CN115160416A - AraC mutant for inducing submarine metal ion Cd (II), constructed submerged microorganism detection sensor and application thereof - Google Patents
AraC mutant for inducing submarine metal ion Cd (II), constructed submerged microorganism detection sensor and application thereof Download PDFInfo
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Abstract
The invention discloses an AraC mutant for inducing submarine metal ions Cd (II), a constructed submerged microorganism detection sensor and application thereof. The AraC mutant is AraCmt-Cd, the amino acid sequence of the AraC mutant is shown as SEQ ID NO.10, and the nucleotide sequence of the coding gene of the AraC mutant is shown as SEQ ID NO. 9. The mutant AraCmt-Cd can specifically bind to metal ion Cd (II) and is induced to activate P BAD A promoter, thereby driving expressionThe free electrons transfer chromoprotein (CymA), and the output of electrochemical signals after metal Cd (II) ions are induced is realized. The metal ion sensing element and the electric signal reporting element-containing submarine detecting microbial sensor constructed by the invention can sense metal ions released by a submarine in water to generate changes of electrochemical signals, so that the submarine is detected in real time.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and molecular biology, and particularly relates to an AraC mutant of an induction submarine metal ion Cd (II), a constructed submerged microbe detection sensor and application thereof.
Background
The submarine is an important component of modern navy and has the characteristics of high concealment and strong hitting power. The submarine main body structure is composed of metal, metal ions can be released around a submarine body through oxidation reduction and the like in a seawater salt solution system, and the metal ions can be sensed by a microorganism-related sensing element and then detected.
In cell-based biosensors, a biological reaction is converted into a physicochemical signal by a sensing module. In these systems, reports such as color development, bioluminescence, and fluorescence are widely used. The reporter gene should be selected with regard to sensitivity and ease of measurement. Since transcription of the reporter gene is controlled by the promoter/operator, the expression level of the reporter gene is designed to be directly proportional to the sensor signal. There are two different reporting methods. (1) The reporter gene encodes an enzyme that catalyzes the formation of a measurable product. For example, β -galactosidase and luciferase catalyze the production of colored products and light, respectively. (2) The protein itself is the product being measured (e.g., green fluorescent protein [ GFP ]). According to the invention, through the construction and optimization of a microbial electric signal reporting element, after an electron metabolism transfer path of microbial induction metal ions is successfully established, detection of electric signals generated by metal induction is realized through electrochemical means such as Cyclic Voltammetry (CV) and timing current by an extracellular electron transfer coupling in-vitro electrochemical device, and the influence of different induction elements, different metal ions and different metal ion concentrations on electrochemical signal intensity is further researched and explored.
Wild type AraC-P BAD The system comprises the following steps: transcription factorThe sub-AraC is capable of responding naturally to L-arabinose, binding of the AraC dimer in the absence of L-arabinose I 1 And O 2 Site at P BAD A DNA loop is formed upstream of the promoter to inhibit transcription; upon binding of L-arabinose, the AraC dimer changes conformation such that it associates with the adjacent I 1 And I 2 Half-site binding activates transcription. The AraC mutant obtained by mutation can bind to compounds other than L-arabinose to activate P BAD Transcriptional activity of the promoter, which allows the AraC mutant to be induced by other compounds. Such AraC-P is compared to a promoter that directly senses a metal ion BAD The system has an amplification effect, and the signal is stronger.
Disclosure of Invention
The invention aims to provide an AraC mutant for inducing submarine metal ions Cd (II), a potential microbe detection sensor constructed by the AraC mutant and application of the potential microbe detection sensor. The AraC mutant AraCmt-Cd can be specifically combined with metal ions Cd (II), and the potential detecting microbial sensor prepared by the AraC mutant AraCmt-Cd realizes the output of electrochemical signals after the metal ions Cd (II) are induced.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides an AraC mutant, which is AraCmt-Cd, and the amino acid sequence of the AraC mutant is shown as SEQ ID NO. 10.
Furthermore, the AraC mutant AraCmt-Cd has the function of obviously inducing submarine metal ions Cd (II).
The invention also provides a coding gene of the AraC mutant, and the coding gene of the AraC mutant has one of the following nucleotide sequences:
(1) A nucleotide sequence shown as SEQ ID NO. 9;
(2) Has more than 95 percent of homology with the nucleotide sequence shown in SEQ ID NO.9, and can encode the nucleotide sequence of the amino acid sequence shown in SEQ ID NO. 10.
