifying the gene OmcS are OmcS-1 and OmcS-2, the nucleotide sequence of the primer OmcS-1 is shown in SEQ ID NO. 9, and the nucleotide sequence of the primer OmcS-2 is shown in SEQ ID NO. 10.

And 2, carrying out PCR amplification on the linearized vector by using a primer by using the PAWP78 plasmid as a template to obtain a linearized vector fragment PAWP78, wherein the primer comprises PAWP78-1 and PAWP78-2, the nucleotide sequence of the PAWP78-1 is shown as SEQ ID NO. 16, and the nucleotide sequence of the PAWP78-2 is shown as SEQ ID NO. 17.

Step 3, cloning the amplified nanowire protein gene segment obtained in the step 1 to the linearized vector segment PAWP78 obtained in the step 2 by a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP 78-OmcS;

step S4, transforming the vector PAWP78-OmcS obtained in the step 3 into E.coli DH5 alpha, and obtaining an expression plasmid PAWP78-OmcS through sequencing verification;

and step 5, electrically converting the over-expression plasmid PAWP78-OmcS obtained in the step 4 into Geobacter sulfurberry PCA for culture to obtain the geobacillus electrogenesis strain PCA/OmcS. The procedure is as in example 1.

Example 3

This example illustrates the construction of the E.coli strain PCA/OmcT. The specific process comprises the following steps:

step 1, amplifying an OmcT gene by using a genome of Geobacter sulfureatedcA as a template to obtain an amplified OmcT gene fragment; the nucleotide sequence of the gene OmcT is shown in SEQ ID NO. 3; the primers used for amplifying the upstream fragment of the gene OmcT are OmcS-1 and OmcT-1, the nucleotide sequence of the primer OmcS-1 is shown as SEQ ID NO. 9, and the nucleotide sequence of the primer OmcT-1 is shown as SEQ ID NO. 11; the primers used for amplifying the downstream fragment of gene OmcT are OmcT-2 and OmcT-3, the nucleotide sequence of primer OmcT-2 is shown in SEQ ID NO. 12, and the nucleotide sequence of primer OmcT-3 is shown in SEQ ID NO. 13.

Step 3, cloning the amplified nanowire protein gene segment obtained in the step 1 to the linearized vector segment PAWP78 obtained in the step 2 by a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP 78-OmcT;

step S4, transforming the vector PAWP78-OmcT obtained in the step 3 into E.coli DH5 alpha, and obtaining an expression plasmid PAWP78-OmcT through sequencing verification;

and 5, electrically transforming the over-expression plasmid PAWP78-OmcT obtained in the step 4 into Geobacter sulfureatedcA for culture to obtain the geobacillus electrogenesis strain PCA/OmcT. The procedure is as in example 1.

Example 4

This example illustrates the construction of the E.coli strain PCA/OmcZ. The specific process comprises the following steps:

step 1, amplifying an OmcZ gene by using a genome of Geobacter sulfureatedcA as a template to obtain an amplified OmcZ gene fragment; the nucleotide sequence of the gene OmcZ is shown in SEQ ID NO. 4; the primers used for amplifying the gene OmcZ are OmcZ-1 and OmcZ-2, the nucleotide sequence of the primer OmcZ-1 is shown in SEQ ID NO. 14, and the nucleotide sequence of the primer OmcZ-2 is shown in SEQ ID NO. 15.

Step 3, cloning the amplified nano-wire protein gene segment obtained in the step 1 to the linearized vector segment PAWP78 obtained in the step 2 by a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP 78-OmcZ;

step S4, transforming the vector PAWP78-OmcZ obtained in the step 3 into E.coli DH5 alpha, and obtaining an expression plasmid PAWP78-OmcZ through sequencing verification;

and 5, electrically converting the over-expression plasmid PAWP78-OmcZ obtained in the step 4 into Geobacter sulfurfurruducens PCA for culture to obtain the geobacillus electrogenesis strain PCA/OmcZ. The procedure is as in example 1.

Comparative example 1

This example illustrates the construction of the non-overexpressing strain E.coli PCA/PAWP 78.

