Copper resistance binary signal conducting system regulation factor mutant, and construction and use thereof

A signal transduction and regulatory factor technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as loss of intracellular substances, weakening of bacterial virulence, energy consumption, etc., to achieve stable and effective expression methods, and reduce bacterial virulence. Effect

Inactive Publication Date: 2010-10-13
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Destruction of this regulation makes CopR activity unregulated, especially the persistent existence of functionally active CopR, which leads to the weakening of bacterial virulence, mainly because active CopR will activate the expression of copA, B, C, D, etc. , causing a large amount of CopA, B, C, D and other proteins to be synthesized; because these proteins can form efflux pumps on the cell membrane, resulting in the loss of intracellular materials and a large amount of energy consumption, thereby weakening the growth and infection capabilities of bacteria

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The anti-copper binary signal transduction system regulator mutant is the base sequence in SEQ ID No.1 of the sequence list.

[0025] Construction of anti-copper dual signaling system regulatory factor mutants:

[0026] 1) Construction of plasmid pLC1:

[0027] Using 100ng of plasmid pEGFP (purchased from Clonetech, USA) as a template, carry out PCR with the following primers (each 0.5uM): PF1 (5'-ATA GAATTCATTTAAAT GCAGCTGGCACGA-3', underlined bases are EcoRI and SwaI sites), PR4 (5'- GGATC CACACAACATACGAGC-3', the underlined base is the BamHI site); PCR conditions are: pre-denatured template DNA at 94°C for 60s, then 94°C for 40s, 54°C for 60s, 72°C for 60s, and after 5 cycles, change to 94°C for 40s , 60°C for 60s, 72°C for 60s, 25 cycles, and then perform PCR amplification under the condition of 72°C extension reaction for 10min. The PCR product was purified with the Tiangen DNA Product Purification Kit and ligated with the carrier pBS-T (purchased from "Tiangen...

Embodiment 2

[0051] The difference from Example 1 is:

[0052] Construction of mutants: 1) Construction of plasmid pLC1: After purification of the PCR amplification product, it was ligated with the carrier pBS-T (purchased from "Tiangen Biochemical Technology Co., Ltd.", Beijing) at room temperature for 3-4 hours, and the ligation mixture was transformed coli DH5α after containing 100ug / ml Anka penicillin (Ap), 40ug / ml Xga1 (5-bromo-4-chloro-3-indole-β-D-lactoside) and 24ug / ml isopropyl-β -D-thiogalactoside (IPTG) LB solid medium was cultured for 20-24 hours, and a white transformant was selected, which was plasmid pBSL1. pBSL1 and plasmid pBR322 (purchased from "New England Biotechnology Co., Ltd.", Beijing) were digested with EcoRI / BamHI to recover 140bp and 3986bp fragments respectively; the two fragments were ligated with T4 DNA ligase for 3-4 hours at room temperature, After the connection solution was transformed into Escherichia coli DH5α, it was cultured on LB solid medium contain...

Embodiment 3

[0057] Anti-copper binary signal transduction system regulatory factor mutant: the anti-copper binary signal transduction system regulatory factor mutant has the effect of reducing bacterial virulence to Pseudomonas fluorescens.

[0058] Expression of regulator mutants in Pseudomonas fluorescens TSS1:

[0059] 1) Construction of bacterial strains TSS1 / pJC104 and TSS1 / JR20: Transform regulatory factor mutant plasmid pJC104 into Escherichia coli S17λπ (purchased from Spain "Biomedal" company) and culture on LB solid medium containing Tc (15ug / ml) After 18-24 hours, pick a transformant and name it S17λπ / pJC104. Cultivate TSS1 and S17λπ / pJC104 overnight in LB liquid medium, then mix the two at a volume ratio of 1:3 (total volume 1ml), centrifuge the mixture (5000g, 4°C, 10min), and collect the bacteria ; suspend the bacterium in 1ml LB liquid medium, get 0.1ml and place it on the solid LB medium and incubate at 30°C for 6-8 hours, then suspend the bacteria in 1ml liquid LB, get 0...

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Abstract

The invention relates to the field of molecular microbiology, in particular to an anti-copper binary signal conduction-system regulating factor mutant, as well as a construction and application thereof. The anti-copper binary signal conduction-system regulating factor mutant is a base sequence in a sequence list SEQ ID No.1. The expression method comprises the following steps that pseuclomones fluorescens TSS1 is used as a template, and a product obtained by the PCR amplification of primers CRF19 and CRR9 is used to connect an expression plasmid pLC1 to obtain a plasmid pLC104, the latter undergoes a restriction digestion by SwaI and is then connected with a plasmid pJR20 to obtain a plasmid pJC104 which is transformed into the pseuclomones fluorescens TSS1 to obtain a strain of TSS1 / Pjc104. The regulating protein obtained from the method provided by the invention is a pseuclomones fluorescens anti-copper systemic regulating factor mutant, the expression of which in the pathogenic pseuclomones fluorescens TSS1 can significantly decrease bacterial virulence.

Description

technical field [0001] The invention relates to the field of molecular microbiology, in particular to an anti-copper binary signal transduction system regulatory factor mutant and its construction and application. Background technique [0002] At present, trace element pollution, including copper pollution, has become a serious worldwide problem. Copper can catalyze the production of harmful peroxides in organisms, thereby causing damage to biofilms and nucleic acid substances, so copper is used for the prevention and control of bacterial diseases. In view of this, many bacteria, especially pathogenic bacteria, have developed one or more anti-copper mechanisms. The discovered anti-copper system usually consists of multiple proteins, including CopA, CopB, CopC, CopD, CopS, and CopR. Among them, CopR is a transcriptional regulatory protein, CopS is a histidine kinase, and CopA, B, C, D, etc. are directly involved in the transport and removal of copper. CopR and CopS constit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/21C12N15/31C12N15/63C12N1/21C12R1/39
Inventor 孙黎胡永华张敏
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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