The invention also provides a latent microbe detecting sensor containing the AraC mutant, wherein the latent microbe detecting sensor simultaneously comprises an encoding gene, an electron transfer chromoprotein gene and a promoter element of the AraC mutant.
Further, the electron transfer chromoprotein gene is a CymA gene with a nucleotide sequence shown as SEQ ID NO. 18.
Furthermore, the amino acid sequence of the electron transfer chromoprotein CymA is shown in SEQ ID NO. 19.
Further, the preparation method of the latent microbe detection sensor comprises the following steps:
(1) Amplifying an electron transfer chromoprotein gene CymA and a promoter element pBAD;
(2) Connecting the amplified fragment in the step (1) with an AraC mutant coding gene fragment in a concentration ratio of 1: 1, and cloning to an expression vector pACYCDuet-1 to obtain a recombinant plasmid pACYC-AraCmt-Cd-PBAD-CymA;
(3) And (3) transforming the recombinant plasmid pACYC-AraCmt-Cd-PBAD-CymA into host escherichia coli to obtain a recombinant strain E.coli/pACYC-AraCmt-Cd-PBAD-CymA, namely the potential detecting microbial sensor.
The invention also provides application of the AraC mutant or the latent microbe detecting sensor in preparing an electrochemical device for detecting submarine metal ions in real time.
Furthermore, the electrochemical device is composed of a working electrode, a reference electrode, a counter electrode and the submarine detection microorganism sensor, can sense metal ions released by the submarine, and outputs electrochemical signals of the submarine detection microorganism sensor through difference after sensing the metal ions, so that the aim of detecting the submarine in real time is fulfilled.
Further, the concentration of metal ions of the submarine detected by the electrochemical device is not lower than 5 mu mol/L.
Further, the submarine metal ions are Cd (II) ions.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention obtains a brand new mutant AraCmt-Cd by screening through a directed evolution method, has the effect of remarkably inducing metal ions Cd (II) of a submarine, can be used for constructing a sensor for detecting latent microorganisms, and is further applied to the submarine and the real-time monitoring of the metal ions of the submarine. The method for screening the AraC mutant can also be suitable for screening other AraC mutants capable of inducing submarine metal ions, and is simple and rapid. The AraC mutant obtained by the invention can induce trace metal ions and has good specificity. The microbial sensor constructed by the invention has a good effect of sensing Cd (II), and can detect trace Cd (II) through an electrochemical signal, so that the microbial sensor has a great application prospect.
Drawings
FIG. 1 shows the fluorescence detection result of the screened mutant AraCmt-Cd inductive metal ion Cd (II).
FIG. 2 is a plasmid map of the constructed vector pACYC-AraCmt-Cd-PBAD-CymA.
FIG. 3 is a diagram of the electrochemical signal output of the constructed latent microbe detecting sensor containing an electric signal element under the condition of the presence or absence of metal ions Cd (II).
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
The examples do not show the specific techniques or conditions, and the techniques described in the literature in the field or the product specifications are followed. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
Example 1: screening of AraC mutants
1. Construction of basic vector pACYCDuet-1-T7-AraC-EGFP-PBAD
Carrying out Polymerase Chain Reaction (PCR) by taking a plasmid pBAD24 (GenBank: X81837.1) as a template and a primer P-AraC-F and a primer P-AraC-R, amplifying an Arac fragment, a primer araBAD-F and a primer araBAD-R, and amplifying an araBAD promoter fragment; and amplifying the EGFP fragment by using a plasmid pEGFP-1 (GenBank: U55761.1) as a template and using a primer p-EGFP-F and a primer p-EGFP-R.
The primer sequences are shown below:
P-AraC-F：
P-AraC-R：
araBAD-F：
araBAD-R：
p-EGFP-F：
p-EGFP-R：
the PCR product was purified by gel recovery using a gel recovery purification kit (Vazyme, cat # DC 301-01).
The plasmid pACYCDuet-1 (Novagen, cat. No. 71147-3) was double-digested with restriction enzyme 1Bgl II (TaKaRa, cat. No. 1606) and restriction enzyme 2Nde I (TaKaRa, cat. No. 1621), and purified for liquid recovery.
The three PCR products were ligated to the digested pACYCDuet-1 plasmid by means of a seamless cloning method using 2 XClon Express Mix (Vazyme, cat # C115) as follows:
the system was incubated at 50 ℃ for 30min. Coli DH5 alpha competence is transformed, the product is coated on an LB solid plate containing 34mg/L chloramphenicol, positive clones are screened by PCR, and recombinant plasmid pACYCDuet-1-T7-AraC-EGFP-PBAD is extracted from the positive clones.
