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Double-tagging microbial cell biosensor for detecting N-acyl homoserine lactone

A technology of acyl homoserine lactone and microbial cells, applied in the field of double-labeled microbial cell sensors

Inactive Publication Date: 2013-07-24
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem that some existing AHL sensors cannot characterize the growth of bacteria, and some cannot characterize the bacteria that do not contain the AHL system. Three components are used: one is the pathogenic bacteria Pseudomonas syringae The B728a AHL synthetase promoter ahlI is the promoter, the cherry red fluorescent protein gene mcherry is the reporter gene, and the detection element P responsive to AHL is constructed ahlI :mcherry, the second is to use the neomycin phosphotransferase promoter nptII as a promoter, and the green fluorescent protein gene gfp as a reporter gene to construct a constitutively expressed green fluorescent protein element P that characterizes the growth of the host nptII :gfp, the third is to use the AHL regulatory protein gene ahlR element of Pseudomonas syringae pathogenic bacteria B728a to detect the influence of AHL on bacteria that do not contain AHL system, and construct a method that can detect AHL and characterize bacteria Two-color fluorescently labeled microbial cell sensor of growth status

Method used

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  • Double-tagging microbial cell biosensor for detecting N-acyl homoserine lactone
  • Double-tagging microbial cell biosensor for detecting N-acyl homoserine lactone
  • Double-tagging microbial cell biosensor for detecting N-acyl homoserine lactone

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Embodiment Construction

[0021] The following examples will further illustrate the present invention

[0022] Step 1: Inoculate a single colony of sensor cells in a 20 mL Erlenmeyer flask, add ampicillin to a final concentration of 50 μg / mL, 37°C, 200 r min -1 Overnight culture;

[0023] Step 2: Take 2 mL of the above bacteria solution into 18 mL of fresh LB medium, add ampicillin to a final concentration of 50 μg / mL, 37°C, 200 rpm -1 Grow to OD 600 =0.6;

[0024] Step 3: Add the 3OC6-HSL solution dissolved in acetonitrile to the culture in the second step, so that the final concentrations are 0, 10, 50, 100, 1000 nmol / L, 37°C, 200 r min -1 Continue to induce culture;

[0025] Step 4: During the 24-hour induction period, take 180 μL of the culture in the third step every 3 hours to a 96-well plate, and use a Varioskan Flash full-wavelength scanning multifunctional microplate reader to detect its green fluorescence under 488 nm excitation light. Emission intensity at 510 nm and cherry red fluore...

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Abstract

The invention relates to a double-tagging microbial cell biosensor for detecting N-acyl homoserine lactone (AHL), and the biosensor is suitable for detection of AHL and characterization of host cells in the environment. The cell biosensor comprises: Escherichia coli used as host cells for carrying recombinant plasmids; recombinant plasmid containing an ahlI promoter responding to AHL, a cherry red fluorescent protein reporter gene mCherry and an AHL regulatory protein gene ahlR; and a neomycin phosphotransferase promoter gene nptII for characterization of host bacteria, a green fluorescent protein reporter gene gfp and a T1-4 pUC19 plasmid of four terminator rrnb tandem sequence. According to the biosensor, constitutive type of expression green fluorescent protein is used to characterize the host bacteria; the response time to AHL is 6 h; and the minimum detectable concentration of AHL is 50 nmol / L. The biosensor has the advantages of high sensitivity, fast response, low cost and simple operation, and can be widely applied to detection of AHL and host characterization in the environment.

Description

technical field [0001] The invention relates to the construction and use of a double-labeled microbial cell sensor responding to N-acyl homoserine lactone in the environment. Background technique [0002] In recent years, it has been found that many bacteria can regulate the expression of related genes by producing, releasing, sensing and responding to chemical signal molecules when the cell density reaches a certain threshold, thereby changing the behavior of bacteria to adapt to environmental changes. Called "quorum sensing" (QS: quorum sensing). Quorum sensing regulates many bacterial behaviors including: bacterial motility, antibiotic production, virulence production, bacterial conjugation, spore formation, biofilm formation, and environmental adaptation. The occurrence of these effects of quorum sensing depends on signaling molecules. There are currently three recognized signaling molecules: small peptides, the main signaling molecule of Gram-positive bacteria; N-acyl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12Q1/02C12R1/19
Inventor 庄国强邓雪梅马安周
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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