The specific process comprises the following steps:

step 1, using a PAWP78 plasmid as a template, and performing PCR amplification on a linearized vector by using primers to obtain a linearized vector fragment PAWP78, wherein the primers comprise PAWP78-1 and PAWP78-2, the nucleotide sequence of the PAWP78-1 is shown in SEQ ID NO. 16, and the nucleotide sequence of the PAWP78-2 is shown in SEQ ID NO. 17.

And 2, transforming the vector PAWP78 obtained in the step 1 into E.coli DH5 alpha, and performing sequencing verification and electric transformation to Geobacter sulfureaterucens PCA for culture to obtain the geobacillus electrogenesis strain PCA/PAWP 78. The procedure is as in example 1.

As shown in fig. 1: the reaction process for Gibson ligation is roughly divided into four steps: firstly, cutting DNA exonclease from the 5' end of DNA to form a cohesive end; then annealing and connecting homologous sequences in the cohesive end; then the DNA polymerase extends in the 5 'to 3' direction of the DNA; finally, DNA ligase closes the gap.

As shown in FIG. 2, which is a map of the PAWP78 plasmid, PAWP78 is an IncP type plasmid containing a Kan resistance gene, and the position indicated by an arrow in the figure is an insertion site of a nanowire gene.

FIG. 3 shows PCR verified gel diagrams of the bacterial solutions of PCA/PAWP78 and PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ, wherein the primers for verifying the insertion sequences on the PAWP78 plasmid are PAWP78-3 and PAWP78-4, the nucleotide sequence of PAWP78-3 is shown in SEQ ID NO. 18, and the nucleotide sequence of PAWP78-4 is shown in SEQ ID NO. 19. The PCR result proves that the engineering strain constructed by the invention is transferred with a correct expression vector.

As shown in FIG. 4, Western bolt verification charts of PCA/PAWP78 and PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ are shown, the engineered strains all express the nanowire proteins, the corresponding bands of the strains highly expressing the nanowire proteins under the same loading are thicker, and the protein expression is actually improved.

In order to better illustrate that the engineering bacteria constructed by the method have better electric output capacity, the following microbiological electrochemical characterization research is carried out on examples 1-5 and comparative example 1.

Pre-preparation of MFC

(1) Pretreatment of the carbon cloth electrode: rinsed overnight with 1M hydrochloric acid, soaked overnight with pure acetone (sealed to prevent acetone evaporation) and then rinsed with sterile deionized water, oven dried and stored until use. Cutting the processed carbon cloth according to the anode and the cathode, wherein the sizes of the cut carbon cloth are as follows: 1cm by 1cm and 2.5cm by 3 cm.

(2) Pretreatment of a Nafion 117 proton exchange membrane: cutting a 20 cm-20 cm proton exchange membrane into a size of 10 cm-10 cm, soaking the cut proton exchange membrane in HCl (1M) overnight for ultraviolet irradiation, and repeatedly washing with sterilized deionized water.

(3) Pretreatment of the MFC reactor: fixing the cut carbon cloth on the lead by using AB glue, then penetrating the lead through a hole reserved on the rubber plug to ensure that the carbon cloth is suspended in the reactor, and sterilizing at high temperature and high pressure (121 ℃, 20 min). After the reactor is cooled, the treated proton exchange membrane is put into the middle of the reactor in a biological safety cabinet, and the reactor is fixed by a clamp.

(4) Preparing an anode culture solution: adding 850mL of ultrapure water into a 1L beaker; adding 20mL of Core medium stock solution, 50mL of Mg/Ca stock solution, 10mL of Trace Mineral stock solution and 1mL of Vitamins stock solution into a beaker in sequence; 1.64g of anhydrous sodium acetate (20mM) was added to the beaker; adjusting pH to 6.8 with NaOH (1M), adding ultrapure water to constant volume of 1L, and adding 2g NaHCO ₃ (ii) a Packaging in anaerobic bottle, and adding CO ₂ /N ₂ (8:2) deoxidizing, sterilizing at 121 ℃ for 20min, and cooling to room temperature for later use.

