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Method for degrading 1,2,3-TCP (trichloropropane) by engineered strain bacillus subtilis

A technology of Bacillus subtilis and engineering bacteria, applied in the biological field, can solve the problems of genetic instability, poor TCP tolerance, and low degradation efficiency

Inactive Publication Date: 2017-08-08
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the constructed strain has the problems of poor TCP tolerance, genetic instability and low degradation efficiency

Method used

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  • Method for degrading 1,2,3-TCP (trichloropropane) by engineered strain bacillus subtilis
  • Method for degrading 1,2,3-TCP (trichloropropane) by engineered strain bacillus subtilis
  • Method for degrading 1,2,3-TCP (trichloropropane) by engineered strain bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Obtaining Enzyme Genes and Relevant Elements Required for Construction of Integrated Vectors

[0063] 1. According to the codon preference optimization of Bacillus subtilis, the haloalkane dehalogenase mutant gene was designed ( dhaA 31, the nucleic acid sequence is shown in SEQ ID NO.1), halohydrin dehalogenase gene ( wxya , the nucleotide sequence is shown in SEQ ID NO.3) and the epoxide hydrolase gene ( echA , the nucleic acid sequence is shown in SEQ ID NO.5).

[0064] The amino acid sequence of haloalkanedehalogenase (DhaA31) is shown in SEQ ID NO.2, the amino acid sequence of halohydrindehalogenase (HheC) is shown in SEQ ID NO.4, and the epoxide hydrolase ( epoxide hydrolase, EchA) amino acid sequence shown in SEQ ID NO.6.

[0065] 2. Insert the above-mentioned synthetic target gene into the plasmid pMD18-T to construct the recombinant plasmid pMD18T- dhaA 31. pMD18T- wxya and pMD18T- echA .

[0066] 3. PCR amplification to obtain the promoter...

Embodiment 2

[0080] Example 2 Construction of Bacillus subtilis integration vector

[0081] 1. Construction of recombinant plasmid pDGIEF- PSphoddhaA 31

[0082] (1) Download the gene sequence encoding plasmid pDGIEF (serial number: DQ358863.1) from the GenBank Data Base database, and synthesize the plasmid pDGIEF at Genaray Company (Shanghai Generay Biotech Co., Ltd), which is the host-wide plasmid pDGIEF.

[0083] (2) Using the recombinant plasmid pMD18T- dhaA 31 as template, apply primers dhaA 31-fw / dhaA 31 -rv Perform PCR amplification to obtain foreign genes dhaA 31; the amplified product was inserted into the plasmid pDGIEF after double digestion Sal I / not I site, construct the recombinant plasmid pDGIEF- dhaA 31.

[0084] The PCR program was as follows: 98°C, 10 min; 98°C, 10 s; 55°C, 10 s; 72°C, 15 s, for 30 cycles; 72°C, 3 min.

[0085] Primer dhaA31-fw (sequence shown in SEQ ID NO.14):

[0086] 5'-ATCCGC GTC GAC ATGTCAGAAATTGGCACAGGCTTTCCGTTTG-3' (the underlined...

Embodiment 3

[0148] Example 3 Construction of Bacillus subtilis engineering bacteria

[0149] 1. Transform the Bacillus subtilis integration vector constructed above into Escherichia coli competent cells, and extract the recombinant plasmid (such as figure 2 and 3 shown):

[0150] (1) Recombinant plasmid pDGIEF- PSphoddhaA 31 Transform Escherichia coli competent cells, extract the recombinant plasmid, and transform the recombinant plasmid pDGIEF- PSphoddhaA 31 After linearization, transform the competent cells of Bacillus subtilis, the recombinant plasmid and the host genome undergo double crossover homologous recombination, and integrate the target gene into the genome amye location. Select positive clones from the LB plate containing spectinomycin, inoculate them into 10 ml LB medium, culture at 37 °C, 200 rpm for 24 h, dilute the culture, and spread it on a solid LB plate containing 0.1 mM IPTG, Filter out IPTG s and spc r A single colony containing the exogenous gene dhaA 3...

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Abstract

The invention discloses a method for degrading 1,2,3-TCP (trichloropropane) by engineered strain bacillus subtilis. According to the method, firstly, alkyl halide dehalogenase genes, halogenohydrin dehalogenase genes and epoxide hydrolase genes which can degrade 1,2,3-TCP and intermediate products of the 1,2,3-TCP are synthesized; the gene sequence and the codase amino acid sequence are respectively shown as SEQ ID NO.1 to 6; then, the bacillus subtilis integrating carrier containing the gene is built; the carrier converts the bacillus subtilis; by a gene ectopic integration and resistance gene knock-out method, the gene is integrated into the bacillus subtilis genome; the engineered strain bacillus subtilis capable of secreting and expressing alkyl halide dehalogenase, halogenohydrin dehalogenase and epoxide hydrolase is obtained. The engineered strain bacillus subtilis has a good degradation effect on TCP and TCP intermediate products; the complete TCP degradation can be realized; the characteristics of high degradation efficiency, high TCP tolerance, stable inheritance and the like are realized; the application prospects are good.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a method for degrading 1,2,3-trichloropropane with Bacillus subtilis engineering bacteria. Background technique [0002] 1,2,3-Trichloropropane (TCP) is a synthetic chlorinated organic compound, which is an important raw material for the manufacture of paints and soil pesticides, and is widely used in industrial and agricultural production. However, TCP has the characteristics of high toxicity and refractory degradation, and its extensive use has caused a large accumulation of this substance in soil and groundwater, posing a huge threat to human health and the ecological environment. [0003] The current methods for degrading TCP include: physical, chemical and biological methods. Physical methods include vacuum filtration and activated carbon adsorption. However, TCP is volatile and difficult to adsorb, so physical methods cannot effectively solve the problem of T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/14C12N1/21C12N15/75A62D3/02C12R1/125A62D101/22
CPCA62D3/02C12N9/14C12N15/75C12Y303/02003C12Y308/01C12Y308/01005A62D2101/22
Inventor 刘玉焕孔伟任广辉
Owner SUN YAT SEN UNIV
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