CN114921396B

CN114921396B - Geobacillus electrogenesis, construction method and application

Info

Publication number: CN114921396B
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: 2023-10-31
Anticipated expiration: 2042-05-31
Also published as: CN114921396A

Abstract

The application provides geobacillus electrogenesis and a construction method and application thereof. The application obtains over-expression plasmid by the seamless cloning method of the nano-wire protein coding gene and PAWP78 through Gibson, and then introduces the over-expression plasmid into strain PCA through electrotransformation to obtain the geobacillus electrogenesis strain over-expressing the nano-wire protein, including strains PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ. The strain of the application carries out anaerobic fermentation for not less than 150 hours in MFC, the maximum output voltage reaches 469mV to 577mV, which is improved by 63 percent compared with the control strain PCA/PAWP78, and the maximum power density reaches 1392mW to 1580mW/m ² The PCA/PAWP78 is improved by 1.62-1.97 times compared with the control strain. It is verified that the nanowire protein can enhance the extracellular electron transfer rate and improve the electric output capacity of the electrogenerator.

Description

Geobacillus electrogenesis, construction method and application

Technical Field

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

Background

Microbial fuel cells (Microbial fuel cells, MFCs) are devices that utilize the catalytic activity of microorganisms to convert chemical energy generated during oxidation of organic matter into electrical energy. The MFCs can be applied to the fields of biological power generation, sewage treatment, biological hydrogen production and the like, and provides a new method for recycling energy and resources.

The power generating bacteria are bacteria with extracellular electronic transfer ((Extracellular electron transfer, EET) capability, and as the most important component in the MFC, the power generating capability directly determines the performance of the MFC.

Improving EET efficiency of electrogenic bacteria is one of the important ways to enhance its electrical output capability. The EET method for improving the electrogenesis bacteria mainly comprises the following steps: promote the expression of c-type cytochromes associated with electron transfer and electron transfer mediators in cell membranes. The electrical output of the engineered strain is significantly improved compared to the wild-type strain, but still at a lower level.

Disclosure of Invention

The application aims at providing a geobacillus electrogenesis and a construction method and application thereof aiming at the defects of the prior art.

The first object of the present application is to provide a geobacillus electrogenerated obtained by Geobacter sulfurreducens PCA over-expressing nanowire protein genes including 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 application is to provide a construction method of the above Geobacillus electricity generating strain, which comprises the following steps

Step S1, amplifying nanowire protein genes by taking a genome of Geobacter sulfurreducens PCA as a template to obtain amplified nanowire protein gene fragments; the amplified nanowire protein gene fragment comprises any one of an amplified gene pilA fragment, an amplified gene OmcS fragment, an amplified gene OmcT fragment or an amplified gene OmcZ fragment, 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 linearization vector by using a PAWP78 plasmid as a template and using a primer to obtain a linearization vector fragment PAWP78;

s3, cloning the amplified nanowire protein gene fragment obtained in the step S1 onto the linearized vector fragment PAWP78 obtained in the step S2 through a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP 78-nanowire protein gene, wherein the vector PAWP 78-nanowire protein gene comprises any one of vector PAWP78-pilA, vector PAWP78-OmcS, vector PAWP78-OmcT or vector PAWP78-OmcZ;

s4, converting 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 PAWP78-OmcZ;

and S5, electrically transforming the over-expression plasmid obtained in the step S4 to Geobacter sulfurreducens PCA, and culturing to obtain the geobacillus electrogenesis, wherein the geobacillus electrogenesis comprises any one of strain PCA/pilA, strain PCA/OmcS, strain PCA/OmcT or strain PCA/OmcZ.

In step S1, 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 fragment 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 as SEQ ID NO. 9, and the nucleotide sequence of the primer OmcS-2 is shown as SEQ ID NO. 10.

Further, in the step S1, 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 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 as SEQ ID NO. 14, and the nucleotide sequence of the primer OmcZ-2 is shown as SEQ ID NO. 15.

Further, in the step S2, primers for performing PCR amplification on the linearization vector comprise PAWP78-1 and PAWP78-2, wherein 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.