(5) Preparing a catholyte: 950mL of ultrapure water was added to a 1L beaker, and 16.462g K was added ₃ [Fe(CN) ₆ ]、8.709g K ₂ HPO ₄ And 6.8045g KH ₂ PO ₄ Adding into a beaker; stirring, adding ultrapure water to constant volume of 1L, packaging into anaerobic bottle, and adding pure N ₂ Oxygen removal is performed.

2. Assembly of MFCs

The well-constructed electrogenic PCA engineering strain is cultured to OD by using NBFA liquid culture medium containing Kana (working concentration 200 mug/mL) ₆₀₀ Taking 50mL of bacterial liquid into a high-speed centrifuge tube in a Coylab anaerobic glove box, centrifuging the bacterial liquid at 4300rpm and 4 ℃ for 10min, removing supernatant in the Coylab anaerobic glove box, and resuspending the bacterial liquid by using 140mL of anode culture solution; opening the cover of the anode chamber of the MFC reactor, removing oxygen in the outlet of a transfer chamber of a Coylab anaerobic glove box, then placing the MFC reactor into the Coylab anaerobic glove box, adding 140mL of resuspended bacterial liquid into the anode chamber of the reactor, adding Kana (working concentration 200 mu g/mL), tightly covering the cover of the anode chamber, and taking the reactor out of the Coylab anaerobic glove box; the cover of the cathode compartment of the MFC reactor was opened on the outside, and 150mL of the cathode was addedAnd (3) covering the anode chamber with the electrode electrolyte, and sealing all hole gaps on the cover with a hot melt adhesive gun. Connecting the lead wires of the anode chamber and the cathode chamber of the MFC reactor by using an external resistor of 2k omega, and after the MFC is assembled, putting the assembled MFC reactor into a constant-temperature incubator at 30 ℃ for culturing.

3. Electrochemical analysis of microorganisms

Real-time monitoring of output voltage: the multi-channel voltage detector PS2024V was connected to a computer, and the positive and negative electrodes of each channel were connected to the external resistance of the MFC, and voltage data was recorded every 30 minutes on the computer.

The operation flow of the measurement of the linear voltammetry (LSV) curve is as follows:

(1) and when the output voltage of the MFC reaches the maximum value and is stable, the externally-connected 2k omega resistor is removed, and the MFC is placed still for discharging for 1 h.

(2) The discharged MFC is connected with a constant potential rectifier of CHI1000C Chenghua, a Working Electrode (WE) is connected with a lead of an anode chamber, a Counter Electrode (CE) is connected with a lead of a cathode chamber, and a Reference Electrode (RE) is connected with a lead of the cathode chamber.

(3) Scanning parameters of the potentiostat are set as follows:

initial potential (V): -0.8V

End potential (V): -0.1V

Initial scan polarization: positive

Scanning rate (V/s): 0.0001

Sampling time (V): 0.001

Rest time(s): 30

Sensitivity (A/V): 1.e ⁰⁰⁴

(4) And after the scanning is finished, storing the data into the computer.

(5) The voltage V value and the current A value in the data are substituted into the formula to obtain the current density and the power density: current Density (mA/m) ² )＝(A×1000)/(1cm ² X 10000), power density (mW/m) ² )＝(V×A)/(1cm ² X 10000 × 1000); plotting with current density as abscissa and power density as ordinate to obtain power density curve, and plotting with current density as abscissa and voltage as ordinate to obtain polar curveAnd (4) forming a curve.

The results are shown in FIG. 5, which is a graph of the MFC output voltage with time for the control strain (PCA/PAWP78) and the engineered strains (PCA/PilA, PCA/OmcS, PCA/OmcT, and PCA/OmcZ), in which the voltage peak of the engineered strain appears and remains at a peak for a while between 100 hours and 180 hours, the voltage peak of the control strain appears and then starts to decrease at 150 hours, and the voltage peak of the engineered strain is 32% to 63% higher than that of the control strain. The engineering strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ have obviously improved electrogenesis activity. The maximum output voltages which can be respectively reached by the engineering strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ are 577mV, 533mV, 551mV and 469mV, which are respectively improved by 63 percent, 50 percent, 55 percent and 32 percent compared with the PCA/PAWP78(355mV) of a control strain.