2. Construction of AraC mutant library
(1) Carrying out error-prone polymerase chain reaction (error-prone PCR), using a vector pACYCDuet-1-T7-AraC-EGFP-PBAD as a template, and amplifying by using primers AraC-F and AraC-R to obtain an M-AraC fragment with a random mutation site, wherein a PCR amplification system is shown as follows:
the PCR procedure was: 3min at 95 ℃;30 × (95 ℃ C. 15s,58 ℃ C. 30s,72 ℃ C. 20 s); 5min at 72 ℃; infinity at 16 ℃.
The primer sequence is as follows:
AraC-F：
AraC-R：
the PCR product M-AraC fragment was purified by gel recovery using a gel recovery purification kit (Vazyme, cat # DC 301-01).
(2) The plasmid pACYCDuet-1-T7-EGFP-PBAD was digested in two ways with restriction enzyme 1Bgl II (TaKaRa, cat 1606) and restriction enzyme 2Nde I (TaKaRa, cat 1621) in the following restriction enzyme systems:
the enzyme digestion system is incubated for 1h at 37 ℃ for gel recovery and purification.
(3) Cloning the M-AraC fragment onto a vector fragment by utilizing seamless cloning, wherein the system is as follows:
the ligation was incubated at 50 ℃ for 30min. The ligation product was transformed into E.coli BL 21-. DELTA.AraC competent cells, spread on LB solid plates containing 34mg/L chloramphenicol, and cultured overnight to obtain a random AraC mutant library.
3. Large Scale screening of Cd (II) -induced AraC mutant libraries
(1) Preliminary screening of the mutant library
The plate obtained above was transferred to an M9Y plate containing 34mg/L chloramphenicol and 10. Mu. Mol/L Cd (II) added thereto through a nitric acid microporous membrane, and the temperature was kept constant at 37 ℃ for 12 hours. When the plate with the transferred membrane has obvious colony growth, the plate is subjected to fluorescence observation through an ultraviolet lamp, and a single colony with obvious fluorescence is obtained by screening, namely the AraC mutant inducing Cd (II).
(2) Rescreening through microplate reader
Single colonies obtained by primary screening were picked and activated by shaking overnight at 37 ℃. The next day, the cells were transferred to 10mL of M9Y medium and cultured to OD 600 And (3) when the concentration is 0.2, adding Cd (II) with the concentrations of 0 mu mol/L, 5 mu mol/L and 10 mu mol/L respectively, putting 200 mu L of the induced bacterial liquid into a 96 micro-porous plate, then putting the micro-porous plate into an enzyme-linked immunosorbent assay (Biotek) for shake culture at 30 ℃, monitoring the fluorescence intensity (RFU) in real time at constant temperature, detecting every 30min, and monitoring for 12h in total.
The results are shown in FIG. 1, and among 20 strains in the primary screening, 1 strain M-16 is screened, and the fluorescence intensity (RFU) of the strain M-16 shows different changes under the action of different concentrations of Cd (II), and the RFU value is higher when the concentration of Cd (II) is higher.
4. Identification of Cd (II) -induced AraC mutant
The strain M-16 with remarkable fluorescence effect is transferred into 10mL LB liquid medium added with 34mg/L chloramphenicol for culture, and is cultured by shaking overnight at 37 ℃. Plasmid extraction was performed using a plasmid extraction kit (Vazyme, cat # DC 201-01). And (3) sending the extracted pACYCDuet-1-T7-AraCmt-Cd-EGFP-PBAD plasmid to a sequencing company for sequencing, and finally determining the mutation sequence of the AraC mutant. The sequencing result shows that the nucleotide sequence of the obtained AraC mutant AraCmt-Cd is shown as SEQ ID NO.9, and the coded amino acid sequence thereof is shown as SEQ ID NO. 10.
Example 2: construction of recombinant strain E.coli/pACYC-AraCmt-Cd-PBAD-CymA
1. Performing PCR by taking the S.oneidensis genome as a template and a primer CymA-F and a primer CymA-R to amplify a CymA fragment; taking plasmid pACYCDuet-1-T7-AraCmt-Cd-EGFP-PBAD as a template, a primer AraCmt-Cd-F/R and a primer pBAD-F/R, carrying out PCR, and respectively amplifying an AraCmt-Cd fragment and a pBAD promoter fragment, wherein the PCR amplification system is shown as follows:
the PCR procedure was: 3min at 95 ℃;30 × (95 ℃ 15s,55 ℃ 15s,72 ℃ 1 min); 5min at 72 ℃; infinity at 16 ℃.