Further, in the step S5, the culture is anaerobic culture at 30 ℃ by using an NBAF culture medium.

A third object of the present application is to provide the use of geobacillus electrogenerated as described above in a microbial fuel cell MFC.

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

The application has the following beneficial effects:

the application adopts the genetic engineering bacteria means to over-express the nanowire protein genes to obtain engineering strainsGeobacillus electrogenerated, strains PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ, have significantly improved electrogenesis activity. The strain of the application is subjected to anaerobic fermentation in MFC for not less than 150h, and the maximum output voltages which can be respectively achieved by the strains PCA/pilA, PCA/omcS, PCA/omcT and PCA/omcZ are 577mV, 533mV, 551mV and 469mV respectively, which are improved by 63%, 50%, 55% and 32% compared with the control strain PCA/PAWP78 (355 mV); strains PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ were able to achieve a maximum output power of 1580mW/m ² 、1502mW/m ² 、1481mW/m ² And 1392mW/m ² Compared with the control strain PCA/PAWP78 (532 mW/m ² ) The lifting is 1.97 times, 1.82 times, 1.78 times and 1.62 times. The over-expression of the nanowire protein obviously improves the electric output capacity of the geobacillus electrogenesis.

Drawings

FIG. 1 is a schematic diagram of the present application employing Giboson seamless cloning;

FIG. 2 is a schematic structural diagram of an over-expression plasmid of the present application;

FIG. 3 is a diagram showing a comparison of PCR verification of bacterial solutions of plasmid-transferred Geobacillus thioreductase PCA engineering strains;

FIG. 4 is a Western bolt verification comparison chart of a plasmid-transferred Geobacillus thioreductase PCA engineering strain;

FIG. 5 is a graph showing comparison of the output voltage results of strains PCA/pilA, PCA/OmcS, PCA/OmcT, PCA/OmcZ of the present application and control strain PCA/PAWP 78;

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

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

FIG. 8 is a graph showing comparison of the results of the alternating impedance results of the strains PCA/pilA, PCA/OmcS, PCA/OmcT, PCA/OmcZ of the present application and the control strain PCA/PAWP78.

Detailed Description

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

The original strain used in the present application, geobacillus Geobacter sulfurreducens PCA, is a typical strain deposited with ATCC: ATCC 51573.

In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available.

Description of sources for biological materials:

1. plasmid source: are commercially available.

2.Geobacter sulfurreducens PCA genome template source: using the Kangzhi bacterial genome DNA kit, 5mL of anaerobic cultured PCA bacterial solution was taken according to the specification to obtain 50uL of PCA genome DNA.

3. Stock solution and culture medium preparation method

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

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

3.3 Trace Mineral stock: into a 1L beaker was added 950mL of ultra-pure water, 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 uniformly, adding ultrapure water to a constant volume of 1L, and preserving at 4 ℃.

3.4 Vitamines stock: into a 1L beaker was added 950mL of ultrapure water, and 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 niacin, 0.005g of pantothenic acid, 0.0001g of vitamin B12, 0.005g of para-aminobenzoic acid and 0.005g of lipoic acid were added to the beaker; stirring uniformly, then fixing the volume to 1L, and preserving at 4 ℃ in dark.

3.5 NBFA liquid medium: 850mL of ultrapure water was added to the 1L beaker; 4.64g (40 mM) fumaric acid was added to the beaker; adjusting the pH to 6.0-6.1 by NaOH (5M); to the beaker, 20mL Core media stock solution, 50mL Mg/Ca stock solution, 10mL Mineral stock solution were added sequentially; 1.64g anhydrous sodium acetate (20 mM) was added to the beaker; adjusting pH to 6.8 with NaOH (5M), adding ultrapure water to 1L, and adding 2g NaHCO ₃ The method comprises the steps of carrying out a first treatment on the surface of the Packaging into anaerobic bottles, and adding CO ₂ /N ₂ And (8) 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 the 1L beaker; 4.64g (40 mM) fumaric acid was added to the beaker; adjusting the pH to 6.0-6.1 by NaOH (5M); to the beaker, 20mL Core media stock solution, 50mL Mg/Ca stock solution, 10mL Mineral stock solution were added sequentially; 1.64g anhydrous sodium acetate (20 mM) was added to the beaker; adjusting pH to 6.8 with NaOH (5M), adding ultrapure water to 1L, and adding 2g NaHCO ₃ The method comprises the steps of carrying out a first treatment on the surface of the Sub-packaging the culture medium into 200-250 mL anaerobic bottles, adding 2g agar powder into each bottle, and mixing with CO ₂ /N ₂ (8:2) deoxygenation, sterilization at 121 ℃,20min, cooling, storage at room temperature, sterilization with high pressure steam at 121 ℃ for 5min before use, dissolution and inversion of plates in Coylab glove box.