As shown in FIG. 6, the engineering strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ can achieve the maximum output power of 1580mW/m ² 、1502mW/m ² 、1481mW/m ² 1392mW/m ² Compared with the control strain PCA/PAWP78(532 mW/m) ² ) The strains are improved by 1.97 times, 1.82 times, 1.78 times and 1.62 times for 150 hours of anaerobic fermentation in MFC, the maximum output voltage reaches 469-577 mV, compared with a control strain PCA/PAWP78, the maximum output voltage is improved by 63%, and the maximum power density reaches 1392-1580 mW/m ² Compared with the control strain PCA/PAWP78, the strain is improved by 1.62-1.97 times.

As shown in FIG. 7, the control strain PCA/PAWP78 showed no redox back peak, while all of the engineered strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ showed significant redox peaks, indicating an increase in conductive substances in the engineered strain MFC. And the peak value of the current density of the engineering strain is higher than that of the control strain, which shows that the catalytic current of the MFC of the engineering strain is also higher than that of the control strain. It was also shown that the MFC constructed with the engineered strain of the present invention had increased conductive material in the anode compartment and the catalytic current of the cells became high.

As shown in FIG. 8, the radii of curves for the engineered strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ were all smaller than those for the control strain PCA/PAWP78, indicating that the inner resistance of MFC in the engineered strain was smaller than that of the control strain PCA/PAWP 78. The alternating current impedance result of the battery is a complete semicircle under ideal conditions, the radius of the semicircle is equal to the resistance of the battery, and the smaller the radius, the smaller the internal resistance of the battery is. It was also shown that the internal cell resistance of MFC constructed with the engineered strain of the invention was significantly less than the control strain PCA/PAWP 78.

In conclusion, the engineering strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ constructed by the invention can enhance the extracellular electron transfer rate by over-expressing the nanowire protein, and the engineering strains constructed by the invention improve the electricity generation and output capacity.

The above is not relevant and is applicable to the prior art.

While certain specific embodiments of the present invention have been described in detail by way of illustration, it will be understood by those skilled in the art that the foregoing is illustrative only and is not limiting of the scope of the invention, as various modifications or additions may be made to the specific embodiments described and substituted in a similar manner by those skilled in the art without departing from the scope of the invention as defined in the appending claims. It should be understood by those skilled in the art that any modifications, equivalents, improvements and the like made to the above embodiments in accordance with the technical spirit of the present invention shall be included in the scope of the present invention.

Sequence listing

<110> geological university in China (Wuhan)

