A copper-sensitive p. putida gene knockout mutant and application thereof

Abstract

The application discloses a copper-sensitive Pseudomonas putida gene knockout mutant and application thereof. The two sets of two-component systems copRS1 and copRS2 of Pseudomonas putida are non-trace knocked out by using a homologous recombination gene knockout technology, and the nucleotide sequences of the copRS1 and copRS2 genes are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively. The mutant obtained by the application has a significantly improved sensitivity to copper ions, and the application of Pseudomonas putida in Cu 2+ detection scenes is widened.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a copper-sensitive Pseudomonas putida gene knockout mutant and its application. Background Technology

[0002] Copper is one of the earliest metals used by humankind, possessing advantages such as good ductility, thermal conductivity, and electrical conductivity. It is widely used in cables, electrical appliances, and electronic components, and can also be used as a raw material to form various high-performance alloys with other metals. Furthermore, copper compounds are used in textile mordants, pool disinfectants, and agricultural fungicides (such as Bordeaux mixture). The widespread use of copper has also led to the generation of large amounts of copper-containing wastewater, mainly including smelting wastewater, printed circuit board etching wastewater, and electroplating wastewater. The discharge of wastewater with excessive copper levels poses a significant hazard to water bodies and soil.

[0003] With rapid industrial development, the ecological environment in many regions has been damaged, and heavy metal pollutants are a significant contributing factor. Major heavy metal pollutants in the environment include mercury, cadmium, lead, chromium, and metalloid arsenic. Copper is also a heavy metal, but due to its relatively low biotoxicity, it receives less attention than the aforementioned highly toxic heavy metals. However, when the concentration of copper ions in the environment exceeds the tolerance range of microorganisms, it will have a significant impact on the environmental microbial ecology. Copper ions can damage the cell walls of microorganisms, and after entering the cells, they can affect the activity of many enzymes, causing metabolic disorders and thus exhibiting antibacterial activity. To resist heavy metal stress, microorganisms in the environment have evolved sophisticated response and resistance mechanisms. Utilizing screened or modified microorganisms to adsorb and immobilize heavy metal ions is an effective way to address heavy metal pollution in water and soil. Furthermore, using modified microorganisms to indicate heavy metal pollution in the environment is also an important means of environmental monitoring. Developing more efficient microbial tools will be beneficial for improving the remediation and monitoring of heavy metal pollution in the environment by microorganisms. Summary of the Invention

[0004] This invention targets *Pseudomonas putida* and its effect on Cu. 2+ It exhibits strong resistance, and a marker-free gene knockout mutant was proposed to improve its performance in monitored environments with Cu. 2+ Potential applications in pollution control.

[0005] The first objective of this invention is to provide a method for processing Cu 2+ The sensitive *Pseudomonas putida* double gene cluster knockout mutant is formed by knocking out the copRS1 gene cluster (sequence shown in SEQ ID NO.1, containing genes copR1 and copS1) and the copRS2 gene cluster (sequence shown in SEQ ID NO.2, containing genes copR2 and copS2) encoding the two-component system in *Pseudomonas putida*.

[0006] Specifically, the mutant strain is formed by the scarless knockout of the coding sequences of the copRS1 gene cluster from position 147 to position 1761 and the copRS2 gene cluster from position 185 to position 1852 in *Pseudomonas putida*.

[0007] The aforementioned *Pseudomonas putida* is *Pseudomonas putida* KT2440 (ATCC 47054).

[0008] The second objective of this invention is to provide a method for constructing a *Pseudomonas putida* copRS1 / copRS2 double gene cluster knockout mutant, comprising the following steps:

[0009] (1) Primers were designed to amplify upstream and downstream sequence fragments of copRS1 and copRS2 using the genome of Pseudomonas putida KT2440 as a template, serving as homologous arms for gene knockout. The primer sequences are as follows:

[0010] upstream amplification primers for copRS1:

[0011] copRS1upS:AGGTACCGCATGCGATATCGAGCTCGCCTTGTCGCCCAGGTTGA;

[0012] copRS1upA: ATCAGCAGGTCGTGGTCGC;

[0013] downstream amplification primers for copRS1:

[0014] copRS1dnS: GCGACCACGACCTGCTGAT CGGCAGGAGGACAGATCAAG;

[0015] copRS1dnA:TTTGTGGAATTCCCGGGAGAGCTC CATTGCCATCCTGCGTACCT;

[0016] upstream amplification primers for copRS2:

[0017] copRS2upS: AGGTACCGCATGCGATATCGAGCTCTTTTCCGCCTTCACCAGAAC;

[0018] copRS2upA:CTTCCCATCCTTTCTAGCCCC;

[0019] downstream amplification primers for copRS2:

[0020] copRS2dnS:GGGGCTAGAAGGATGGGAAG GGCAACCATTGACCCACAAC;

[0021] copRS2dnA:TTTGTGGAATTCCCGGGAGAGCTC GGCTCTGACCGTAGGTATCG.

[0022] (2) The upstream and downstream fragments of copRS1 and copRS2 were simultaneously ligated into the linearized plasmid pDS3.0 digested with SacI via recombinase, and then transformed into Escherichia coli S17-1λpir by heat shock to obtain pDS-copRS1 and pDS-copRS2.