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

Example 1

This example illustrates the construction of the Geobacillus electrogenerated strain PCA/pilA. The specific process comprises the following steps:

step 1, amplifying a pilA gene by taking a Geobacter sulfurreducens PCA genome 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 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 fragment 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 PCR reaction system is as follows: 45. Mu.L of gold plate Mix (high fidelity DNA polymerase of the Optimago family), 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 were:

the reaction system of Gibson is: 10. Mu.L Gibson Mix (2X, NEB), 3. Mu.L insert DNA, 2. Mu.L vector fragment DNA, 5. Mu.L deionized water. (molar ratio between DNA fragments is controlled to be 1:1)

The reaction conditions of Gibson are: incubate at 50℃for 30min, then preserve the samples at-20 ℃.

And 2, carrying out PCR amplification on the linearized vector by using a PAWP78 plasmid as a template and using a primer 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 fragment obtained in the step 1 onto the linearization vector fragment PAWP78 obtained in the step 2 through a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP78-pilA;

s4, converting the vector PAWP78-pilA obtained in the step 3 into E.coli DH5 alpha, and obtaining an expressed plasmid PAWP78-pilA through sequencing verification;

and 5, electrically converting the over-expression plasmid PAWP78-pilA obtained in the step 4 into Geobacter sulfurreducens PCA for culture to obtain the geobacillus electrogenerated strain PCA/pilA. The specific process is as follows: 1mL of frozen PCA wild strain is inoculated into 10mL of NBFA liquid culture medium in a Coylab anaerobic glove box, 5mL of bacterial liquid is transferred into 50mL of NBFA liquid culture medium when the bacterial liquid is OD600 = 0.3 in 30 ℃,10 mL of bacterial liquid is transferred into 100mL of NBFA liquid culture medium when the bacterial liquid is OD600 = 0.3 in 30 ℃, and the bacterial strain grows to OD600 = 0.3; the bacterial liquid and the high-speed centrifuge tube of the last step are placed on ice in a Coylab anaerobic glove box, and the bacterial liquid is taken into the high-speed centrifuge tube to be centrifuged, 4300 Xg, 4 ℃ for 10min. Removing the supernatant on ice, and re-suspending the thalli by using PCA EB buffer with the same volume as the supernatant; repeating the operation of the previous step on ice, cleaning thalli twice, and then re-suspending 100mL of thalli collected by centrifugation by using 200 mu L of PCA EB buffer to obtain competent cells; 1000ng of plasmid was added to 100. Mu.L PCA competent cells, gently mixed and transferred to a cuvette; the electrotransport converter is set to be 1.47KV for electrotransport conversion; after electric conversion, 1mL of NBFA liquid culture medium is added into the electric shock cup, then all liquid in the electric shock cup is sucked out by a syringe and added into 10mL of NBFA liquid culture medium, and anaerobic recovery culture is carried out for 24 hours at 30 ℃; concentrating the thallus of the previous step to 100 mu L, then coating on an NBFA solid plate containing Kana (working concentration 200 mu g/mL), and carrying out anaerobic culture at 30 ℃; when single colony grows on the NBFA solid plate, picking the single colony in a Coylab anaerobic glove box to an NBFA liquid culture medium containing Kana (working concentration is 200 mu g/mL), and carrying out anaerobic culture at 30 ℃; and (3) taking 500 mu L of bacterial liquid for centrifugation, removing supernatant, re-suspending bacterial cells with 20 mu L of sterilized deionized water, and carrying out PCR (polymerase chain reaction) verification by using the re-suspended bacterial liquid as a template to obtain a bacterial strain PAWP78-pilA with plasmids correctly introduced.

