Application of biphenyl dioxygenase in degrading polychlorinated biphenyl

A technology of biphenyl dioxygenase and hydroxybiphenyl dioxygenase, which is applied in the application field of biphenyl dioxygenase in the degradation of polychlorinated biphenyls, can solve the problems of low degradation efficiency and improve the degradation capacity Effect

Inactive Publication Date: 2012-02-01
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition to bacteria, some fungi can also degrade low-concentration PCBs, but the degra...

Method used

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  • Application of biphenyl dioxygenase in degrading polychlorinated biphenyl
  • Application of biphenyl dioxygenase in degrading polychlorinated biphenyl
  • Application of biphenyl dioxygenase in degrading polychlorinated biphenyl

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Construction of Pseudomonas fluorescens expression gene vector with optimized codon-biased dihydroxybiphenyl dioxygenase gene

[0022] (1) Test method:

[0023] The characteristics of the Pseudomonas fluorescens expression vector pFB1690 with pGM promoter and t1t2 terminator constructed by the Genetic Engineering Research Office of Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences are as follows: the expression of the target gene is controlled by the pGM promoter and t1t2 terminator, Km resistance gene as a selection marker.

[0024] After double digestion with BamHI and Sac I from the cloning vector plasmid containing the preferred codon-optimized dihydroxybiphenyl dioxygenase gene, the DNA recovery kit recovers about 900 bp of the dihydroxybiphenyl dioxygenase gene fragment. The fragment was connected with the corresponding enzyme-cut vector, and the correct Pseudomonas fluorescens expression gene vector pFB1690 was obtained after identifi...

Embodiment 2

[0028] Genetic transformation of Pseudomonas fluorescens expressing gene vector

[0029] (1) Test method:

[0030] 1. Competent state preparation

[0031] (1) Streak culture on a 2YT plate at a culture temperature of 29°C.

[0032] (2) Pick a single colony of fresh Agrobacterium with a diameter of 2-3 mm on the 2YT solid medium, and spread the colony by Vortex shaking in a sterile eppendorf containing 1 mL of sterile 2YT culture medium.

[0033] (3) In 5 mL of 2YT culture medium, culture overnight at 29° C. and 275 rpm.

[0034] (4) Dilute 5mL of bacterial liquid into 500mL of 2YT liquid culture medium, and cultivate to OD at 29°C 550 0.5~0.7(2~5×10 8 cells / mL).

[0035] (5) Pour the bacterial solution into a pre-cooled centrifuge tube, centrifuge at 2600g for 12-15 minutes at 4°C, and pour off the supernatant carefully.

[0036] (6) Sufficiently resuspend the bacteria with an equal volume of sterile water pre-cooled at 4°C, centrifuge at 3500g for 10 minutes, and pour o...

Embodiment 3

[0049] HPLC Determination of Degradation of Dihydroxybiphenyl

[0050] (1) Test method:

[0051] Steps for detecting bacterial degradation of biphenyl by HPLC:

[0052] (1) Add 100 mL of LB culture medium to a 200 mL Erlenmeyer flask, add 100 μL of the strain to be selected, and cultivate overnight at 28°C on a shaker at 120 rpm.

[0053] (2) On the second day, when the bacteria grow well, draw 1 mL and add 10 μL of dihydroxybiphenyl (dissolved in 0.5 μg / μL methanol) prepared in advance. Let stand at room temperature for 2 minutes.

[0054] (3) Pipette 500 μL of bacterial liquid into Dorf tubes (2 tubes, namely 1000 μL), add 500 μL of ether respectively, and mix well (it is recommended to vibrate for 5-10 minutes in an oscillator), so that dihydroxybiphenyl can be fully extracted by ether , aspirated the supernatant, and placed in a new Dorf tube (combined into one tube).

[0055] (4) Finally, add 500 μL of methanol into the Dorf tube, mix well, and perform rotary evaporatio...

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Abstract

The invention relates to application of biphenyl dioxygenase in degrading polychlorinated biphenyl, which comprises the following steps of: firstly, constructing an expressing gene carrier of pseudomonas fluorescens of a dioxydiphenyl dioxygenase gene optimized by preferred codons; secondly, inheriting and transforming pseudomonas fluorescens of the expressing gene carrier containing the dioxydiphenyl dioxygenase optimized by the preferred codons; and finally, determining the result of degrading dioxydiphenyl by the obtained transformant. The invention can be used for preparing gene engineering bacteria of microbes for degrading polychlorinated biphenyl.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to the application of a biphenyl dioxygenase in degrading polychlorinated biphenyls. Background technique [0002] Polychlorinated biphenyls (Polychlorinated biphenyls) are a class of persistent organic pollutants (POPs), which have certain harmful effects on natural ecosystems and human health. Polychlorinated biphenyls, as a kind of persistent organic pollutants with wide distribution and large pollution, have great potential harm to the environment. It has become increasingly important to remediate the environmental pollution caused by PCBs. At present, there are mainly two methods: non-biodegradation and biodegradation. Although there are many methods for the degradation of PCBs, most of them are in the experimental stage, especially the biodegradation method, which has great prospects, but there is still a long way to go. Although the non-biodegradatio...

Claims

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Application Information

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IPC IPC(8): C12N15/78C12N15/53C12N1/21A62D3/02C12R1/39A62D101/22
Inventor 熊爱生姚泉洪彭日荷帅建军田永生赵伟付晓燕金晓芬朱波许晶韩红娟陈晨
Owner SHANGHAI ACAD OF AGRI SCI
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