[0023] (3) pDS-copRS1 was introduced from Escherichia coli S17-1λpir into Pseudomonas putida via conjugation transfer, and the strain was spread on LB plates containing gentamicin and chloramphenicol to obtain the first homologous recombination strain.

[0024] (4) After expanding the culture of the strains that underwent the first homologous recombination, the strains that underwent the second homologous recombination were screened using a sucrose-containing medium. Single colonies were isolated on LB plates containing chloramphenicol and verified by PCR using primers copRS1wyzS and copRS1wyzA to obtain the copRS1 knockout mutant strain. The primer sequences are as follows:

[0025] copRS1wyzS:TCGGTGAACATCTTGCCTTG;

[0026] copRS1wyzA:TGGTGATGGTGGTGAACGAC.

[0027] (5) pDS-copRS2 was introduced from Escherichia coli S17-1λpir into the copRS1 mutant strain of Pseudomonas putida via conjugation transfer. The resulting copRS1 mutant strain was obtained by plating on LB plates containing gentamicin and chloramphenicol.

[0028] (6) After expanding the copRS1 mutant strain resulting from the first homologous recombination, the strain resulting from the second homologous recombination was screened using a sucrose-containing medium. Single colonies were isolated on LB plates containing chloramphenicol, and PCR verification was performed using primers copRS2wyzS and copRS2wyzA to obtain the copRS1 / copRS2 double knockout mutant strain. The primer sequences are as follows:

[0029] copRS2wyzS:CATCAGGTCCTGAAGCATGG;

[0030] copRS2wyzA: CGAGGCAGAACAATGGTGTG.

[0031] The third objective of this invention is to provide a *Pseudomonas putida* copRS1 / copRS2 double gene cluster knockout mutant strain for use in Cu 2+ Applications in detection. The mutant strain is sensitive to Cu. 2+ It has high sensitivity and can be determined based on the growth status of the strain or as a Cu 2 + The host bacteria of the induction reporter vector are applied to Cu 2+ Scenarios for concentration level detection.

[0032] The present invention has the following beneficial effects:

[0033] The *Pseudomonas putida* copRS1 / copRS2 double-gene cluster knockout mutant strain provided by this invention has a positive effect on Cu. 2+ The sensitivity of *Pseudomonas putida* to Cu was significantly improved compared to the wild-type strain (*Pseudomonas putida* KT2440), thus broadening the range of *Pseudomonas putida* strains in Cu. 2+ Applications in detection. Attached Figure Description

[0034] Figure 1 This is a PCR verification result diagram of the *Pseudomonas putida* copRS1 / copRS2 double gene cluster knockout mutant strain. (Lane: 1. Marker III; 2. Amplification using wild-type strain DNA as template with primers copRS1wyzS and copRS1wyzA; 3. Amplification using wild-type strain DNA as template with primers copRS2wyzS and copRS2wyzA; 4. Amplification using copRS1 / copRS2 double gene cluster knockout mutant strain DNA as template with primers copRS1wyzS and copRS1wyzA; 5. Amplification using copRS1 / copRS2 double gene cluster knockout mutant strain DNA as template with primers copRS2wyzS and copRS2wyzA).

[0035] Figure 2 It is the results of different concentrations of Cu in wild-type strains of *Pseudomonas putida* and *Pseudomonas putida* copRS1 / copRS2 double gene cluster knockout mutants (ΔcopRS1 / copRS2). 2+ Growth curve in culture medium. Detailed Implementation

[0036] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0037] Example 1

[0038] 1. Construction of copRS1 and copRS2 gene cluster knockout vectors

[0039] The genomic information of *Pseudomonas putida* KT2440 (ATCC 47054) was obtained from the NCBI database: NC_002947.4. The nucleotide sequence of the copRS1 gene cluster is shown in SEQ ID NO.1, with a length of 2030 bp; the nucleotide sequence of the copRS2 gene cluster is shown in SEQ ID NO.2, with a length of 2084 bp. Primers were designed based on copRS1 and copRS2 and their upstream and downstream sequences, and high-fidelity DNA polymerase was used. Max DNAPolymerase (Takara Bio) was used to perform PCR amplification using the genome of *Pseudomonas putida* KT2440 as a template to obtain homologous arms for gene knockout. The amplification product of primers copRS1upS and copRS1upA (616 bp, nucleotide sequence shown in SEQ ID NO. 3) was used as the upstream homologous arm of copRS1; the amplification product of primers copRS1dnS and copRS1dnA (691 bp, nucleotide sequence shown in SEQ ID NO. 4) was used as the downstream homologous arm of copRS1; the amplification product of primers copRS2upS and copRS2upA (544 bp, nucleotide sequence shown in SEQ ID NO. 5) was used as the upstream homologous arm of copRS2; and the amplification product of primers copRS2dnS and copRS2dnA (616 bp, nucleotide sequence shown in SEQ ID NO. 6) was used as the downstream homologous arm of copRS2. Meanwhile, plasmid pDS3.0 (its nucleotide sequence is shown in SEQ ID NO.7) was extracted using a plasmid extraction kit (Sangon Biotech) and digested with restriction endonuclease SacI (Takara Bio). The PCR amplification products and the digested pDS3.0 were subjected to agarose gel electrophoresis and gel extraction for DNA purification using a gel extraction kit (Sangon Biotech).