The primer sequences are shown below:
CymA-F：
CvmA-R：
AraCmt-Cd-F：
AraCmt-Cd-R：
pBAD-F：
pBAD-R：
the PCR product was purified by gel recovery using a gel recovery purification kit (Vazyme, cat # DC 301-01).
2. The pACYCDuet-1 plasmid was double-digested with restriction enzyme 1Bgl II (TaKaRa, cat # 1606) and restriction enzyme 2Xho I (TaKaRa, cat # 1635) in the following restriction enzymes:
the enzyme digestion system is incubated for 1h at 37 ℃ for gel recovery and purification.
3. Three PCR products were ligated to the digested pACYCDuet-1 vector by means of seamless cloning using 2 × Clon Express Mix (Vazyme, cat # C115) as follows:
the system was incubated at 50 ℃ for 30min. Transforming the E.coli BL 21-delta AraC competence, coating the E.coli BL 21-delta AraC competence on an LB solid plate containing 34mg/L chloramphenicol, screening positive clones by PCR, extracting a recombinant plasmid pACYC-AraCmt-Cd-PBAD-CymA from the positive clones (figure 2), and identifying the recombinant plasmid pACYC-AraCmt-Cd-PBAD-CymA by sequencing, wherein the nucleotide sequence of the plasmid pACYC-AraCmt-Cd-PBAD-CymA is shown in SEQ ID NO. 17.
Example 3: application of recombinant strain E.coli/pACYC-AraCmt-Cd-PBAD-CymA in detection of metal ions Cd (II)
(1) Construction of detection device
A three-electrode electrochemical system is adopted, wherein a working electrode, a reference electrode and a counter electrode are respectively a glassy carbon electrode, an Ag/AgCl electrode and a platinum wire, and a PBS (phosphate buffer solution) containing various metal ions is taken as an electrolyte; firstly, the system is in an open circuit state for 1h, the scanning speed is set to be 1mV/s, the scanning range of an anode test is-0.7 to +0.2V (vs. SCE), and the scanning range of a cathode test is-0.4 to +0.4V (vs. SCE); repeating each scan for 3 cycles, typically with the curves of the second and third scans substantially coinciding, and taking the data of the third scan for analysis; culturing the constructed potential detecting microbial sensor E.coli/pACYC-AraCmt-Cd-PBAD-CymA, collecting thalli, adding into an electrochemical system, and performing CV test and signal acquisition.
(2) Detection with electrical signal as output
And (3) introducing a buffer solution with the concentration content of metal ion Cd (II) of 5 mu mol/L into the constructed device, detecting the electric signal output change of the microbial fuel cell by using an electrochemical workstation, and detecting once every 1 hour for 12 hours in total.
As shown in FIG. 3, the result shows that the signal output of the biosensor containing the signal reporting element is significantly different from that of the sensor without metal ions when 5. Mu. Mol/L of metal ions Cd (II) exist, the detection voltage reaches over 0.25V after 6 hours, and the current density is reduced to a steady value after all the metal ions are eliminated in the later period.
The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding claims.
