CN110724656A - Electricity-producing Shewanella recombinant strain, construction method and application - Google Patents

Abstract

The invention discloses an electricity-producing Shewanella recombinant strain, a construction method and application thereof, wherein the construction method of the electricity-producing Shewanella recombinant strain comprises the following steps: knocking out a target gene SO _3941 of wild Shewanella oneidensis MR-1 to obtain an electric Shewanella recombinant strain delta 3941, wherein the nucleotide sequence of the gene SO _3941 is shown as SEQ ID NO 15. The electro-generating Shewanella recombinant strain delta 3941 constructed by the invention is a gene-deficient strain, the strain can improve the concentration of intracellular cyclic di-GMP (cyclic di-GMP), and the increase of the c-di-GMP can promote the cell surface adhesion and the formation of a biological membrane, thereby enhancing the extracellular electron transfer rate and improving the power density of a microbial fuel cell.

Description

Electricity-producing Shewanella recombinant strain, construction method and application

Technical Field

The invention relates to the technical field of biological energy, in particular to an electricity-generating Shewanella recombinant strain, a construction method and application thereof.

Background

Energy shortage and environmental pollution are increasingly serious problems faced by China nowadays, so that energy development, environmental waste treatment and renewable utilization of energy in the process become a great challenge for sustainable development of modern society of China. Scientists are constantly looking for new technical solutions, one of which is Microbial Fuel Cells (MFC) for the production of new alternative energy sources and environmental waste management devices, and their importance is nowadays increasingly emerging.

MFC is a device that converts chemical energy in organic matter into electrical energy using an electrogenic microorganism as an anode catalyst. The electricity generating capability of microorganisms is greatly different, the electricity generating microorganisms determine the function and the application of MFC, Shewanella oneidensis (Shewanella oneidensis) belongs to one of the microorganisms which are widely used for generating electricity in MFC and the research on the metabolic pathway and the extracellular electron transfer pathway is relatively clear. Shewanella oneidensis MR-1 (Shewanella MR-1 for short) is the most widely studied strain in Shewanella in terms of genomic sequence annotation and genetic characterization. The strain can transfer electrons to an anode in a microbial fuel cell without adding an external medium, and becomes one of model organisms for researching how microorganisms generate current in MFCs.

Studies have shown that microbial Extracellular Electron Transfer (EET) is a complex process influenced by a variety of cellular components. Wherein the thickness of the biological membrane directly influences the strength of the extracellular electron transfer and influences the power density of the biological fuel cell. However, the wild type Shewanella strain MR-1 has weak cell adhesion and cannot form a thick and stable biofilm. Therefore, constructing Shewanella strains which are easy to form biofilms can not only compensate for some defects of Shewanella, but also improve the electrochemical effect of MFC.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an electricity-generating Shewanella recombinant strain.

The second purpose of the invention is to provide a construction method of the electricity-generating Shewanella recombinant strain.

The third purpose of the invention is to provide the application of the electricity-generating Shewanella recombinant strain.

The technical scheme of the invention is summarized as follows:

a method for constructing an electricity-generating Shewanella recombinant strain comprises the following steps: knocking out a target gene SO _3941 of wild Shewanella oneidensis MR-1 to obtain an electric Shewanella recombinant strain delta 3941, wherein the nucleotide sequence of the gene SO _3941 is shown as SEQ ID NO 15.

The electricity-producing Shewanella recombinant strain delta 3941 constructed by the method.

The electrogenic Shewanella recombinant strain delta 3941 can be used for electrogenesis.

The invention has the advantages that:

the electro-generating Shewanella recombinant strain delta 3941 constructed by the invention is a gene-deficient strain, the strain can improve the concentration of intracellular cyclic di-GMP (cyclic di-GMP), and the increase of the c-di-GMP can promote the cell surface adhesion and the formation of a biological membrane, thereby enhancing the extracellular electron transfer rate and improving the power density of a microbial fuel cell.

Drawings

FIG. 1 shows a method for gene amplification in the primer design and the homologous region.

FIG. 2 is a graph showing comparison of the generated voltage of Shewanella MR-1 and an electric Shewanella recombinant strain delta 3941 MFC.

FIG. 3 is an electrochemical characterization chart.