[0040] The upstream and downstream homologous arms of copRS1 and copRS2 were ligated to linearized pDS3.0, respectively, using a seamless cloning kit (Takara Bio). The reaction system is as follows:

[0041]

[0042] 10 μL of the recombinant ligation product was mixed with E. coli S17-1λpir chemocompetent cells, incubated on ice for 30 min, then heat-shocked in a 42℃ water bath for 90 s. After cooling on ice, 700 μL of LB medium was added for resuscitation and cultured for 1 h. After centrifugation, most of the supernatant was removed, and the bacterial cells were resuspended and spread on LB plates containing 20 μg / mL gentamicin. The cells were cultured overnight at 37℃. Single colonies were picked and PCR was performed using primers copRS1upS / copRS1dnA and copRS2upS / copRS2dnA, respectively, to obtain the vectors pDS-copRS1 and pDS-copRS2. The obtained plasmids were sent to Qingke Biotechnology Co., Ltd. for sequencing verification.

[0043] The primer sequences are as follows:

[0044] copRS1upS:AGGTACCGCATGCGATATCGAGCTCGCCTTGTCGCCCAGGTTGA;

[0045] copRS1upA: ATCAGCAGGTCGTGGTCGC;

[0046] copRS1dnS: GCGACCACGACCTGCTGATCGGCAGGAGGACAGATCAAG;

[0047] copRS1dnA:TTTGTGGAATTCCCGGGAGAGCTC CATTGCCATCCTGCGTACCT;

[0048] copRS2upS: AGGTACCGCATGCGATATCGAGCTCTTTTCCGCCTTCACCAGAAC;

[0049] copRS2upA:CTTCCCATCCTTTCTAGCCCC;

[0050] copRS2dnS:GGGGCTAGAAGGATGGGAAGGGCAACCATTGACCCACAAC;

[0051] copRS2dnA:TTTGTGGAATTCCCGGGAGAGCTC GGCTCTGACCGTAGGTATCG.

[0052] The nucleotide sequence of the copRS1 gene cluster is shown in SEQ ID NO.1, specifically:

[0053] ATGAAACTGCTGATCGTCGAAGACCAGGCCCGCACCGGCCAGTACCTGAGCCAAGGCCTGAGCGAGGCCGGGTTTGCCACCGAATTGGCCACCGATGGCGAGACCGGCCAGTTCCTCGCCCTGACCGGCGACCACGACCTGCTGATCCTCGACGTGATGCTGCCCGGCCGCGACGGCTGGCAGATCCTTCAGGCCGTGCGCCAGGCTGGCCTGGATACGCCAGTGCTGTTCCTGACCGCCCGCGACGCCGTCGAGGATCGCGTGCATGGCCTGGAACTGGGCGCCGACGACTACCTGGTCAAGCCGTTCGCCTTTTCCGAGCTGCTGGCCCGGGTACGCAGCCTGCTGCGCCGTGGCACCAGCCCGAGCCAGGACACGATCCTGAGCCTGGCTGACCTGCGCCTGG