Example 2

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

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

Step 3, cloning the amplified nanowire protein gene fragment obtained in the step 1 onto the linearization vector fragment PAWP78 obtained in the step 2 through a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP78-OmcS;

s4, converting the vector PAWP78-OmcS obtained in the step 3 into E.coli DH5 alpha, and obtaining an expressed plasmid PAWP78-OmcS through sequencing verification;

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

Example 3

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

step 1, amplifying an OmcT gene by using a Geobacter sulfurreducens PCA genome 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 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.

Step 3, cloning the amplified nanowire protein gene fragment obtained in the step 1 onto the linearization vector fragment PAWP78 obtained in the step 2 through a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP78-OmcT;

s4, converting the vector PAWP78-omcT obtained in the step 3 into E.coli DH5 alpha, and obtaining an expressed plasmid PAWP78-omcT through sequencing verification;

and 5, electrotransformation of the over-expression plasmid PAWP78-omcT obtained in the step 4 to Geobacter sulfurreducens PCA for culture to obtain the geobacillus electrogenerated strain PCA/omcT. The procedure is as in example 1.

Example 4

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

step 1, amplifying an OmcZ gene by taking a Geobacter sulfurreducens PCA genome 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 as SEQ ID NO. 14, and the nucleotide sequence of the primer OmcZ-2 is shown as SEQ ID NO. 15.

Step 3, cloning the amplified nanowire protein gene fragment obtained in the step 1 onto the linearization vector fragment PAWP78 obtained in the step 2 through a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP78-OmcZ;

s4, converting the vector PAWP78-OmcZ obtained in the step 3 into E.coli DH5 alpha, and obtaining an expressed plasmid PAWP78-OmcZ through sequencing verification;

and 5, electrotransformation of the over-expression plasmid PAWP78-omcZ obtained in the step 4 to Geobacter sulfurreducens PCA for culture to obtain the geobacillus electrogenerated strain PCA/omcZ. The procedure is as in example 1.

Comparative example 1

This example illustrates the construction of the Geobacillus electrogenerated strain PCA/PAWP78 without overexpression.

The specific process comprises the following steps:

step 1, PCR amplification of a linearization vector is carried out by using a PAWP78 plasmid as a template and a primer to obtain a linearization 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.

And 2, converting the vector PAWP78 obtained in the step 1 into E.coli DH5 alpha, and performing sequencing verification, and performing electrotransformation into Geobacter sulfurreducens PCA for culture to obtain the geobacillus electrogenerated strain PCA/PAWP78. 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, DNA exonuclease cuts from the 5' -end pair of DNA to form a sticky end; homologous sequences in the cohesive ends are then annealed; the DNA polymerase then extends in the 5 'to 3' direction of the DNA; finally, the DNA ligase blocks the gap.

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

As shown in FIG. 3, the bacterial liquid PCR verification gel diagrams of PCA/PAWP78 and PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ are shown, the primers for verifying the insertion sequence on the PAWP78 plasmid are PAWP78-3 and PAWP78-4, the nucleotide sequence of PAWP78-3 is shown as SEQ ID NO. 18, and the nucleotide sequence of PAWP78-4 is shown as SEQ ID NO. 19. PCR results prove that the engineering strain constructed by the application is transferred into a correct expression vector.

As shown in FIG. 4, which shows Western bolt verification graphs of PCA/PAWP78 and PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ, the engineered strains all expressed nanowire proteins, and the strains highly expressing nanowire proteins under the same loading conditions had thicker bands, indicating that the protein expression levels were indeed improved.

In order to better illustrate that the engineering bacteria constructed by the application have better electric output capacity, microbial electrochemical characterization researches are carried out on examples 1-5 and comparative example 1.

Preparation of MFC

(1) Pretreatment of carbon cloth electrodes: rinsing with 1M hydrochloric acid overnight, soaking in pure acetone overnight (note sealing, preventing acetone volatilization), rinsing with sterile deionized water, and oven drying and preserving. Cutting the treated carbon cloth according to the anode and the cathode respectively to the following sizes: 1cm by 1cm and 2.5cm by 3cm.