Detailed Description

Original strain Shewanella oneidensis MR-1 is short: wild-type Shewanella MR-1, ATCC 700550 strain purchased from ATCC (U.S. https:// www.atcc.org /) at 9 months 2010.

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

Example 1

A method for constructing an electricity-generating Shewanella recombinant strain comprises the following steps: knocking out a target gene SO _3941 of wild Shewanella MR-1 to obtain an electricity-producing Shewanella recombinant strain delta 3941, wherein the nucleotide sequence of the gene SO _3941 is shown as SEQ ID NO 15.

The specific construction method comprises the following steps:

knocking out a wild Shewanella MR-1 target gene SO _3941 by using att site-specific recombination and a double-exchange deletion method.

(1) Primer design and gene amplification:

obtaining genome sequence of Shewanella MR-1 from NCBI database, designing three pairs of primers, 3941-LF (SEQ ID NO.4) and 3941-LR (SEQ ID NO.5), 3941-5-O (SEQ ID NO.6) and 3941-5-I (SEQ ID NO.7), 3941-3-O (SEQ ID NO.8) and 3941-3-I (SEQ ID NO.9), according to target gene SO _3941 and upstream and downstream gene sequences thereof, amplifying upstream homologous regions and downstream homologous regions of target gene in vitro, wherein the upstream homologous regions and the downstream homologous regions comprise partial target gene sequences, the amplification length is about 500-1000bp, and connecting the upstream and downstream homologous regions together through fusion PCR to form fusion homologous region fragments. As shown in fig. 1. Wherein, specific sites attB1 and attB2 are respectively added at the 5 'ends of the primers 3941-5-O and 3941-3-O, and a homologous complementary sequence (Linker) with about 20bp is added at the 5' ends of the primers 3941-5-I and 3941-3-I.

The nucleotide sequence of the upstream homologous region is shown as SEQ ID NO. 1;

the nucleotide sequence of the downstream homologous region is shown as SEQ ID NO. 2;

the nucleotide sequence of the fusion homologous region is shown as SEQ ID NO. 3;

the nucleotide sequences of the specificity sites attB1 and attB2 are respectively shown as SEQ ID NO.10 and SEQ ID NO. 11;

the nucleotide sequence of the homologous complementary sequence (Linker) is shown as SEQ ID NO. 12;

(2) specifically recombining and integrating the fusion fragment obtained in the step (1) to a suicide plasmid pHG1.0 vector (the nucleotide sequence is shown as SEQ ID NO. 13) through att sites, transferring the constructed plasmid into escherichia coli WM3064 (a commercial strain), transferring the WM3064 and the Shewanella MR-1 in a combined manner, and transferring the constructed plasmid into the Shewanella MR-1. Shewanella MR-1 needs to be cultured in LB medium at 30 ℃ whereas Escherichia coli WM3064, the growth of which needs to be cultured at 37 ℃ with the addition of 0.3M/mL DAP (2, 6-diaminopimelic acid) in LB medium.

a. att site-specific recombination reaction system (5 uL):

fusion fragment 1 uL: (_～75ng)

pHG1.0:1uL(_～75ng)

BP clonase II enzyme mix:1uL

H₂O:2uL

b. Gateway BP clonase II enzyme reaction program:

mixing the above components, reacting at 25 deg.C for 5 hr, and cooling to 4 deg.C for storage;

c. WM3064 chemical conversion step:

1. 50ul WM3064 competent cells were taken from-80 ℃ freezer and placed on ice for 10 min;

2. uniformly mixing the reaction compound in the step b and WM3064 competent cells in a centrifugal tube of 1.5mL, and carrying out ice bath for 30 min;

3. then thermally shocking for 90s at 42 ℃ in a water bath kettle, and carrying out ice bath again for 5 min;

4. adding 1mL LB medium containing 0.3M/mL DAP to the system, culturing at 37 deg.C and 150rpm for 1 hr;

5. coating 200uL of culture solution on an LB plate (containing 15ug/mL gentamicin), and culturing at 30 ℃ for 12 hours;

6. single colonies were picked for PCR verification and the correct WM3064 was verified for conjugal transfer with Shewanella MR-1.

