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Organic solvent responded expression vector and application thereof in biological catalysis

A technology of organic solvents and expression vectors, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, bacteria, etc., can solve the problems of limited application and high price

Active Publication Date: 2013-09-11
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only a small number of genetic manipulation tools can be used in organic solvent-tolerant Pseudomonas putida, and most of the existing manipulation tools are based on the E. coli lactose operon, which requires the use of expensive IPTG as an inducer , which limits their application in industrial and environmental fields

Method used

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  • Organic solvent responded expression vector and application thereof in biological catalysis
  • Organic solvent responded expression vector and application thereof in biological catalysis
  • Organic solvent responded expression vector and application thereof in biological catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Construction of Organic Solvent Responsive Expression Vectors pMMS and pMMRS

[0044] promoter P srp Directing the expression of the organic solvent tolerance gene in strain S12 is a promoter that can be induced by a variety of organic solvent compounds. Most of these compounds are relatively cheap, and many of them are widely used in industry, such as n-hexane , cyclohexane and toluene. The heterologous expression system based on this promoter will undoubtedly be a good substitute for the lac promoter expression system. This embodiment uses the above P srp The promoter replaced the original promoter of pMMB66EH, and the expression vector pMMS ( figure 1 ). To this end, a reporter gene, chloramphenicol resistance gene cat, was inserted into the multiple cloning site of the wide host range vector pMMB66EH to construct pMMB66EH-cat. Then perform double digestion (MluI+EcoRI), and insert the promoter fragment extended by PCR from the S12 genome using Psrp.f (MluI) and...

Embodiment 2

[0050] Vector availability and host range

[0051] In order to further verify the correct host range of the vector construction, in this example, the vector constructed above was also introduced into each Pseudomonas in Table 1, and it was checked whether the vector could endow the host bacteria with the presence of an inducer (organic solvent). With chloramphenicol resistance, the results show that Pseudomonas containing pMMS-cat can grow on LB medium containing 5g / l chloramphenicol when the inducer is present, which shows that the constructed vector contains the correct target promoter sub, and has the ability to initiate the expression of downstream genes under solvent-induced conditions.

[0052] The impact of carrier pMMS-cat and pMMRS-cat on the chloramphenicol sensitivity of each bacterial strain when table 4 inducer (organic solvent) exists

[0053]

[0054] The results in Table 4 show that the vector pMMRS-cat can also endow various strains with resistance ...

Embodiment 3

[0056] Construction of dibenzothiophene desulfurization bacteria

[0057] Biological desulfurization is an environmentally friendly and energy-saving method for desulfurization of fossil fuels and a potential alternative to chemical desulfurization. The biological desulfurization of petroleum and petroleum refined fuel oil is generally carried out using an oil-water two-phase system, which is a type of two-phase biocatalysis. Fuel oil is a mixture of various organic solvent compounds, such as alkanes and aromatic hydrocarbons. Therefore, there is the same problem as the biphasic reaction in biological desulfurization, that is, the toxicity of organic solvents to cells. Previously, a recombinant solvent-tolerant desulfurization strain A4 was constructed with pMMB66EH, which contained the carrier pMMABCD, which was specifically described in the Chinese example patent application number 200610044410.1, the publication number CN1861785, and the publication date was November 15, 20...

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Abstract

The invention relates to an organic solvent responded expression vector and application thereof in biological catalysis, belonging to the field of biotechnology; the organic solvent response broad host constitutive expression vector comprises a vector pMMS and a vector pMMRS, and consists of a DNA element, a replicor RSF1010, a selection marker gene bla, multiple cloning sites, an intergenic sequence and an organic solvent response promoter Psrp; and the expression vector is characterized in that the nucleotide sequence of the pMMS is shown by SEQ ID No. 1, and the nucleotide sequence of the pMMRS is shown by SEQ ID No. 2. The invention has advantages in biological catalysis, especially in double-liquid-phase biological catalysis. The invention is better in economy and effectiveness, and ensures engineering bacteria constructed by organic solvent induction vector to be better than genetic engineering bacteria constructed by common isopropyl beta D thiogalactopy ranoside (IPTG) induction vector under the condition of high-concentration organic solvent exists.

Description

technical field [0001] The present invention relates to an organic solvent expression vector and its application in the field of biotechnology, specifically an organic solvent-responsive promoter P srp The constructed organic solvent-responsive expression vector and its application in biocatalysis. Background technique [0002] Biocatalysis has broad application potential in the synthesis of fine chemicals and bioremediation. As an energy-saving, environmentally friendly, safe and effective method, whole-cell catalysis is attracting more and more attention. Organic solvents are often encountered in biocatalysis. For example, in the process of biocatalysis, in order to increase the solubility of reactants or avoid the decomposition of products, it is often necessary to introduce a two-liquid phase reaction containing organic solvents; in many polluted areas that need bioremediation, there are also various kinds of bioremediation. Saturation concentration of organic solvent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/70C12N15/78C12N1/21
Inventor 许平陶飞马翠卿刘艳华苏斐徐友强李福利
Owner SHANGHAI JIAOTONG UNIV
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