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

(3) Pretreatment of MFC reactor: and fixing the cut carbon cloth on a lead by using AB glue, then passing the lead through a hole reserved on a rubber plug to suspend the carbon cloth in a reactor, and sterilizing at high temperature and high pressure (121 ℃ for 20 min). After the reactor is cooled, the treated proton exchange membrane is placed in the middle of the reactor in a biosafety cabinet, and the reactor is fixed by a clamp.

(4) Preparation of an anode culture solution: 850mL of ultrapure water was added to the 1L beaker; to the beaker were added sequentially 20mL Core media stock, 50mL Mg/Ca stock, 10mL Trace Mineral stock, and 1mL Vitamins stock; 1.64g anhydrous sodium acetate (20 mM) was added to the beaker; adjusting pH to 6.8 with NaOH (1M), adding ultrapure water to a volume of 1L, and then adding 2g NaHCO ₃ The method comprises the steps of carrying out a first treatment on the surface of the Packaging into anaerobic bottles, and adding CO ₂ /N ₂ And (8) deoxidizing, sterilizing at 121 ℃ for 20min, and cooling to room temperature for later use.

(5) Configuration of catholyte: 950mL of ultrapure water was added to the 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 a volume of 1L, packaging into anaerobic bottles, and adding pure N ₂ Oxygen removal is performed.

2. Assembly of MFC

Culturing the constructed electrogenesis PCA engineering strain to OD by using NBFA liquid culture medium containing Kana (working concentration 200 mu g/mL) ₆₀₀ =0.5, 50mL of bacterial liquid was taken in a Coylab anaerobic glove box into a high-speed centrifuge tube, centrifuged at 4300rpm at 4 ℃ for 10min, the supernatant was removed in the Coylab anaerobic glove box, and the bacterial cells were resuspended in 140mL of anodic culture; the lid of the anode chamber of the MFC reactor was opened to allow it to be placed in the Coylab anaerobic glove box after deoxygenation in the transfer compartment of the Coylab anaerobic glove box, 140mL of resuspended bacterial liquid was added to the anode chamber of the reactor and K was addedana (working concentration 200. Mu.g/mL), the lid of the anode chamber was closed, and the reactor was taken out of the Coylab anaerobic glove box; the lid of the cathode chamber of the MFC reactor was opened outside, 150mL of catholyte was added thereto, the lid of the anode chamber was closed, and all the hole gaps on the lid were sealed with a hot melt glue gun. The MFC reactor was connected to the anode and cathode compartments by a 2kΩ external resistor, the MFC was assembled, and the assembled MFC reactor was placed in a 30℃incubator for cultivation.

3. Microbial electrochemical analysis

Real-time monitoring of output voltage: the multichannel voltage detector PS2024V was connected to a computer, and the anode and cathode of each channel were connected to the external resistor of the MFC, and voltage data were recorded every 30 minutes on the computer.

Determination of Linear voltammetry (LSV) curves the procedure is as follows:

(1) And when the output voltage of the MFC reaches the maximum value and is stable, removing the external 2k omega resistor, and standing and discharging for 1h.

(2) The discharged MFC is connected with a Shanghai Chenhua CHI1000C potentiostat, 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) The potentiostat scan parameters were set as follows:

initial potential (V): -0.8V

Termination potential (V): -0.1V

Initial scan polarization: positive)

Scan rate (V/s): 0.0001

Sampling time (V): 0.001

Rest time(s): 30

Sensitivity (a/V): 1.e ⁰⁰⁴

(4) After the scanning is completed, the data is stored in a computer.

(5) Bringing the voltage V value and the current A value in the data into a formula can 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 x 1000); the power density curve is obtained by plotting current density as abscissa and power density as ordinate, and the polarization curve is obtained by plotting current density as abscissa and voltage as ordinate.