(3) And (3) culturing the recombinant strain obtained in the step (2) on an LB (containing 15ug/mL gentamicin) plate, selecting a single colony for PCR verification, and obtaining the recombinant strain with the suicide plasmid integrated into the MR-1 genome through resistance screening and colony PCR verification. The primers used in colony PCR were 3941-LF (on the MR-1 genome) and F primer (on the suicide plasmid pHG1.0). The nucleotide sequence of the primer F primer is shown as SEQ ID NO. 14;

(4) culturing the strains verified to be correct in the step (3) in LB (containing 15ug/mL gentamicin) liquid culture medium overnight, then transferring the strains to non-resistant LB culture medium according to the proportion of 1% to culture until the OD value is about 0.4, coating 200uL of the strains on an LB plate which does not contain NaCl and contains 10% of sucrose by mass concentration, and culturing for 12 hours at 30 ℃. Then picking single colony to carry out PCR verification and carrying out sequencing verification; through sucrose negative screening and colony PCR verification, the SO _3941 gene knockout Shewanella delta 3941 is obtained. The correct recombinant strains were verified and stored in a-80 ℃ freezer for subsequent experiments.

Example 2: electricity-producing Shewanella recombinant strain delta 3941 and wild-type Shewanella MR-1MFC electricity production

1. Activation of bacterial strains

The electro-shewanella recombinant strain delta 3941 and the wild-type shewanella MR-1 were taken out from a refrigerator at-80 ℃ and cultured overnight in LB medium at 30 ℃ and 200rpm, respectively. The overnight culture broth was transferred to a new LB medium at a rate of 1% for 10 hours at 30 ℃ under 200rpm, measured for OD 600, calculated for volume (OD 600 in MFC: 0.5), centrifuged at 5000rpm for 10 minutes, resuspended in anolyte, and added to the MFC anode compartment.

2. MFC power generation

The experimental setup used a two-chamber MFC,110mL anode chamber (containing 110mL of both the thalli and anolyte) and 110mL cathode chamber, the size of the anode carbon cloth electrode was 1cm × 1cm, the size of the cathode carbon cloth electrode was 2.5cm × 3cm, the two chambers were separated by proton exchange membrane, which was soaked overnight with 1M hydrochloric acid aqueous solution before use, and washed three times with sterile distilled water.

The anolyte contained 6g/L disodium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 0.5g/L sodium chloride, 1g/L ammonium chloride, 1mM magnesium sulfate, 0.1mM calcium chloride, 20mM sodium lactate, 5% LB medium, and the balance water. The catholyte comprised 50mM potassium ferricyanide, 50mM dipotassium hydrogen phosphate and 50mM potassium dihydrogen phosphate, the balance being water. MFC is placed in a 30 ℃ incubator, and the anode and the cathode are connected with an external resistor of 2K omega.

3. Analysis of electrochemical effects

Cyclic Voltammetry (CV) was performed using silver chloride as a reference electrode, and scanned at a sweep rate of 1mV/s using a multichannel electrochemical workstation CHI 1000C. Linear Sweep Voltammetry (LSV) was swept from-0.87V to-0.1V at a sweep rate of 0.1mV/s, using a multichannel electrochemical workstation CHI 1000C.

4. Results

As can be seen from figure 2, the electrogenesis recombinant Shewanella delta 3941MFC has better electrogenesis performance, the highest output voltage is 113.3mV, and is improved by 30 percent compared with the original strain wild Shewanella MR-1;

the extracellular electron transfer efficiency of MFC can be further studied by bioelectrochemical analysis, and FIG. 3 shows a Linear Sweep Voltammogram (LSV) with a sweep rate of 0.1mV/s, i.e., a polarization curve, from which it can be seen that the maximum current density for MFC power generation using recombinant shewanella bacteria Δ 3941 is about 344mA/m²Maximum power density of 107.5mW/m²Relative toThe original strains, wild Shewanella MR-1, are greatly improved.