<120> geobacillus electrogenesis and construction method and application thereof

<141> 2022-05-28

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 462

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of PilA

<400> 1

agaggagcca gtgacgaaaa tcgtcagaca caagtgacga aataggtggt gaaggggtag 60

gttgaagcgg ttgcgttgtg taacgtgctg aaattgtagc catgtataag ttggttcggc 120

ttttgctatg ttcacgataa cgtttaagga ttaaacggat aattggccaa ttacccccat 180

accccaacac aagcagcaaa aagaagaaag gagacactta tgcttcagaa actcagaaac 240

aggaaaggtt tcacccttat cgagctgctg atcgtcgttg cgatcatcgg tattctcgct 300

gcaattgcga ttccgcagtt ctcggcgtat cgtgtcaagg cgtacaacag cgcggcgtca 360

agcgacttga gaaacctgaa gactgctctt gagtccgcat ttgctgatga tcaaacctat 420

ccgcccgaaa gttaattgat taaatacata ctggaggaaa cc 462

<210> 2

<211> 1654

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of OmcS

<400> 2

aaaatacctc ctggcgatct gtgtcggact ccggttggcg gtaaggaggt gccggggccc 60

cctttcaggc ggaccccggc ggaatcacac aacatcagat tgtggcaaga ttagtccttg 120

gcgtggcact tgttgcagag agcgcgctgg taaggagcaa acttgtcagc agtacggcca 180

tagtaggcag cggtcatctc gttgaccgaa cggccctgga gggacgaggt attggggtcc 240

gtaccataga tcgagttgcc ggaagcatcg gcgatggtcg tgaactcgta tgccaggttg 300

aagcgggtca tgctgtcgaa gcccgaagcg tgagcgcggt ggcaggagag gcagttcacg 360

ttgctggtgg cgtcggcgcc ggtcagcgcg gtgtcgtcga tcttggcgtg acccttgagc 420

acggtgtagt cggcagtgcc ttcctcgaac ggagcaaggg acaggtaggc ggaagcctgg 480

gtaccggtca ggtcgccgga cttcttgtag gagttgtaca gaccggcgat ggtagcaccg 540

aacttggcgc cgttgccggc cgggtgacgc aggttggtcg ggtacgcgct gttgtggatg 600

tcggtgtggc agttggcgca ccactcggac atgccctggc cgtaggcaac gcgggtctga 660

gtcgtagctt ccgtccggtt gtaggtggac ggagcaacgg cggccggtac ctggttggcg 720

aaggcatagg aaccgctcag ggacttgggc tggtagccgg tgccgcccag gatgcggtat 780

gcaccaacgg cgccccacgc ggtgggatcg ttgctgttct ggtaggaacc gctgttcttg 840

atggggagac cggtggtggc gatgctgccg tcaacaaaac gacgatactt cccgtgggga 900

tcgtggcagc tggagcagtg aagctggttg gccggatagg taccgcccgg ggccgtggtc 960

agggtggtgt cggcaacata gttgtagtcg ccggcaacga tgttgtggcc tttgcgctcg 1020

ccttcgctcg tgttgagacc acgaacgttc caggtgtagg tcttcttcac ccagccgaag 1080

tcgccgcccg gggtcatctg gagcggagcg gtaccggcag gcatatcagc ttcagcggtg 1140

gagatgtggt agctggaagg accggtgtca ccggcgtgct ggtggcagtt cagacaggac 1200

gagctctggg tggcgccctg gagcagcatg gggccggtgg tgaactgggc agtggcgctg 1260

ttcatgactg cgccgcccag cgagttgtgc atcgtgtggc acccttcgca ctcggcaacg 1320

ccgccggagt ggaaagcgaa cgccgcgggg gcgctcatga gaagtgctgc tgctgccacg 1380

gaaagactta ctttcatccc ctttttcatc atttcctcca ttttggttgg tttctccggc 1440

aacccggccg tcccgctcca gcggcacccg tcggctttgt gtccccacct tgcgatgggt 1500

ttactctttt cggaggaaat ttggtttcgc ctttcggctc gtgagtgttg tgcctgcaat 1560

gcaatacaaa atggtcttca gtgcatcccc agcgttttgt ggtccctcca cctcccctcc 1620

aaggcgggat gccggcctga gttacgactc gcga 1654

<210> 3

<211> 1429

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of OmcT

<400> 3

ggcacgctca aacccctcga ccggacaggc gccggaaacc gaggggtgtt tcaccggata 60

gtggatcgat tgctggaaaa aaggcggcca cgaccggccg gaggccggcc gtgaccagaa 120

ctacatgaca tatgaatcag tctttggcat gacacttgtt gcacaggacc cgttgatacg 180

gggcgaagtt ggtggggggt cttccatagt acgcctgctg cgtttcggca acggtccgtc 240

cctgggcctg ggctgcgttg gtgaccgggt cgggccacac ggggttgcct gacgcatcgg 300

ccacggtcat gaactcgttg ccgaggccgt accgcatcat gctgtcaaag ccggatgcgt 360

