Phenol degrading and regulating gene and acting promoter thereof

A phenol degradation and promoter technology, applied in the field of promoters, can solve problems such as gene cloning and regulation mechanism that are not involved in the regulation of phenol degradation

Inactive Publication Date: 2009-09-16
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In short, the research of patent 021488142 focuses on the cloning of phenol degradation functional genes (structural genes), and does not involve the cloning of phenol degradation regulation genes and the research on the regulation mechanism

Method used

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  • Phenol degrading and regulating gene and acting promoter thereof
  • Phenol degrading and regulating gene and acting promoter thereof
  • Phenol degrading and regulating gene and acting promoter thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Cloning and sequence analysis of the nucleotide sequence of SEQ ID NO: 1 in Acinetobacter calcoaceticus PHEA-2.

[0053] The present invention clones the sequence of SEQ ID NO: 1 located upstream of the phenol hydroxylase gene by reverse PCR method, and the specific process is as follows. Use EcoRI (Takala Company) to digest 5ug of Acinetobacter calcoaceticus PHEA-2 chromosomal DNA, digest the product T4 DNA ligase (NEB Company) at 10°C for overnight self-ligation, and design the reverse direction according to the cloned phenol hydroxylase gene sequence Primers, using the self-ligated product as a template to amplify a 3kb fragment, which was recovered and ligated into a T vector (Takala Company). Sequencing found that the cloned sequence extended the known phenol hydroxylase gene sequence upstream by 1961bp, including a complete open reading frame of 1671bp, encoding 556 amino acids. The protein SEQ ID NO: 3 encoded by sequence SEQ ID NO: 1 is found to be a ...

Embodiment 2

[0054] Example 2 Expression of SEQ ID NO: 3 polypeptide in Escherichia coli and construction of whole cell biosensor.

[0055] (1) Construction of the prokaryotic expression vector of SEQ ID NO:1.

[0056] The two ends of the sequence of SEQ ID NO: 1 were introduced into the NdeI and SalI restriction sites by PCR method, and the amplified sequence was digested with NdeI and SalI (Takala Company) and then ligated with the same expression vector pET28a (NEB Company) The recombinant expression vector pEMR was obtained.

[0057] (2) Construct the test expression vector of SEQ ID NO:2.

[0058] The two ends of the sequence of SEQ ID NO: 2 were introduced into XhoI and PstI restriction sites by PCR method. The amplified sequence was digested with XhoI and PstI (Takala Company) and then connected to the upstream of the lacZ gene without a promoter of the promoter detection vector pPR9Tt (Pedro Miguel Santos, 2001) digested with the same enzymes, and the phenol hydroxylase promoter ...

Embodiment 3

[0061] Example 3 Co-transformation bacteria PEMRP-14 sensed the range of phenolic compounds.

[0062]Using 28 different phenolic compounds as inducers to induce PEMRP-14 bacteria, and measuring the activity of β-galactosidase, it was found that phenol, catechol, o-cresol and 2-chlorophenol can be encoded by SEQ ID NO: 1 Protein recognition ( image 3 ).

[0063] The specific method is as follows: pick freshly cultivated colonies, shake the bacteria at 37 degrees overnight, and insert 1% inoculum into 29 test tubes of LB medium the next day, shake until the OD600 reaches about 0.6, and insert different phenols into each tube The compound, with a final concentration of 0.3mM, was incubated for 2 hours, and 29 different samples were taken out to measure the enzyme activity of β-galactosidase. The determination method was referred to "Molecular Cloning" (Sambrook et al., 1989). The results showed that MphR could recognize four compounds, phenol, catechol, o-cresol and 2-chloroph...

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Abstract

The invention discloses a phenol degrading and regulating protein and a nucleotide sequence for coding the protein. The invention also discloses an acting phenol hydroxylase promoter of the phenol degrading and regulating protein and a functional binding site for the phenol degrading and regulating protein in the sequence of the promoter. The invention constructs a recombinant vector including the gene and a testing expression vector of the phenol hydroxylase promoter, and implants the two vectors into Escherichia coli. As proved by tests, the invention expresses the regulating gene in the Escherichia coli to activate the expression of the phenol hydroxylase promoter, thus acting as a whole-cell biological sensor to detect phenolic compounds including phenol, pyrocatechol, orthocresol, dichlorophen and the like.

Description

Technical field: [0001] The present invention relates to phenol degradation regulation gene and its function promoter. Specifically, it relates to a nucleotide coding sequence of a phenol degradation regulation protein and a nucleotide coding sequence of a phenol hydroxylase promoter acting on it. The present invention also relates to the application of the two nucleotide coding sequences in the biological degradation of phenol and the biological treatment and monitoring of phenolic pollutants. Background technique: [0002] Phenolic compounds such as phenol are often found in industrial wastewater, causing huge direct and potential harm to the environment. [0003] Biodegradation is a very environmentally friendly technology for treating pollutants such as phenol. Chinese patent 021488142 "Phenol Hydroxylase Gene and Its Application" screened and isolated Acinetobacter calcoaceticus PHEA-2, which has the ability to degrade phenol. The strain can convert phenol into catec...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/70C07K14/22C12Q1/68C12Q1/04C02F3/00C02F101/34
Inventor 张维彭子欣陈明林敏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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