Table 1: primer and sequence thereof related to the invention

Sequence listing

<110> leading edge technology research institute of Tianjin university

<120> electricity-generating Shewanella recombinant strain, construction method and application

<160>15

<170>SIPOSequenceListing 1.0

<210>1

<211>954

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ggggacaagt ttgtacaaaa aagcaggctg ccctcccaat tacaacatac agataacatt 60

taattgatag aagcacataa gatttataag gacaagacaa caaataacct tcactgaaaa 120

ataaatgaag catcaaaaga tgaataatgc atttatttat aaataaaaat tttattagac 180

agatagttaa ctcactttat taaacgctaa cttgttttaa tccgtttatc ctaaacacaa 240

attgacagct tattatttaa ccgtcaaatt taagggctac cataccaaat aatggtttta 300

tctcaatttg acctaatgtc tatgctatag tgacgatgac attcactcat tgctgacatt 360

cctttgaaca aaatcgctct ctttgtcgat gtgcaaaata tttattacac ctgtcgcgag 420

gcatatcagc gccaatttaa ctatcgcaaa ctgtggcaac atttaagcac acagggagaa 480

attgtcagtg ctgtcgctta tgccattcat cggggcgatg atggccaatt aaagttccaa 540

gatgccttaa ggcatatcgg ctttgagcta aaactaaagc cctttattca acgcagtgat 600

ggatcggcaa aaggagattg ggatgtaggg ataacgattg atgtacttga tgctgccccc 660

aatgtagata cagtaatact gctttctggc gatggtgatt ttgcgatatt gcttgagaaa 720

ataacgcaaa aatatggcgt aaaagcagag gtctatggtg tacctcaact gacggctaaa 780

gctctgatgg atgccacaac gcaatttaac ccgattgatg atgttttact gctctaacag 840

ctataaacct taagtttgaa actcttgggt tgataaatga taaaaattat ctatgtcagc 900

caacaatata ctgtaaacac cagcatctaa ctgcccgctt cagtggctaa cacc 954

<210>2

<211>752

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gcttcagtgg ctaacaccgc cgtggcaacg gctaacaacg catgcctcac atccatgtag 60

actttgaagt catggggcag aaaggcattc attgaatttt cctaattaat tatttgttcc 120

tcgctaaaat atagtcaata ccttgataat tagctgcaaa atacaaaaaa gacctgaaat 180

ttcaggtctt ttctatcgat attaaataga acttatcaca caatgaagtt agacctctag 240

cgtcctctta cgacggccgc gttttactaa ggagtagtaa aacagtggtg tcagaatgag 300

accaaagata gtgactccaa tcatacctgc aaataccgcc actcccatgg cctgacgcat 360

ctcagcgccc gcccccgtcg aaaacaccat aggcaccact cccatgataa aggcgataga 420

agtcatcaag atggggcgca gccttaagcg tgccgcctca agaattgact ccataacctc 480

cataccatgg tcttgtttct ctttggcaaa ttcgacaatc agaatcgcgt tcttcgtcgc 540

taagcccacg aggacgataa gaccaatctg ggtgaaaata ttattatctc caccatagag 600

aagcacacca ctgagcgccg agagtaaggt cattgggata attaagataa tcgccaatgg 660

taaacttaaa ctctcgtatt gcgccgcgag caccataaac accagcacaa tcaccagcgg 720

gaaagcctgc ttttttgtac aaacttgtcc cc 752

<210>3

<211>1688

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ggggacaagt ttgtacaaaa aagcaggctg ccctcccaat tacaacatac agataacatt 60

