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Method for constructing trichloropropane degrading bacterium by taking pseudomonas putida as original strain

A technology of Pseudomonas putida, trichloropropane, applied in the field of environmental technology restoration

Active Publication Date: 2017-05-31
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

No strains have been isolated from the environment that utilize TCP under aerobic conditions

Method used

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  • Method for constructing trichloropropane degrading bacterium by taking pseudomonas putida as original strain
  • Method for constructing trichloropropane degrading bacterium by taking pseudomonas putida as original strain
  • Method for constructing trichloropropane degrading bacterium by taking pseudomonas putida as original strain

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Experimental program
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Effect test

Embodiment 1

[0018] Embodiment 1, take Pseudomonas putida as the construction of the trichloropropane degrading bacteria of bacterial strain

[0019] Liquid medium used in the experiment:

[0020] Luria-Bertani (LB) medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, water 1L, pH7.0, sterilized at 121°C for 20 minutes for later use.

[0021] Kanamycin (Kan): Weigh 1g of Kanamycin into a 100mL beaker, add 90mL of sterilized ddH 2 O water, stir well to dissolve, dilute to 100mL, filter and sterilize, aliquot (0.5-1mL / part), store at -20°C, and set aside.

[0022] Pentafluorouracil (5-FU): Weigh 0.0375 g of pentafluorouracil into a 1.5 mL EP tube, and dissolve it in 500 μL of dimethyl sulfoxide solution at a concentration of 106 μg / mL.

[0023] The three genes dhaA31, hheC and echA in the trichloropropane degradation pathway were cloned from the host bacteria Rhodococcus rhodochrous NCIMB 13064 (GenBank accession no.AF060871.1) and Agrobacterium radiobacter AD1 (GenBank accession n...

Embodiment 2

[0027] Embodiment 2, optimize the expression of trichloropropane degradation pathway in host bacteria

[0028] In view of the fact that none of the three genes inserted in Example 1 are transcribed, we optimized the codons of the three genes dhaA31, hheC and echA according to the codon preference of Pseudomonas putida. The optimized codon sequences are shown in SEQID NO.1, respectively. Shown in SEQ ID NO.2 and SEQ ID NO.3, the promoter and RBS in the original strain were replaced at the same time, and the promoter and RBS ( image 3 ). The strain construction and RT-PCR detection were the same as in Example 1. RT-PCR test results such as Figure 4 As shown, the three exogenous genes were all correctly transcribed.

Embodiment 3

[0029] Embodiment 3, verify the function of trichloropropane degrading engineering bacteria

[0030] Liquid media used in the experiments:

[0031] SMM medium: Na 2 HPO 4 12H 2 O 5.4g, KH 2 PO 4 1.4g, (NH) 2 SO 4 0.5g, MgSO 4 ·7H 2 O 0.2g, 2mL trace elements, 1L water, pH7.0, sterilized at 121°C for 20 minutes for later use.

[0032] Trace element solution composition: 0.1M HCl 1000mL, MnSO 4 4H 2 O 0.86g, ZnSO 4 ·7H 2 O 0.2g, CoSO 4 ·7H2 O0.28g, CuSO 4 ·5H 2 O 0.25g, FeSO 4 ·7H 2 O 3.6g, sterilized by filtration after preparation, and sterilized at 121°C for 20 minutes for later use.

[0033] The correct transcribed recombinant bacterium that obtains in embodiment 2 is with OD 600 The inoculum size of =0.05 was inserted into 100 mL of SMM medium, and was connected with 2 mM / L trichloropropane. The trichloropropane of 2mM can degrade more than 99% in 24h, proves the efficient expression ( Figure 5 ).

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Abstract

The invention discloses a method for constructing a trichloropropane degrading bacterium by taking pseudomonas putida as an original strain, belongs to the technical field of environmental restoration, and relates to a method for stably and efficiently expressing three genes dhaA31, hheC and echA in a trichloropropane degrading pathway in the pseudomonas putida and practical application of the engineering bacterium. The method comprises the following steps of: optimizing a codon of a foreign gene; replacing a prompter and a ribosome bind site of an original gene; and using a traceless insertion method to respectively integrate optimized expression cassettes into a genome of the pseudomonas putida, so as to obtain the engineering bacterium which is capable of stably and efficiently expressing trichloropropane degrading genes. The engineering bacterium is inoculated into an industrial sewage small experimental unit and is capable of degrading 2Nm / L of trichloropropane in two days. According to the outstanding performance of the engineering bacterium in the small experimental unit, the engineering bacterium has huge potential in the large-scale treatment of industrial polluted wastewater.

Description

technical field [0001] The invention belongs to the field of environmental technology restoration, and specifically relates to a method for constructing a trichloropropane-degrading bacterial strain using Pseudomonas putida as a starting strain. Background technique [0002] The synthetic substance 1,2,3-trichloropropane TCP has a worldwide output of up to 5,000 tons / year, and is used by chemical companies as a solvent, a precursor of soil fumigants, and as a structural unit for the synthesis of other compounds, such as di Allyl chloride and polysulfones. TCP is also a by-product during the synthesis of epichlorohydrin. In its large production, TCP is often found in industrial and hazardous waste sites. A recent case of TCP contamination of drinking water in California demonstrates the urgent need for efficient removal of TCP, a toxic carcinogen, from the environment. To date no strains have been isolated from the environment that utilize TCP under aerobic conditions. ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/78C12R1/40
CPCC12N9/14C12N15/78C12N2800/22C12Y303/02003C12Y308/01C12Y308/01005
Inventor 杨超宫婷宋存江徐晓庆左振强刘瑞华
Owner NANKAI UNIV
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