[0054] ACCTGATCCGCCGCCGCGCCGAGCGTGCCGGGCTGCGCATCGACCTGACCGCCAAGGA

[0055] GTTCTCACTCCTTGAGCTGCTGCTGCGCCGCCAGGGTGAGGTGCTGCCAAAATCGCTGA

[0056] TCGCCTCGCAGGTGTGGGACATGAACTTCGACAGCGACACCAATGTGATCGAAGTGGC

[0057] CATCCGCCGCCTGCGCCTGAAAATCGACGACCCGCACCCCAACAAGCTGATCCACACCG

[0058] TCCGCGGCATGGGTTACGTACTCGAAGAGCGCACCGAGTGATGCGCCGGGTTTCCCTCG

[0059] GCAGCCGCCTGGCCCTGCTGTTCGCCGCCTGCACCGCCACCGTCTCGCTGGGCGCCGGT

[0060] CTGCTGTTCAGCCGGGCCAGCGAGCAGCACTTCGTCGAACTCGACCAACAACTGCTGG

[0061] ACTCGCGCCTGTCATTGTTTCGGACGCAACTGGCTGGCGTCAGCACGGCTGATGAGCTC

[0062] CAGGCCCGCCTGCCGGCCCTGCGCGATGAACTGAGTCACCAGGCCGACCTGGCCCTGC

[0063] GCATCAGCGCCAGCAATGGCGCCACCTGGTTCGAAAGCCGCAGCGGGCTGCCTCATGC

[0064] AGCGCAGGCCACCGGGCTGGCCACCCTGCATGCGCCAGGCATCGACTACCGCAGCCTG

[0065] TCCGTGCCCCTGACACAGGGCGCCATGCAGTCGCCGCGACTGACCCTGTACCTCGACAT

[0066] CACCCACCACCAGCACTTCCTGCAGGGCATGCAACGACTGATCTGGCTGACGGTCGGCC

[0067] TGTCGGCGCTAATCACCGCACTATTGGGTGCCTGGGCCGCCCGCAGCGGCCTGCGCCCA

[0068] TTGCGGCAGATGGGCCAGGTGGCCGCCAGCGTGTCAGCGCGCTCCCTCACCACCCGTCT

[0069] GCCGGTGGCGCAGATGCCCGAAGAGTTGGCCGAGCTGGCCAGCAGCATGAACGCGATG

[0070] CTGCAGCGCCTGGACGATGCCTTCCAGCGCCTGTCAGCGTTTTCGGCCGATATCGCCCA

[0071] CGAGTTACGTACGCCGCTGTCCAACCTGCTGACGCACACCCAGGTCACCCTCACCCGCC

[0072] CACGCAGCCTCGAAGAATACCGAGAGGCACTGCATGGCAACCTGGAAGAACTGCAATG

[0073] GATGGCGCAGATGATCAACGACATGCTGTTTCTGGCCAAGGCCGACCACGGCCTGCTGG

[0074] TACCGGGGGACGCGCCTTTGGCGCTGCATGACGAGGTGGATGCGCTGCTGGAGTACTAC

[0075] GCGCCTCTGGCAGAAGACAGCGACGTGCAGATGCTGCGCGAGGGTGAAGCGGTGCTGC

[0076] ACGGCGACCAGCATATGCTGCGCCGGGCGCTGTCCAACCTGCTGGACAACGCCATGCGT

[0077] TTTACCCCGGCAGGAGGACAGATCAAGGTAACCCTGGGGCCGGGGCCGACGATCAACG

[0078] TGGCCAATACCGGGCTGGCTATCGACCCGGCCGCACTACCGCGGCTTTTCGACCGCTTCT

[0079] ACCGGGTGGACCCGGCTCGACGGGAAGGCAGCAGCGAGCATGCGGGGCTGGGCCTGG

[0080] CGATTACCCGGTCGATCGTGCAGGCCCATGGCGGATGCATACGGGCGGAGTGCGAGGGT

[0081] GGGTGGACCCGATTCGTGATCGAGTTCACTCAAGACCGGTAA.

[0082] The nucleotide sequence of the copRS2 gene cluster is shown in SEQ ID NO. 2, specifically:

[0083]

[0084] The nucleotide sequence of the upstream homology arm of copRS1 is shown in SEQ ID NO. 3, specifically:

[0085] aggtaccgcatgcgatatcgagctcGCCTTGTCGCCCAGGTTGAATTCGTGCGGCAGCTTGCCCTCGTTGACCAGCAAGAAGCGCACCGTCTCGCCGGCTTTTACCTGCAGGCTTTTTGGCTCGAAGGCGATGTCCTTGAGCACCACGTTGACCGTACGGGTGGCCTTGGCCGCCGGGGCAGGCTCGCCAAAGGCGAAGCTCTGCGCCTGGCCAGCGAAAGCCGGCAGGCTGCCCAGGGCAAGGGTGGCGGTAATGAACAGGTGTTTCATGATGACTCCGGGAATACACAGCAGGATGAGGCCACATTAGCCGGGGGCGACTGGCAGCTACCTGACAGGCACATTACAACTTTGTCAGCTTGGGCGAGCGGGCACATCGTCACGTATCATTTCAGCCATCCCATTGCCCCTGTGCTAGCGGTAAGGCGGCGCCATAGGCTACGCACAATCTTTCAGAATATGGACAGCGCATGAAACTGCTGATCGTCGAAGACCAGGCCCGCACCGGCCAGTACCTGAGCCAAGGCCTGAGCGAGGCCGGGTTTGCCACCGAATTGGCCACCGATGGCGAGACCGGCCAGTTCCTCGCCCTGACCGGCGACCACGACCTGCTGAT。

[0086] The nucleotide sequence of the downstream homology arm of copRS1 is shown in SEQ ID NO. 4, specifically:

[0087] GCGACCACGACCTGCTGATCGGCAGGAGGACAGATCAAGGTAACCCTGGGGCCGGGGCCGACGATCAACGTGGCCAATACCGGGCTGGCTATCGACCCGGCCGCACTACCGCGGCTTTTCGACCGCTTCTACCGGGTGGACCCGGCTCGACGGGAAGGCAGCAGCGAGCATGCGGGGCTGGGCCTGGCGATTACCCGGTCGATCGTGCAGGCCCATGGCGGATGCATACGGGCGGAGTGCGAGGGTGGGTGGACCCGATTCGTGATCGAGTTCACTCAAGACCGGTAATCCTGGGGCGCTTTACGCCCCCAGGATCTCAGGCAGACATCAAACCGCCAGGCCAATCGCCGGCTGCACCTGCGACGCCCGGTAGGCCGGGTAAATGGTCGCCAGGAAACTCATCACCAGGCCGGCGGTGCAGATGATCGCCACATCCCCCCACTGCAACTGCGACGGCAAGCTGCTGACGAAGTACACATCCGACGTAAAGATGTGTTGCCCGCTCACCCGCTCCAGCCAGCCGACGATCTGGCTGACATTGAACGCCGCAATCACGCCCAGTACACCACCAATCAGCGTGCCGACAATGCCGATCAGGCTGCCCTGGACCATGAACGTACCCATGATCTGCGCCGGCGTGGCGCCCAAGGTACGCAGGATGGCAATGGAGCTCTCCCGGGAATTCCACAAA。