taattgatag aagcacataa gatttataag gacaagacaa caaataacct tcactgaaaa 120

ataaatgaag catcaaaaga tgaataatgc atttatttat aaataaaaat tttattagac 180

agatagttaa ctcactttat taaacgctaa cttgttttaa tccgtttatc ctaaacacaa 240

attgacagct tattatttaa ccgtcaaatt taagggctac cataccaaat aatggtttta 300

tctcaatttg acctaatgtc tatgctatag tgacgatgac attcactcat tgctgacatt 360

cctttgaaca aaatcgctct ctttgtcgat gtgcaaaata tttattacac ctgtcgcgag 420

gcatatcagc gccaatttaa ctatcgcaaa ctgtggcaac atttaagcac acagggagaa 480

attgtcagtg ctgtcgctta tgccattcat cggggcgatg atggccaatt aaagttccaa 540

gatgccttaa ggcatatcgg ctttgagcta aaactaaagc cctttattca acgcagtgat 600

ggatcggcaa aaggagattg ggatgtaggg ataacgattg atgtacttga tgctgccccc 660

aatgtagata cagtaatact gctttctggc gatggtgatt ttgcgatatt gcttgagaaa 720

ataacgcaaa aatatggcgt aaaagcagag gtctatggtg tacctcaact gacggctaaa 780

gctctgatgg atgccacaac gcaatttaac ccgattgatg atgttttact gctctaacag 840

ctataaacct taagtttgaa actcttgggt tgataaatga taaaaattat ctatgtcagc 900

caacaatata ctgtaaacac cagcatctaa ctgcccgctt cagtggctaa caccgccgtg 960

gcaacggcta acaacgcatg cctcacatcc atgtagactt tgaagtcatg gggcagaaag 1020

gcattcattg aattttccta attaattatt tgttcctcgc taaaatatag tcaatacctt 1080

gataattagc tgcaaaatac aaaaaagacc tgaaatttca ggtcttttct atcgatatta 1140

aatagaactt atcacacaat gaagttagac ctctagcgtc ctcttacgac ggccgcgttt 1200

tactaaggag tagtaaaaca gtggtgtcag aatgagacca aagatagtga ctccaatcat1260

acctgcaaat accgccactc ccatggcctg acgcatctca gcgcccgccc ccgtcgaaaa 1320

caccataggc accactccca tgataaaggc gatagaagtc atcaagatgg ggcgcagcct 1380

taagcgtgcc gcctcaagaa ttgactccat aacctccata ccatggtctt gtttctcttt 1440

ggcaaattcg acaatcagaa tcgcgttctt cgtcgctaag cccacgagga cgataagacc 1500

aatctgggtg aaaatattat tatctccacc atagagaagc acaccactga gcgccgagag 1560

taaggtcatt gggataatta agataatcgc caatggtaaa cttaaactct cgtattgcgc 1620

cgcgagcacc ataaacacca gcacaatcac cagcgggaaa gcctgctttt ttgtacaaac 1680

ttgtcccc 1688

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

caaccgcaag tgcttcagac 20

<210>5

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

atcggactaa agcgaaacag c 21

<210>6

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ggggacaagt ttgtacaaaa aagcaggctg ccctcccaat tacaacatac 50

<210>7

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ggtgttagcc actgaagcgg gcagttagat gctggtgt 38

<210>8

<211>48

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ggggaccact ttgtacaaga aagctgggtt tcccgctggt gattgtgc 48

<210>9

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gcttcagtgg ctaacaccgc cgtggcaacg gctaacaac 39

<210>10

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ggggacaagt ttgtacaaaa aagcaggct 29

<210>11

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ggggaccact ttgtacaaga aagctgggt 29