gggcgcggtg gcaggagagg cacatgaccc ggtccgtgga gatggggccg gcagttgacg 420

tggtctgggc cttgagcgcg ttcaggtcgt gggtgttgtc gctctggaac ggcaccagcg 480

aggtgaacgc cgtatccacg gcgcccgtca ggttgcccga cttcttgtac gagttgtaga 540

tctgggccac gaaggggcca aggttctgat cggccgggtg aaccagggtg ccgaaggtgg 600

tgtgcatctg cccgtggcag ttggcgcacc agagggacat gctccggccg taggcgaccc 660

gcgtgtcgct ggtggcttcc gagcggttgt agctgttggg ggccacggcg tagggggccg 720

cgaacatgaa ggcatcgccg ccgctcaggg acttgggctt gtacccggca ccgcccagga 780

ggcgataaac gccgacggca gtggtggcat cgggaacagc gccgtaggag ccggagctcc 840

tgatcggctt gccggtcttg gcctggacgc ccagtgaggt gatccggtag gtgccgtggg 900

ggtcatggca gctgatgcag ctgaactggt tggccgggaa gggggtgttc atgctccccg 960

gggccgcggt gatggtgctg tcggccacgt agttgtaatc ggcggccacg atgttgtggc 1020

ccttgcgctc gccgtggctc cactccgtgg cggcaccggc gcgcggcacc cagctgtagg 1080

tcttcttgag ccagccgaaa tcgccgcccg gcgtgagctg gagcggcgga gagccggcgg 1140

gcatgtcggc ttctgcggta ctgatgtggt agctgctggg ccccgtgtca ccggcatgct 1200

ggtgacagtt gaggcacgac gagctctggt ccgtcccctg gagcagataa gggccggtgg 1260

tgaactgggc agtagcgttg ttcatctgct ggccgcccag ggagttgtgc atggtgtggc 1320

atccctcgca ctctgccacg ccacctgagt ggaaagccca tcccgtgccg gttgcgccga 1380

gaaccaggag cgccgctgct gtgacgggaa gagtcttgaa gcgtttcat 1429

<210> 4

<211> 2051

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of OmcZ

<400> 4

ggtgcacgta tcccttcacg ttcgacgatg gtgaatcaca tcgcgaatgc atgtaaatga 60

tgtctgggga ggagatggag ggcaggggaa cgaatcccct gccccgttgg tcggaaaggt 120

attaccgttt gactttcttc ggagccgcca ggattttgcc ggagagcgga ccgttgatgg 180

tttttacggt cacgtagccg ttggttgcaa agtccgggct ggtggcaacc accttggtgt 240

cgctccagga aatgacgttt gcctgggtgg ttccggcgta cacaccgata ccggcgtcgt 300

actcggagct cggtgccggt ccgaaaccgg tgccggtaat ggtaagggtc ttgccagtcg 360

caagggtggc ggaggcaatg atccgcgccg gcgcgacggt cagcttggca aggttgctga 420

ccttgttggc cttggtgatg cggagctcgt agacgccttc aacgagggcg ggtacggaca 480

ccttgatctc gctttcggtg accgagaagg gagtgagtgt caggctcgtg ctgccgctga 540

cgagcgcaac agtgggctga taggtggtca cgccgtcggg tccgacgttg acgaagctgg 600

agccgacgat ggtcaggacg gcttccttgc ccgcggtgac cgtgtagctg ctctggccgt 660

tgatggcggg cacggtggca ttggtgtagg gcgaggaatt accgaaccag gaccagtggc 720

agccctggca gtcccagttg ttgccgatgt ggccccagcc cagatcctcc agccccggct 780

tgaccgtgcc gaggttggcg gcattggggc tgtccttctg gatattgtgg agcgagttca 840

cgccgtggca gacctcgcac tggcggatcg gtacattgga ggacgtattg tggcagaggt 900

tgcagtcggt gatgccggtg ccgtggtgag tatcctggtt gctgaagatc gggcggaccg 960

tattggtctt cggatcgatg gcgttgggtg cggcctggtg gcatgcttcg cagccctgaa 1020

cgataacaac attgccatcc gtagcggtta cagacctgcc gctcggcatc ggagtgacgg 1080

agcttgcgct gtaggtcgga atgtagtggc cgtcaagcgg gttgtcgatg aagttgccgt 1140

ggcagtactt gcagtccttg gcaacagcgg cgggagaggt gtggtgcgga gtctgggtgt 1200

ggcagttgaa acagtttctg aaatcctgga aggtaaaacc gcccgagccg tcgggaacca 1260

tgacgtggca accggtggca agggtcgggg ggacagtgcc ggaggtattg atgcagcttg 1320

ccggcggcgt aaccgtgttg atcagggcgt gatgctgctg gacgagcacc gtgtcgctga 1380

cgtgacactc gagacagtcg gctttggtca ggttgggaaa cttggtatcg tagatcccca 1440

ggaactggtt taccggcggg ggcggaacag ctgcacccac cattgctgcg ccggttaaga 1500

caacggctgc gagcgatgcg ccaatcagta cctttttctt cattcctttc tgctcctttc 1560

ttgtgaagac tccattatga aacattacat tcaaagcgat tgacgtattc cttcctcctt 1620