[0088] The nucleotide sequence of the upstream homology arm of copRS2 is shown in SEQ ID NO. 5, which is specifically:

[0089] aggtaccgcatgcgatatcgagctcTTTTCCGCCTTCACCAGAACCGCTTCGGCTTTGTCGCGAAGCTGCTGTATCCGCTCTAATGCCCGATCGCTTCCACCTTCAGCAAAAACGGCCGACGACAGCAGCATCGATGTAGCCACAAGCAAAAGTCTGATCGATTTCATGGGACTCTCTCCATCAGGGGGTTGAAACCATTGCTACCGATAACGGCAGGCGTGACTGCAATGCCCGTCTTCAATGACCGTAGACGGCAGGCCCTGACAGCTGGCTGATACCAACATTACTTTTACGTCAGCTTCTGGTTAGCTCCTTTTTTTGCTGCACACTGGATGTGTACACCTTTGGAAGCTGAGCACATGAAACTACTCGTAGCTGAGGATGAACCGAAAACTGGTGCTTATTTACAACAGGGCCTTGCAGAGGCGGGATTCACTGTTGACCGGGTCCTCACGGGTACCGATGCCCTTCAACATGCACTCAGCGAAAGCTATGACCTGCTGATACTCGACGTGATGATGCCGGGGCTAGAAGGATGGGAAG。

[0090] The nucleotide sequence of the downstream homology arm of copRS2 is shown in SEQ ID NO. 6, which is specifically:

[0091] GGGGCTAGAAGGATGGGAAGGGCAACCATTGACCCACAACACATCAACAAGATTTTTGATCGCTTCTACCGGGCCGACCCGGCAAGGCGAGAGGGGGGACCTAGCAATGCTGGGCTTGGTCTGGCCATTACCCGCTCCATCGTCAAAGCACATGATGGAAAAGTCTGGTGCACCTCGTCCGAAGGGGTTACAGCATTCAATTTCATTTTTCCTGCCGCCCACCGAACAGCTCACATTCGCACGCGGCATTAATGCTGCGGTAATCGCTTTGGCCTACACTCGACCATCATGATTCGATTCCTACGACTGCTAACCATGTTGATGCTCGTGGTCGCGCTTCCTCTCCATGGGATGGCGGGAATCGACTCACCCATAGAACCATGCCCGATGCAGTCTCAGGGGATGGATATGATGGCAGGCATGGACCACGATTGTTGCCAGGATCAAGACAACGGAAAGCCCAACCAACACTCATCCAAAGCCTGCAAAGTCGGCCAGGAATGTAGAACTGCAAGCACACTGCAGGTCAGCATGGTCGAGCCCGCACTCACCTTCGCCACTACACGTCCAGTCGATACCTACGGTCAGAGCCGAGCTCTCCCGGGAATTCCACAAA。

[0092] The nucleotide sequence of plasmid pDS3.0 is shown in SEQ ID NO. 7, which is specifically:

[0093]

[0094] AGGGGACAGGCGAGAGACGATGCCAAAGAGCTACACCGACGAGCTGGCCGAGTGGGT

[0095] TGAATCCCGCGCGGCCAAGAAGCGCCGGCGTGATGAGGCTGCGGTTGCGTTCCTGGCG

[0096] GTGAGGGCGGATGTCGAGGCGGCGTTAGCGTCCGGCTATGCGCTCGTCACCATTTGGGA

[0097] GCACATGCGGGAAACGGGGAAGGTCAAGTTCTCCTACGAGACGTTCCGCTCGCACGCC

[0098] AGGCGGCACATCAAGGCCAAGCCCGCCGATGTGCCCGCACCGCAGGCCAAGGCTGCGG

[0099] AACCCGCGCCGGCACCCAAGACGCCGGAGCCACGGCGGCCGAAGCAGGGGGGCAAGG

[0100] CTGAAAAGCCGGCCCCCGCTGCGGCCCCGACCGGCTTCACCTTCAACCCAACACCGGA

[0101] CAAAAAGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGC

[0102] GGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCG

[0103] GGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTG

[0104] TTGGGCGCCATCTCCTTGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGAC

[0105] ACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACA

[0106] AGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAG

[0107] TCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTAC

[0108] TGAGAGTGCACCATAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC

[0109] AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGC

[0110] CTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGC

[0111] TGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC

[0112] CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT

[0113] ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT

[0114] GTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGC

[0115] TCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGT

[0116] TAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCAT

[0117] GGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTG

[0118] ACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTC

[0119] TTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCA

[0120] TCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCC

[0121] AGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC

[0122] GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG

[0123] ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG

[0124] GTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGT

[0125] TCCGCGCACATTTCCCCGAAAAGTGCCACCTGCAGTTCACTTACACCGCTTCTCAACCC

[0126] GGTACGCACCAGAAAATCATTGATATGGCCATGAATGGCGTTGGATGCCGGGCAACAGC

[0127] CCGCATTATGGGCGTTGGCCTCAACACGATTTTACGTCACTTAAAAAACTCAGGCCGCA

[0128] GTCGGTAACCTCGCGCATACAGCCGGGCAGTGACGTCATCGTCTGCGCGGAAATGGACG

[0129] AACAGTGGGGCTATGTCGGGGCTAAATCGCGCCAGCGCTGGCTGTTTTACGCGTATGAC

[0130] AGTCTCCGGAAGACGGTTGTTGCGCACGTATTCGGTGAACGCACTATGGCGACGCTGGG

[0131] GCGTCTTATGAGCCTGCTGTCACCCTTTGACGTGGTGATATGGATGACGGATGGCTGGCC

[0132] GCTGTATGAATCCCGCCTGAAGGGAAAGCTGCACGTAATCAGCAAGCGATATACGCAGC

[0133] GAATTGAGCGGCATAACCTGAATCTGAGGCAGCACCTGGCACGGCTGGGACGGAAGTC

[0134] GCTGTCGTTCTCAAAATCGGTGGAGCTGCATGACAAAGTCATCGGGCATTATCTGAACAT

[0135] AAAACACTATCAATAAGTTGGAGTCATTACCAAAAGGTTAGGAATACGGTTAGCCATTTG

[0136] CCTGCTTTTATATAGTTCATATGGGATTCACCTTTATGTTGATAAGAAATAAAAGAAAATG

[0137] CCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATTGTCCTTGTTC

[0138] AAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTTGG

[0139] CGCAAACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTGTCATATAGCTTGTAATCACG

[0140] ACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTA

[0141] GGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCCAGTT

[0142] AAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTC

[0143] ATTTTTGATCCGCGGGAGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTC

[0144] GCGCGTTCAATTTCATCTGTTACTGTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCA

[0145] GTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTTTGCTAACTCAGCCGTGC

[0146] GTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCC

[0147] ATAGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTT

[0148] GCTTCAAATACTAAGTATTTGTGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTAT

[0149] GGTTGTCGCCTGAGCTGTAGTTGCCTTCATCGATGAACTGCTGTACATTTTGATACGTTTT

[0150] TCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTGTC

[0151] TGATGCTGATACGTTAACTTGTGCAGTTGTCAGTGTTTGTTTGCCGTAATGTTTACCGGA

[0152] GAAATCAGTGTAGAATAAACGGATTTTTCCGTCAGATGTAAATGTGGCTGAACCTGACCA

[0153] TTCTTGTGTTTGGTCTTTTAGGAGAATCATTTGCATCGAATTTGTCGCTGTCTTAAAG

[0154] ACGCGGCCAGCGTTTTTCCAGCTGTCAATAGAAGTTTCGCCGACTTTTTGATAGAACATG

[0155] TAAATCGATGTGTCATCCGCATTTTTAGGATCTCCGGCTAATGCAAAGACGATGTGGTAG

[0156] CCGTGATAGTTTGCGACAGTGCCGTCAGCGTTTTGTAATGGCCAGCTGTCCCAAACGTC

[0157] CAGGCCTTTTGCAGAAGAGATATTTTTAATTGTGGACGAATCGAATTCAGGAACTTGATA

[0158] TTTTTCATTTTTTTGCTGTTCAGGGATTTGCAGCATATCATGGCGTGTAATATGGGAAATG

[0159] CCGTATGTTTCCTTATATGGCTTTTGGTTCGTTTCTTTCGCAAACGCTTGAGTTGCGCCTC

[0160] CTGCCAGCAGTGCGGTAGTAAAGGTTAATACTGTTGCTTGTTTTGCAAACTTTTTGAGT

[0161] TCATCGTTCATGTCTCCTTTTTTATGTACTGTGTTAGCGGTCTGCTTCTTCCAGCCCCTCCT

[0162] GTTTGAAGATGGCAAGTTAGTTACGCACAATAAAAAAAGACCTAAAATATGTAAGGGGT

[0163] GACGCCAAAGTATACACTTTGCCCTTTACACATTTTAGGTCTTGCCTGCTTTATCAGTAAC

[0164] AAACCCGCGCGATTTACTTTTCGACCTCATTCTATTAGACTCTCGTTTGGATTGCAACTGG

[0165] TCTATTTTCCTCTTTTGTTTGATAGAAAATCATAAAAGGATTTGCAGACTACGGGCCTAAA

[0166] GAACTAAAAAATCTATCTGTTTCTTTTCATTCTCTGTATTTTTTATAGTTTCTGTTGCATGG

[0167] GCATAAAGTTGCCTTTTTAATCACAATTCAGAAAATATCATAATATCTCATTTCACTAAATA

[0168] ATAGTGAACGGCAGGTATATGTGATGGGTTAAAAAGGATCGATCCTCTAGAGTCGACGC

[0169] GTCGATCCGGTGATTGATTGAGCAAGCTAGCTTTATGCTTGTAAACCGTTTTGTGAAAAA

[0170] ATTTTTAAAATAAAAAAGGGGACCTCTAGGGTTCCCCAATTAATTAGTAATATAATCTATTA

[0171] AAGGTCATTCAAGGTCATCCACCGGATCAATTCCCCTGCTCGCGCAGGCCTGGGGTGCC

[0172] AAGCTCTCGGGTAACATCAAGGCCCGATCCTTGGAGCCCTTGCCCTCCCGCACGATGAT

[0173] CGTGCCGTGATCGAAATCCAGATCCTTGACCCGCAGTTGCAAACCCTCACTGATCCGCG

[0174] CGTCGAATTGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGG

[0175] GTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGC

[0176] GGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCT

[0177] CATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAG

[0178] ACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTA

[0179] AGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTC

[0180] TTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGC

[0181] TACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGT

[0182] CAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAG

[0183] GGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAAC

[0184] ATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTG

[0185] TACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGC

[0186] GTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTAC

[0187] GAACCGAACAGGCTTATGTCAATTCGAGAATTAATTCGACGCGCAGATCAGTTGGAAGA

[0188] ATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTAACAA

[0189] TTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCGTTAGATGCACTAAG

[0190] CACATAATTGCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGCGTGC

[0191] ATAATAAGCCCTACACAAATTGGGAGATATATCATGAAAGGCTGGCTTTTTCTTGTTATCG

[0192] CAATAGTTGGCGAAGTAATCGCAACATCCGCATTAAAATCTAGCGAGGGCTTTACTAAGC

[0193] TGATCCGGTGGATGACCTTTTGAATGACCTTTAATAGATTATATTACTAATTAATTGGGGA

[0194] CCCTAGAGGTCCCCTTTTTTATTTTAAAAATTTTTTCACAAAACGGTTTACAAGCATAAA

[0195] GCTAGCTTGCTCAATCAATCACCGGATCGACGCGTCGACGGATCCCAAGCTTCTTCTAGAGGTACCGCATGCGATATCGAGCTTCCCGGGAATTCCACAAA.

[0196] 2. Use pDS-copRS1 and pDS-copRS2 to knock out copRS1 and copRS2 respectively.

[0197] First, pDS-copRS1 was introduced from *Escherichia coli* S17-1λpir into *Pseudomonas putida* KT2440 via conjugative transfer. Specifically, *E. coli* S17-1λpir (donor bacteria) containing pDS-copRS1 was activated and cultured in LB medium containing 20 μg / mL gentamicin, and *Pseudomonas putida* KT2440 (recipient bacteria) was activated and cultured in LB medium containing 25 μg / mL chloramphenicol. 1 mL of each bacterial culture was collected by centrifugation, washed once with antibiotic-free LB medium, centrifuged again, and the supernatant was discarded to remove antibiotics. The bacterial cells were then resuspended in 500 μL of LB medium at a donor-recipient bacteria ratio of 3:1 and incubated statically at 28°C for 12 h. 100 μL of the resuspended bacterial cells was then spread onto LB plates containing 40 μg / mL gentamicin and 25 μg / mL chloramphenicol and incubated overnight at 28°C. Single colonies were picked and PCR amplified using primers copRS1wyzS and copRS1wyzA for verification. If no PCR product was found, plasmid pDS-copRS1 was inserted into the upstream or downstream region of copRS1 via homologous recombination. The correct strains that underwent the first homologous recombination were first inoculated into antibiotic-free LB medium and then transferred to LB medium containing 10% sucrose for 24 hours. Single colonies were isolated by streaking the culture onto multiple LB plates and incubated overnight at 28°C. The best-growing single colonies were then picked and PCR amplified using primers copRS1wyzS and copRS1wyzA for verification. No amplification product indicated that the second recombination had not occurred; an amplification product fragment of 2.9 kb indicated restoration to the wild-type strain; and an amplification product fragment of 1.3 kb indicated a copRS1 knockout mutant. Using the same method described above, pDS-copRS2 was used to knock out copRS2 in the copRS1 mutant strain. PCR amplification was performed using primers copRS2wyzS and copRS2wyzA for verification. Amplified products with a fragment size of 2.9 kb represented a restored wild-type strain, while those with a fragment size of 1.2 kb represented a successfully knocked-out copRS1 / copRS2 double-gene cluster knockout mutant. Correct mutant amplification products are shown below. Figure 1 As shown.

[0198] The primer sequences for identification are as follows:

[0199] copRS1wyzS:TCGGTGAACATCTTGCCTTG;

[0200] copRS1wyzA:TGGTGATGGTGGTGAACGAC;

[0201] copRS2wyzS:CATCAGGTCCTGAAGCATGG;

[0202] copRS2wyzA: CGAGGCAGAACAATGGTGTG.

[0203] 3. The effect of *Pseudomonas putida* copRS1 / copRS2 double gene cluster knockout mutant on Cu 2+ Tolerance testing

[0204] A *Pseudomonas putida* copRS1 / copRS2 double-gene cluster knockout mutant strain was cultured in LB medium, and its resistance to Cu was evaluated by growth curve analysis. 2+ Tolerance, the specific steps are as follows:

[0205] (1) Wild-type strain of *Pseudomonas putida* (*Pseudomonas putida* KT2440) and *Pseudomonas putida* copRS1 / copRS2 double gene cluster knockout mutant strain were inoculated into LB medium and cultured at 28°C with shaking at 180 rpm until OD. 600 ≈1, collect bacterial cells by centrifugation and suspend the cells in LB medium, then dilute to OD. 600 ≈0.2.

[0206] (2) Add a 1M CuCl2 solution to LB medium to prepare a medium containing 16mM CuCl2. Perform a 2-fold serial dilution in a 96-well plate, leaving 75μL in each well, and then add 75μL of the diluted bacterial solution from step (1) to each well.