<210>12

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

gcttcagtgg ctaacacc 18

<210>13

<211>10381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta 60

aaacacaaca tatccagtca ctatgaatca actacttaga tggtattagt gacctgtagt 120

cgactaagtt ggcagcatca cccgacgcac tttgcgccga ataaatacct gtgacggaag 180

atcacttcgc agaataaata aatcctggtg tccctgttga taccgggaag ccctgggcca 240

acttttggcg aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg 300

aaataagatc actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag 360

ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 420

aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 480

tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct 540

ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag 600

acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa 660

ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca 720

tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta 780

ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa 840

acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc 900

aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc tgtgatggct 960

tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg 1020

cgtaaacgcc gcgtggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg 1080

ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag 1140

gtatgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa 1200

ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt 1260

cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg 1320

tttattgaaa tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa 1380

ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat 1440

tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga 1500

taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat 1560

gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag 1620

ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc 1680

aggctccctt atacacagcc agtctgcagg tcgatacagt agaaattaca gaaactttat 1740

cacgtttagt aagtatagag gctgaaaatc cagatgaagc cgaacgactt gtaagagaaa 1800

agtataagag ttgtgaaatt gttcttgatg cagatgattt tcaggactat gacactagcg 1860

tatatgaata ggtagatgtt tttattttgt cacacaaaaa agaggctcgc acctcttttt 1920

cttatttctt tttatgattt aatacggcat tgaggacaat agcgagtagg ctggatacga 1980

cgattccgtt tgagaagaac atttggaagg ctgtcggtcg actaagttgg cagcatcacc 2040

cgaagaacat ttggaaggct gtcggtcgac tacaggtcac taataccatc taagtagttg 2100

attcatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa 2160

atctaattta atatattgat atttatatca ttttacgttt ctcgttcagc ttttttgtac 2220

aaagttggca ttataaaaaa gcattgctca tcaatttgtt gcaacgaaca ggtcactatc 2280

agtcaaaata aaatcattat ttggggcccg agatccatgc tagcgtgagc tcgatatcgc 2340

atgcggtacc tctagaagaa gcttgggatc cgtcgacgcg tcgatccggt gattgattga 2400

gcaagctagc tttatgcttg taaaccgttt tgtgaaaaaa tttttaaaat aaaaaagggg 2460

acctctaggg tccccaatta attagtaata taatctatta aaggtcattc aaaaggtcat 2520

ccaccggatc agcttagtaa agccctcgct agattttaat gcggatgttg cgattacttc 2580

gccaactatt gcgataacaa gaaaaagcca gcctttcatg atatatctcc caatttgtgt 2640

agggcttatt atgcacgctt aaaaataata aaagcagact tgacctgata gtttggctgt 2700

gagcaattat gtgcttagtg catctaacgc ttgagttaag ccgcgccgcg aagcggcgtc 2760

ggcttgaacg aattgttaga cattatttgc cgactacctt ggtgatctcg cctttcacgt 2820

agtggacaaa ttcttccaac tgatctgcgc gtcgaattaa ttctcgaatt gacataagcc 2880

tgttcggttc gtaaactgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc 2940

ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt 3000

ttttttgtac agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga 3060

tgtttgatgt tatggagcag caacgatgtt acgcagcagc aacgatgtta cgcagcaggg 3120

cagtcgccct aaaacaaagt taggtggctc aagtatgggc atcattcgca catgtaggct 3180

cggccctgac caagtcaaat ccatgcgggc tgctcttgat cttttcggtc gtgagttcgg 3240

agacgtagcc acctactccc aacatcagcc ggactccgat tacctcggga acttgctccg 3300

tagtaagaca ttcatcgcgc ttgctgcctt cgaccaagaa gcggttgttg gcgctctcgc 3360

ggcttacgtt ctgcccaagt ttgagcagcc gcgtagtgag atctatatct atgatctcgc 3420

agtctccggc gagcaccgga ggcagggcat tgccaccgcg ctcatcaatc tcctcaagca 3480

tgaggccaac gcgcttggtg cttatgtgat ctacgtgcaa gcagattaca gtgacgatcc 3540

cgcagtggct ctctatacaa agttgggcat acgggaagaa gtgatgcact ttgatatcga 3600

cccaagtacc gccacctaac aattcgttca agccgagatc ggcttcccgg ccaattcgac 3660

gcgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 3720

atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 3780

gcacccagcc tgcgcgagca ggggaattga tccggtggat gaccttttga atgaccttta 3840

atagattata ttactaatta attggggacc ctagaggtcc ccttttttat tttaaaaatt 3900

ttttcacaaa acggtttaca agcataaagc tagcttgctc aatcaatcac cggatcctct 3960

agagtcgacg cgtcgactct agaggatcga tcctttttaa cccatcacat atacctgccg 4020

ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 4080

aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 4140

attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 4200

agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 4260

aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 4320

gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 4380

tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 4440

agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 4500

ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 4560

ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 4620

agcaaaaaaa tgaaaaatat caagttcctg aattcgattc gtccacaatt aaaaatatct 4680

cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 4740

tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 4800

atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 4860

aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 4920

aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 4980

tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 5040

ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 5100

tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 5160

acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 5220

aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 5280

tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 5340

ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 5400

agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 5460

taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca 5520

ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 5580

ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 5640

tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 5700

ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 5760

cagacaaaca atcaacgttt gcgccaagct tcctgctgaa catcaaaggc aagaaaacat 5820

ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 5880

aaagaaaatg ccgatatcct attggcattt tcttttattt cttatcaaca taaaggtgaa 5940

tcccatatga actatataaa agcaggcaaa tggctaaccg tattcctaac cttttggtaa 6000

tgactccaac ttattgatag tgttttatgt tcagataatg cccgatgact ttgtcatgca 6060

gctccaccga ttttgagaac gacagcgact tccgtcccag ccgtgccagg tgctgcctca 6120

gattcacgtt atgccgctca attcgctgcg tatatcgctt gctgattacg tgcagctttc 6180

ccttcaggcg ggattcatac agcggccagc catccgtcat ccatatcacc acgtcaaagg 6240

gtgacagcag gctcataaga cgccccagcg tcgccatagt gcgttcaccg aatacgtgcg 6300

caacaaccgt cttccggaga ctgtcatacg cgtaaaacag ccagcgctgg cgcgatttag 6360

ccccgacata gccccactgt tcgtccattt ccgcgcagac gatgacgtca ctgcccggct 6420

gtatgcgcga ggttaccgac tgcggcctga gttttttaag tgacgtaaaa tcgtgttgag 6480

gccaacgccc ataatgcggg ctgttgcccg gcatccaacg ccattcatgg ccatatcaat 6540

gattttctgg tgcgtaccgg gttgagaagc ggtgtaagtg aactgcaggt ggcacttttc 6600

ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 6660

cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 6720

gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 6780

ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 6840

tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 6900

aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg 6960

ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 7020

agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 7080

gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 7140

gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 7200

gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 7260

cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 7320

ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 7380

cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 7440

gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 7500

cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 7560

tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttat 7620

ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct 7680

atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc 7740

ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 7800

ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc aaggagatgg 7860

cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca 7920

tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag 7980

caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatccttt 8040

ttgtccggtg ttgggttgaa ggtgaagccg gtcggggccg cagcgggggc cggcttttca 8100

gccttgcccc cctgcttcgg ccgccgtggc tccggcgtct tgggtgccgg cgcgggttcc 8160

gcagccttgg cctgcggtgc gggcacatcg gcgggcttgg ccttgatgtg ccgcctggcg 8220

tgcgagcgga acgtctcgta ggagaacttg accttccccg tttcccgcat gtgctcccaa 8280

atggtgacga gcgcatagcc ggacgctaac gccgcctcga catccgccct caccgccagg 8340

aacgcaaccg cagcctcatc acgccggcgc ttcttggccg cgcgggattc aacccactcg 8400