Sequence listing
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<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ttctccatgg taccctcgag atgaactggc gtgcactatt 40
<210> 12
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gcagcggttt ctttaccaga ttatcctttt ggatatgggt g 41
<210> 13
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gatatacata tggcagatct ttatgacaac ttgacggcta ca 42
<210> 14
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tggtttctta tatccaattg atggctgaag cgcaaaatga 40
<210> 15
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ttcagccatc aattggatat aagaaaccaa ttgtccatat tg 42
<210> 16
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gttcatctcg agggtaccat ggagaaacag tagagagt 38
<210> 17
<211> 5000
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60
gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag 120
ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag 180
taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt 240
gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata 300
tggcagatct ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac 360
gaaactcgct cgggctggcc ccggtgcatt ttttaaatac tcgcgagaaa tagagttgat 420
cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtagtg ctcaaaagca 480
gcttcgcctg actaatgcgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct 540
ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga 600
tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat 660
tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct 720
caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 780
tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaacccg 840
tattggcagg tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 900
aaacccactg gatataccat tcgcgagcct ccggatgacg accgtagtga tgaatcttac 960
ctggcgggaa cagcaaaata tcacccggtc ggcaggcaaa ttctcgtccc tgatttttca 1020
ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 1080
cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 1140
cgttaaacga gtatcccggc agcaggggat cattttgcgc ttcagccatc aattggatat 1200
aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 1260
tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 1320
aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 1380
attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 1440
atcctacctg acgcttttta tcgcaactct ctactgtttc tccatggtac cctcgagatg 1500
aactggcgtg cactatttaa acctagcgca aaatattcaa ttctggcgct gatggtcgtc 1560
ggtattgtta tcggcgtagt gggttatttc gcaacccaac aaaccttaca tgcgacaagt 1620
acggatcagt tctgtatgtc ctgtcacagc aaccattcac ttaaggatga agttcttgca 1680
tctgcccatg gtggtggtcg tgcgggtgtg accgttcaat gtcaagactg tcacttacca 1740
catggccctg tagattattt gatcaaaaaa atcattgtat ctaaagactt atatggtttc 1800
ttaacgattg atggctttaa cactcaagct tggttagatg aaaaccgtaa agagcaagcc 1860
gatctagcac ttaagtattt ccgcagtaat gactccgcta actgtcaaca ctgccatact 1920
cgcatttatg aaaaccagcc agaaactatg aagcctatgg ctgtcagaat gcacactaat 1980
aactttaaga aagatccaga agccagaaaa acctgtgttg actgccataa aggtgtagct 2040
cacccatatc caaaaggata atctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag 2100
cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa 2160
taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaacct 2220
caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag 2280
caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg 2340
ctttcgaatt tctgccattc atccgcttat tatcacttat tcaggcgtag caccaggcgt 2400
ttaagggcac caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta 2460
ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacagacgg catgatgaac 2520
ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatagtgaa 2580
aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac 2640
ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag 2700
gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc 2760
gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca 2820
agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac ggaactccgg 2880
atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt 2940
tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca 3000
ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac 3060
ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga 3120
taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct 3180
tacgtgccga tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac 3240
agggacacca ggatttattt attctgcgaa gtgatcttcc gtcacaggta tttattcggc 3300
gcaaagtgcg tcgggtgatg ctgccaactt actgatttag tgtatgatgg tgtttttgag 3360
gtgctccagt ggcttctgtt tctatcagct gtccctcctg ttcagctact gacggggtgg 3420
tgcgtaacgg caaaagcacc gccggacatc agcgctagcg gagtgtatac tggcttacta 3480
tgttggcact gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca 3540
ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct cactgactcg 3600
ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc 3660
ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt 3720
tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc 3780
gaaacccgac aggactataa agataccagg cgtttcccct ggcggctccc tcgtgcgctc 3840
tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca 3900
ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg 3960
aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4020
cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta 4080
gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct 4140
cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg 4200
ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct 4260
caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca atttatctct 4320
tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc atgttagtca 4380
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 4440