gccacctcct ttcggcatgt catgtcccct cactctggca aattcaatgc atggttctgc 1680

gactaaaagg cgctgcatcg gacaaccgca gcaggacgca tacacattga ccggtagacc 1740

cgcatgtctc tgcgtccggc gatgcgcagg ccggaatggt cttcgggcag atttttcaga 1800

tggtacctgg ctgtgttcag ggggcgagca gggccgggat atcgcatcac atatatcatt 1860

aagtaggata aacacaactg tgtactgtag ctattcagag ctttttgtca acatactttt 1920

aaaggccatt ttaacgttca gcaacaaatt agcaacacac atacttgcaa tcccgccaga 1980

caagaatttg acgttctctt ccgccaagct cgcctgatac gctattttac tgcatataac 2040

tagcagcgac a 2051

<210> 5

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PilA-1

<400> 5

agaggagcca gtgacga 17

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PilA-2

<400> 6

tccttaaacg ttatcgtg 18

<210> 7

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PilA-3

<400> 7

ttacccccat accccaa 17

<210> 8

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PilA-4

<400> 8

ggtttcctcc agtatgt 17

<210> 9

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OmcS-1

<400> 9

aaaatacctc ctggcg 16

<210> 10

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OmcS-2

<400> 10

tcgcgagtcg taactca 17

<210> 11

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OmcT-1

<400> 11

tttctccggc aaccc 15

<210> 12

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OmcT-2

<400> 12

ggcacgctca aacccct 17

<210> 13

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OmcT-3

<400> 13

atgaaacgct tcaagac 17

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OmcZ-1

<400> 14

tgtcgctgct agttatatgc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OmcZ-2

<400> 15

ggtgcacgta tcccttcacg 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PAWP78-1

<400> 16

ttgtcgggaa gatgcgtgat 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PAWP78-2

<400> 17

cagctcactc aaaggcggta 20

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PAWP78-3

<400> 18

cgggtttcgc cacctctga 19

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PAWP78-4

<400> 19

tggacgagtc ggaatcgcag a 21

Claims

1. An electrogenic bacillus, wherein the electrogenic bacillus is obtained by expressing a nanowire protein gene through Geobacter sulfurreduce PCA, and the nanowire protein gene comprises any one of a gene PilA, a gene OmcS, a gene OmcT or a gene OmcZ; the nucleotide sequence of the gene PilA is shown as SEQ ID NO. 1, the nucleotide sequence of the gene OmcS is shown as SEQ ID NO. 2, the nucleotide sequence of the gene OmcT is shown as SEQ ID NO. 3, and the nucleotide sequence of the gene OmcZ is shown as SEQ ID NO. 4.

2. The method for constructing the geobacillus for electricity generation according to claim 1, which comprises the following steps:

s1, amplifying the gene of the nano-conductive line protein by taking the genome of the Geobacter sulfurducencencA as a template to obtain an amplified gene segment of the nano-conductive line protein; the amplified nanowire protein gene segment comprises any one of an amplified gene PilA segment, an amplified gene OmcS segment, an amplified gene OmcT segment or an amplified gene OmcZ segment, wherein the nucleotide sequence of the gene PilA is shown as SEQ ID NO. 1, the nucleotide sequence of the gene OmcS is shown as SEQ ID NO. 2, the nucleotide sequence of the gene OmcT is shown as SEQ ID NO. 3, and the nucleotide sequence of the gene OmcZ is shown as SEQ ID NO. 4;