gccagctcgt cggtgtagct ctttggcatc gtctctcgcc tgtcccctca gttcagtaat 8460

ttcctgcatt tgcctgtttc cagtcggtag atattccaca aaacagcagg gaagcagcgc 8520

ttttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc 8580

tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc 8640

caacggcgtc agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc 8700

ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg 8760

ccggctaccg ccggcgtaac agatgagggc aagcggatgg ctgatgaaac caagccaacc 8820

aggaagggca gcccacctat caaggtgtac tgccttccag acgaacgaag agcgattgag 8880

gaaaaggcgg cggcggccgg catgagcctg tcggcctacc tgctggccgt cggccagggc 8940

tacaaaatca cgggcgtcgt ggactatgag cacgtccgcg agctggcccg catcaatggc 9000

gacctgggcc gcctgggcgg cctgctgaaa ctctggctca ccgacgaccc gcgcacggcg 9060

cggttcggtg atgccacgat cctcgccctg ctggcgaaga tcgaagagaa gcaggacgag 9120

cttggcaagg tcatgatggg cgtggtccgc ccgagggcag agccatgact tttttagccg 9180

ctaaaacggc cggggggtgc gcgtgattgc caagcacgtc cccatgcgct ccatcaagaa 9240

gagcgacttc gcggagctgg tgaagtacat caccgacgag caaggcaaga ccgagcgcct 9300

gggtcacgtg cgcgtcacga actgcgaggc aaacaccctg cccgctgtca tggccgaggt 9360

gatggcgacc cagcacggca acacccgttc cgaggccgac aagacctatc acctgctggt 9420

tagcttccgc gcgggagaga agcccgacgc ggagacgttg cgcgcgattg aggaccgcat 9480

ctgcgctggg cttggcttcg ccgagcatca gcgcgtcagt gccgtgcatc acgacaccga 9540

caacctgcac atccatatcg ccatcaacaa gattcacccg acccgaaaca ccatccatga 9600

gccgtatcgg gcctaccgcg ccctcgctga cctctgcgcg acgctcgaac gggactacgg 9660

gcttgagcgt gacaatcacg aaacgcggca gcgcgtttcc gagaaccgcg cgaacgacat 9720

ggagcggcac gcgggcgtgg aaagcctggt cggctggatc cggccacgat gcgtccggcg 9780

tagaggatct gaagatcagc agttcaacct gttgatagta cgtactaagc tctcatgttt 9840

cacgtactaa gctctcatgt ttaacgtact aagctctcat gtttaacgaa ctaaaccctc 9900

atggctaacg tactaagctc tcatggctaa cgtactaagc tctcatgttt cacgtactaa 9960

gctctcatgt ttgaacaata aaattaatat aaatcagcaa cttaaatagc ctctaaggtt 10020

ttaagtttta taagaaaaaa aagaatatat aaggctttta aagcttttaa ggtttaacgg 10080

ttgtggacaa caagccaggg atgtaacgca ctgagaagcc cttagagcct ctcaaagcaa 10140

ttttgagtga cacaggaaca cttaacggct gacatgggaa ttccacatgt ggaattccac 10200

atgtggaatt gtgagcggat aacaatttgt ggaattcccg ggagagctcg agctgcagct 10260

ggatggcaaa taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat 10320

aagcaatgct ttcttataat gccaactttg tacaagaaag ctgaacgaga aacgtaaaat 10380

g 10381

<210>14

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

agggatgtaa cgcactgaga agc 23

<210>15

<211>1269

<212>DNA

<213> Shewanella oneidensis (shewanella oneidensis)

<400>15

atgaatgcct ttctgcccca tgacttcaaa gtctacatgg atgtgaggca tgcgttgtta 60

gccgttgcca cggcattgga ttttgttggg gtagatgatt tgcaccacgg ccatagggtt 120

gcctatatgg cttatgaatg tgcaagtgtg ctcggctggc ctgatgaaaa aaagcagttc 180

gcctattttg cggggcttat ccatgattgc ggcgtttcct cctccgaaga acacttacgt 240

ctacttaaat taatgcaacc ggaagatgcc cattgtcata gcaaacgtgg ctatgaggca 300

ttgcttgaat gcccgatttt agatgttttt gctcctatcg ttctatacca ccacacgcct 360

tggcttgagc tgcaatccca tgatctttct gtctttgagc gtgatattgc tgcactgatt 420

ttcttagcgg atcgtaccga ctttttaagg gccagatata cgcatggttg ccatgaggag 480

ctgattacat tgcatgaaag tatggttgct gagaatttat tggctcactg cggaacctta 540

tttgaaccag aaatggtcaa tgctatgtgc caactggtta agaaggatgg cttttggtac 600

aacatggatg caactcatat tgagttgtta ggtttggagt tcaaagctaa tcacttttac 660

gacaaagagt tagatatcgg aggtgtgaag caattagcgc gatttttagc tcgaatagtc 720

gatgctaaga gtccctttac atttcaccat tcagaaaaag tcgcattgtt agcaaaatta 780

gtggcaaagg actgcggtat atcagatacc gatgctgaat tattatacgt cgcgggatta 840

ttacatgatg tcggtaagct taaaacgcct gacctattgc tgcataaaga aggcaaatta 900

acgaaggaag agtattcgat tgtcaaacgt catactgttg atacagaaca tactttacat 960

cgttttttcc ccaagtcggt gattggcgaa tgggcctcaa atcaccatga gcgtctcgat 1020

ggttctggct atccttttag taagcggcag gaacaattag atttaccttc gcgtattctt 1080

gctgtagtcg atgtttttca ggcgctgact caaaagcgac catatcgagg ctctttgtcg 1140

ctatcagaga tttacgacat tatgcagccc atggtcgata aggggcagtt agatgctggt 1200

gtttacagta tattgttggc tgacatagat aatttttatc atttatcaac ccaagagttt 1260

caaacttaa 1269

Claims (3)

1. A method for constructing an electricity-generating Shewanella recombinant strain is characterized by comprising the following steps: knocking out a target gene SO _3941 of wild Shewanella oneidensis MR-1 to obtain an electric Shewanella recombinant strain delta 3941, wherein the nucleotide sequence of the gene SO _3941 is shown as SEQ ID NO 15.

2. An electrogenic shewanella recombinant strain Δ 3941 constructed by the method of claim 1.

3. Use of the power-producing shewanella recombinant strain of claim 2 to produce electricity Δ 3941.

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