As a result, as shown in FIG. 5, which is a graph showing the change with time of the MFC output voltage of the control strain (PCA/PAWP 78) and the engineering strain (PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ), the engineering strain exhibited a voltage peak between 100 hours and 180 hours and maintained the peak for a period of time, the control strain exhibited a voltage peak at 150 hours and then began to drop, and the voltage peak of the engineering strain was 32% -63% higher than that of the control strain. Engineering strains PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ, which have significantly improved electrogenesis activity. The maximum output voltages achievable by the engineering strains PCA/PilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ were 577mV, 533mV, 551mV and 469mV respectively, which were increased by 63%, 50%, 55% and 32% compared to the control strain PCA/PAWP78 (355 mV).

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

As shown in FIG. 7, the control strain PCA/PAWP78 showed no reverse redox peak, while all of the engineering strains PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ showed significant redox peaks, indicating an increase in the conductive material in the engineering strain MFC. And the peak value of the current density of the engineering strain is higher than that of the control strain, which indicates that the catalytic current of the engineering strain MFC is also higher than that of the control strain. It was also shown that MFC constructed with the engineered strain of the application increased conductive species in the anode chamber and the catalytic current of the cells was high.

As shown in FIG. 8, the radii of the curves for engineering strains PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ were all smaller than those of the control strain PCA/PAWP78, indicating that the internal resistance of the engineering strain MFC was smaller than that of the control strain PCA/PAWP78. 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 is, the smaller the internal resistance of the battery is. It was also shown that the internal cell resistance of the MFC constructed with the engineered strain of the application was significantly less than the control strain PCA/PAWP78.

In conclusion, the engineering strains PCA/pilA, PCA/OmcS, PCA/OmcT and PCA/OmcZ constructed by the application can enhance extracellular electron transfer rate through overexpression of nanowire proteins, and the engineering strains constructed by the application improve electricity generation and output capacity.

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

While certain specific embodiments of the present application have been described in detail by way of example, it will be appreciated by those skilled in the art that the foregoing examples are provided for the purpose of illustration only and are not intended to limit the scope of the application, and that various modifications or additions and substitutions to the described specific embodiments may be made by those skilled in the art without departing from the scope of the application or exceeding the scope of the application as defined in the accompanying claims. It should be understood by those skilled in the art that any modification, equivalent substitution, improvement, etc. made to the above embodiments according to the technical substance of the present application should be included in the scope of protection of the present application.

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Sequence listing

<110> university of Chinese geology (Wuhan)

<120> Geobacillus electrogenesis, construction method and application

<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. Use of Geobacillus electrogenerated in microbial fuel cell MFC, characterized in that Geobacillus electrogenerated is produced byGeobacter sulfurreducens The PCA overexpression nanowire protein gene is obtained, and the nanowire protein gene is a gene pilA; the nucleotide sequence of the gene pilA is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the time for anaerobic fermentation of the geobacillus electrogenerated in MFC is not less than 150 hours.

3. The method for constructing geobacillus electrogenesis according to claim 1, which comprises the following steps:

s1, toGeobacter sulfurreducensAmplifying the nanowire protein gene by taking the genome of PCA as a template to obtain an amplified nanowire protein gene fragment; the amplified nanowire protein gene fragment comprises an amplified gene pilA fragment, and the nucleotide sequence of the gene pilA is shown as SEQ ID NO. 1;

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

s3, cloning the amplified nanowire protein gene fragment obtained in the step S1 onto the linearized vector fragment PAWP78 obtained in the step S2 through a Gibson seamless cloning method to obtain a recombinant plasmid, namely a vector PAWP 78-nanowire protein gene, wherein the vector PAWP 78-nanowire protein gene comprises a vector PAWP78-pilA;

s4, converting 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 an over-expression plasmid PAWP78-pilA;

s5, electrically converting the over-expression plasmid obtained in the step S4 intoGeobacter sulfurreducensPCA culture to obtain Geobacillus electrogenes PCA/pilA.

4. The construction method according to claim 3, 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 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 fragment 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.

5. The construction method according to claim 3, wherein in step S2, the primers for performing PCR amplification of the linearized vector 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.

6. The method according to claim 3, wherein in step S5, the culture is anaerobic culture at 30℃using NBAF medium.