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 4500
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 4560
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 4620
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 4680
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 4740
tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 4800
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4860
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4920
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4980
<210> 18
<211> 564
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atgaactggc gtgcactatt taaacctagc gcaaaatatt caattctggc gctgatggtc 60
gtcggtattg ttatcggcgt agtgggttat ttcgcaaccc aacaaacctt acatgcgaca 120
agtacggatc agttctgtat gtcctgtcac agcaaccatt cacttaagga tgaagttctt 180
gcatctgccc atggtggtgg tcgtgcgggt gtgaccgttc aatgtcaaga ctgtcactta 240
ccacatggcc ctgtagatta tttgatcaaa aaaatcattg tatctaaaga cttatatggt 300
ttcttaacga ttgatggctt taacactcaa gcttggttag atgaaaaccg taaagagcaa 360
gccgatctag cacttaagta tttccgcagt aatgactccg ctaactgtca acactgccat 420
actcgcattt atgaaaacca gccagaaact atgaagccta tggctgtcag aatgcacact 480
aataacttta agaaagatcc agaagccaga aaaacctgtg ttgactgcca taaaggtgta 540
gctcacccat atccaaaagg ataa 564
<210> 19
<211> 187
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Met Asn Trp Arg Ala Leu Phe Lys Pro Ser Ala Lys Tyr Ser Ile Leu
1 5 10 15
Ala Leu Met Val Val Gly Ile Val Ile Gly Val Val Gly Tyr Phe Ala
20 25 30
Thr Gln Gln Thr Leu His Ala Thr Ser Thr Asp Gln Phe Cys Met Ser
35 40 45
Cys His Ser Asn His Ser Leu Lys Asp Glu Val Leu Ala Ser Ala His
50 55 60
Gly Gly Gly Arg Ala Gly Val Thr Val Gln Cys Gln Asp Cys His Leu
65 70 75 80
Pro His Gly Pro Val Asp Tyr Leu Ile Lys Lys Ile Ile Val Ser Lys
85 90 95
Asp Leu Tyr Gly Phe Leu Thr Ile Asp Gly Phe Asn Thr Gln Ala Trp
100 105 110
Leu Asp Glu Asn Arg Lys Glu Gln Ala Asp Leu Ala Leu Lys Tyr Phe
115 120 125
Arg Ser Asn Asp Ser Ala Asn Cys Gln His Cys His Thr Arg Ile Tyr
130 135 140
Glu Asn Gln Pro Glu Thr Met Lys Pro Met Ala Val Arg Met His Thr
145 150 155 160
Asn Asn Phe Lys Lys Asp Pro Glu Ala Arg Lys Thr Cys Val Asp Cys
165 170 175
His Lys Gly Val Ala His Pro Tyr Pro Lys Gly
180 185
Claims (10)
1. An AraC mutant is characterized in that the AraC mutant is AraCmt-Cd, and the amino acid sequence of the AraC mutant is shown as SEQ ID NO. 10.
2. The AraC mutant according to claim 1, wherein the AraC mutant AraCmt-Cd has a function of significantly inducing submarine metal ions Cd (II).
3. The AraC mutant coding gene of claim 1, having one of the following nucleotide sequences:
(1) A nucleotide sequence shown as SEQ ID NO. 9;
(2) Has more than 95 percent of homology with the nucleotide sequence shown in SEQ ID NO.9, and can encode the nucleotide sequence of the amino acid sequence shown in SEQ ID NO. 10.
4. The mutant AraC of claim 1, wherein the mutant AraC encodes a gene, an electron transfer chromoprotein gene, and a promoter element.
5. The latent microbe detection sensor of claim 4, wherein the electron transfer chromoprotein gene is a CymA gene having a nucleotide sequence shown in SEQ ID No. 18.
6. The latent microbe sensor of claim 4, wherein the method of making the latent microbe sensor comprises the steps of:
(1) Amplifying an electron transfer chromoprotein gene CymA and a promoter element pBAD;
(2) Connecting the amplified fragment in the step (1) with an AraC mutant coding gene fragment in a concentration ratio of 1: 1, and cloning the fragment into an expression vector to obtain a recombinant plasmid;
(3) And transforming the recombinant plasmid into competent cells to obtain a recombinant strain, namely the latent microbe detecting sensor.
7. Use of the AraC mutant of claim 1 or the submersible microbial sensor of claim 4 in the preparation of an electrochemical device for the real-time detection of submarines.
8. The application of claim 7, wherein the electrochemical device comprises a working electrode, a reference electrode, a counter electrode and the submarine detection microbial sensor, and can sense metal ions released by a submarine, and output electrochemical signals of the submarine detection microbial sensor through difference after sensing the metal ions, so as to achieve the purpose of detecting the submarine in real time.
9. The use according to claim 8, wherein the electrochemical device detects submarine metal ions at a concentration of not less than 5 μmol/L.
10. The use of claim 8, wherein the submarine metal ions are Cd (II) ions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210643360.8A CN115160416A (en) | 2022-06-08 | 2022-06-08 | AraC mutant for inducing submarine metal ion Cd (II), constructed submerged microorganism detection sensor and application thereof |
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|---|---|---|---|
| CN202210643360.8A CN115160416A (en) | 2022-06-08 | 2022-06-08 | AraC mutant for inducing submarine metal ion Cd (II), constructed submerged microorganism detection sensor and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116284276A (en) * | 2023-02-28 | 2023-06-23 | 江苏省中国科学院植物研究所 | Escherichia coli regulatory protein AraC mutant protein AraCm and application thereof |
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2022
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116284276A (en) * | 2023-02-28 | 2023-06-23 | 江苏省中国科学院植物研究所 | Escherichia coli regulatory protein AraC mutant protein AraCm and application thereof |
| CN116284276B (en) * | 2023-02-28 | 2023-10-03 | 江苏省中国科学院植物研究所 | Escherichia coli regulatory protein AraC mutant protein AraCm and application thereof |
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