[0207] (3) Place the 96-well plate in a microplate reader, incubate at 28°C, and measure the absorbance at 600 nm every 1 hour to evaluate the strain's resistance to Cu using a growth curve. 2+ Tolerance.

[0208] Experimental results: such as Figure 2 As shown, wild-type *Pseudomonas putida* resists 2 mM Cu 2+ It exhibits strong tolerance, and its growth is unaffected. However, the *Pseudomonas putida* copRS1 / copRS2 double-gene cluster knockout mutant strain is susceptible to Cu. 2+ The sensitivity is significantly improved when Cu 2+ Increasing the concentration to 0.5 mM can have a significant impact on growth, when Cu 2+The strain cannot grow at a concentration of 2 mM.

[0209] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for treating Cu 2+ A susceptible *Pseudomonas putida* double-gene cluster knockout mutant, characterized by: It is from *Pseudomonas putida* copRS1 Gene clusters and copRS2 Gene cluster knockout; copRS1 The sequence of the gene cluster is shown in SEQ ID NO.1; copRS2 The sequence of the gene cluster is shown in SEQ ID NO.

2.

2. The mutant strain according to claim 1, characterized in that, It is copRS1 The coding sequence of the gene cluster from position 147 to position 1761 and copRS2 Knockout of the coding sequence between positions 185 and 1852 of the gene cluster.

3. The mutant strain according to claim 1, characterized in that, The aforementioned *Pseudomonas putida* is *Pseudomonas putida* KT2440.

4. A method for constructing a mutant strain according to claim 1 or 2, characterized in that, Includes the following steps: (1) Using the genome of *Pseudomonas putida* as a template, primers were used to amplify the genome of *Pseudomonas putida*. copRS1 Upstream and downstream fragments were amplified using primers. copRS2 Upstream and downstream segments serve as homologous arms for gene knockout; (2) Each copRS1 and copRS2 The upstream and downstream fragments were simultaneously ligated into the linearized plasmid pDS3.0 digested with SacI via recombinase, and then transformed into E. coli S17-1λpir by heat shock to obtain pDS-copRS1 and pDS-copRS2. (3) pDS-copRS1 was introduced from Escherichia coli S17-1λpir into Pseudomonas putida via conjugation transfer, and the resulting strain was plated on LB plates containing gentamicin and chloramphenicol to obtain the first homologous recombination strain. (4) After expanding the culture of the strains that underwent the first homologous recombination, the strains that underwent the second homologous recombination were screened using a sucrose-containing medium. Single colonies were isolated on LB agar plates containing chloramphenicol, and PCR verification was performed using primers to obtain the results. copRS1 Knockout mutants; (5) pDS-copRS2 was introduced from Escherichia coli S17-1λpir via conjugative transfer. copRS1 The knockout mutant strains were plated on LB agar plates containing gentamicin and chloramphenicol to obtain the first homologous recombination. copRS1 mutant strain; (6) The first homologous recombination copRS1 After the mutant strain was expanded, strains undergoing a second homologous recombination were screened using sucrose-containing medium. Single colonies were isolated on LB agar plates containing chloramphenicol, and PCR verification was performed using primers to obtain... copRS1 / copRS2 Double knockout mutant.

5. The construction method according to claim 4, characterized in that, The primers mentioned in step (1) are specifically: copRS1 Upstream amplification primers: copRS1upS: AGGTACCGCATGCGATATCGAGCTCGCCTTGTCGCCCAGGTTGA; copRS1upA: ATCAGCAGGTCGTGGTCGC; copRS1 Downstream amplification primers: copRS1dnS: GCGACCACGACCTGCTGATCGGCAGGAGGACAGATCAAG; copRS1dnA:TTTGTGGAATTCCCGGGAGAGCTC CATTGCCATCCTGCGTACCT; copRS2 Upstream amplification primers: copRS2upS: AGGTACCGCATGCGATATCGAGCTCTTTTCCGCCTTCACCAGAAC; copRS2upA:CTTCCCATCCTTTCTAGCCCC; copRS2 Downstream amplification primers: copRS2dnS:GGGGCTAGAAGGATGGGAAG GGCAACCATTGACCCACAAC; copRS2dnA:TTTGTGGAATTCCCGGGAGAGCTC GGCTCTGACCGTAGGTATCG.

6. The construction method according to claim 4, characterized in that, The primers mentioned in step (4) are specifically: copRS1wyzS:TCGGTGAACATCTTGCCTTG; copRS1wyzA:TGGTGATGGTGGTGAACGAC.

7. The construction method according to claim 4, characterized in that, The primers mentioned in step (6) are specifically: copRS2wyzS:CATCAGGTCCTGAAGCATGG; copRS2wyzA: CGAGGCAGAACAATGGTGTG.

8. The construction method according to claim 4, characterized in that, The aforementioned *Pseudomonas putida* is *Pseudomonas putida* KT2440.

9. The mutant strain according to any one of claims 1-3 in Cu 2+ Applications in detection.

10. The application according to claim 9, characterized in that, The mutant strain is resistant to Cu 2+ It has high sensitivity and can be determined based on the growth status of the strain or as a Cu 2+ The host bacteria of the induction reporter vector are applied to Cu 2+ Scenarios for concentration level detection.

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