s2, carrying out PCR amplification on a linearized vector by using a primer by using the PAWP78 plasmid as a template to obtain a linearized vector fragment PAWP 78;

s3, cloning the amplified nano-lead protein gene segment obtained in the step S1 to the linearized vector segment PAWP78 obtained in the step S2 by a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP 78-nano-lead protein gene, wherein the vector PAWP 78-nano-lead protein gene comprises any one of a vector PAWP78-PilA, a vector PAWP78-OmcS, a vector PAWP78-OmcT or a vector PAWP 78-OmcZ;

s4, transforming the vector PAWP 78-nanowire protein gene obtained in the step S3 into E.coli DH5 alpha, and obtaining an over-expression plasmid PAWP 78-nanowire protein gene through sequencing verification, wherein the over-expression plasmid PAWP 78-nanowire protein gene comprises any one of an over-expression plasmid PAWP78-PilA, an over-expression plasmid PAWP78-OmcS, an over-expression plasmid PAWP78-OmcT or an over-expression plasmid PAWP 78-OmcZ;

s5, electrically transforming the overexpression plasmid obtained in the step S4 into Geobacter sulfurreducens PCA for culture to obtain the Geobacter electrogenesis, wherein the Geobacter electrogenesis comprises any one of the strain PCA/PilA, the strain PCA/OmcS, the strain PCA/OmcT or the strain PCA/OmcZ.

3. The method according to claim 2, wherein in step S1, the primers used for amplifying the upstream fragment of the gene PilA are PilA-1 and PilA-2, the nucleotide sequence 3 of the primer PilA-1 is shown in SEQ ID No. 5, and the nucleotide sequence of the primer PilA-2 is shown in SEQ ID No. 6; the primers used for amplifying the downstream segment of the gene PilA are PilA-3 and PilA-4, the nucleotide sequence of the primer PilA-3 is shown as SEQ ID NO. 7, and the nucleotide sequence of the primer PilA-4 is shown as SEQ ID NO. 8.

4. The construction method according to claim 2, characterized in that in step S1, the primers used for the amplification gene OmcS are OmcS-1 and OmcS-2, the nucleotide sequence of the primer OmcS-1 is shown in SEQ ID NO. 9, and the nucleotide sequence of the primer OmcS-2 is shown in SEQ ID NO. 10.

5. The construction method according to claim 2, wherein in step S1, the primers used for amplifying the upstream fragment of gene OmcT are OmcS-1 and OmcT-1, the nucleotide sequence of the primer OmcS-1 is represented by SEQ ID NO. 9, and the nucleotide sequence of the primer OmcT-1 is represented by SEQ ID NO. 11; the primers used for amplifying the downstream fragment of the gene OmcT are OmcT-2 and OmcT-3, the nucleotide sequence of the primer OmcT-2 is shown as SEQ ID NO. 12, and the nucleotide sequence of the primer OmcT-3 is shown as SEQ ID NO. 13.

6. The construction method according to claim 2, wherein in step S1, the primers used for amplifying the gene OmcZ are OmcZ-1 and OmcZ-2, the nucleotide sequence of the primer OmcZ-1 is shown in SEQ ID NO. 14, and the nucleotide sequence of the primer OmcZ-2 is shown in SEQ ID NO. 15.

7. The method of claim 2, wherein in step S2, the primers for carrying out PCR amplification on the linearized vector include PAWP78-1 and PAWP78-2, the nucleotide sequence of the PAWP78-1 is shown in SEQ ID NO. 16, and the nucleotide sequence of the PAWP78-2 is shown in SEQ ID NO. 17.

8. The method according to claim 2, wherein the culturing in step S5 is performed anaerobically at 30 ℃ in NBAF medium.

9. Use of the geobacter electrogenesis bacterium of claim 1 in a microbial fuel cell MFC.

10. The use according to claim 9, wherein the geobacter electrogenesis is anaerobically fermented in MFC for not less than 150 hours.