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Wild esterase B3 genetically engineered bacteria and building method and application thereof

A technology of genetically engineered bacteria and construction methods, applied in the field of wild-type esterase B3 genetically engineered bacteria and its construction, can solve problems such as organic phosphorus pollution and achieve effective degradation

Inactive Publication Date: 2010-10-13
石元亮 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the long-term application of pesticides has also caused serious pollution of soil, groundwater, surface water and even the atmosphere by organic phosphorus, which has become a serious social problem.

Method used

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  • Wild esterase B3 genetically engineered bacteria and building method and application thereof
  • Wild esterase B3 genetically engineered bacteria and building method and application thereof
  • Wild esterase B3 genetically engineered bacteria and building method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1) Extraction of the plasmid DNA of the Culex cDNA library resistant to organophosphate insecticides: the plasmid pUC-b3 (purchased from Queen's University, Canada) was transformed into Escherichia coli DH5α, and the single colony was inoculated in a medium containing Ampicillin / ml ampicillin in 5ml liquid LB medium, cultivate overnight on a shaker at 37°C, take 1.5ml of the culture and centrifuge to collect the bacteria. Extract plasmid DNA as a PCR reaction template (plasmid DNA extraction operation according to F. Osper, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Compiled Molecular Biology Experiment Guide" New York John Wiley & Sons Publishing House, 2005 Fourth Edition P20).

[0022] 2) Design primers encoding the whole gene sequence b3 of wild-type esterase B3: design a pair of primers with NcoI and XhoI restriction sites and 5' end protection bases respectively as follows:

[0023] wB3-F: 5'-CCACCATGGATGAGTTTGGAAAGC-3'

[0024] wB3-R: 5'-AGGCTCGAGT...

Embodiment 2

[0055] Determination of esterase activity in fermentation broth of wild-type esterase B3 genetically engineered bacteria:

[0056] 1) α-naphthyl acetate (α-NA) and β-naphthyl acetate (β-NA) are the specific substrates of esterase B1. Since the product amount of the enzymatic reaction is directly proportional to the OD value, if the esterase activity of the engineered bacteria is stronger, the OD value will be larger.

[0057] 2) heterologous expression of the wild-type esterase B3 protein in Escherichia coli BL21 (DE 3): the wild-type esterase B3 engineering bacterium single bacterium colony obtained in embodiment 1 is contained in 40ug / ml kanamycin In liquid LB medium, activate the culture overnight, transfer to the next stage with 1% inoculum, and culture on a shaker at 37°C until OD 600 was 0.6, added IPTG with a final concentration of 1.0 mmol / L, induced expression at 30°C for 6 hours, and collected the fermentation broth.

[0058] 3) Add the engineered bacteria solution...

Embodiment 3

[0063] Determination of the degradation activity of wild-type esterase B3 engineering bacteria broken protein solution on organophosphorus pesticides in vegetable leaves:

[0064] 1) heterologous expression of the wild-type esterase B3 protein in Escherichia coli BL21 (DE 3): the wild-type esterase B3 engineering bacteria single bacterium colony obtained in Example 1 was contained in 40ug / ml kanamycin In liquid LB medium, activate the culture overnight, transfer to the next stage with 1% inoculum, and culture on a shaker at 37°C until OD 600 was 0.6, added IPTG with a final concentration of 1.0 mmol / L, induced expression at 30°C for 6 hours, and collected the fermentation broth.

[0065] 2) Centrifuge the above-mentioned fermented bacterial liquid at 6,000g for 10 minutes at 4°C to collect the bacterial cells, resuspend the collected bacterial cells in 50 ml of PBS solution per 100 ml of fermentation liquid, and ultrasonically disrupt the bacterial cells at 4°C until the cells...

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Abstract

The invention relates to genetic engineering and protein expression technology, in particular to wild esterase B3 genetically engineered bacteria and a building method and application thereof. The wild esterase B3 genetically engineered bacteria have a base sequence shown as SEQ ID No.1. The building method comprises the following steps of: performing polymerase chain reaction (PCR) amplification by taking a complete sequence in a culex tarsalis cDNA library as a template and a DNA fragment with NcoI and XhoI restriction enzyme cutting sites and 5'end protection basic groups respectively as a primer; performing digestion on the PCR amplification product by using a restriction enzyme; cloning the digested product to a prokaryotic expression vector; and transforming the product to an Escherichia coli expression host, wherein the expression product is the wild esterase B3 genetically engineered bacteria. The wild esterase B3 genetically engineered bacteria after being fermented can degrade organic phosphorus, organic chlorine, carbamate and pyrethroid compounds. In the method, simple Escherichia coli is used as the genetically engineered bacteria, so that a large number of esterase B3 proteins with biological activity are produced and microbiological culture capable of degrading organic phosphorus compounds is obtained; and the pathogenicity of the microbiological culture is low and can be used for bioremediation of an area which is polluted by organic phosphorus.

Description

technical field [0001] The invention relates to genetic engineering and protein expression technology, in particular to a wild-type esterase B3 genetically engineered bacterium and its construction method and application. Background technique [0002] In the process of agricultural production, spraying chemical pesticides to control diseases and insect pests is still an indispensable measure. Severely excessive pesticide residues often cause acute poisoning in humans, and long-term accumulation of small amounts of pesticide residues in the human body can also cause many chronic diseases. According to statistics, 75.4% of pesticide poisoning is caused by organophosphorus pesticides. The reasons for exceeding the standard residues of pesticides include the use of highly toxic pesticides such as methamidophos and parathion on crops, the use of pesticides in excessive concentrations, and the spraying of pesticides during the harvest period. For agricultural products with exces...

Claims

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Application Information

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IPC IPC(8): C12N1/00C12N1/21C12N15/70A62D3/02A62D101/28A62D101/22A62D101/26A62D101/04C12R1/19
Inventor 石元亮张惠文卢宗云
Owner 石元亮
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