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A method for determining the mechanism of action of antibiotics using a bioluminescent reporter system

A reporting system, bioluminescence technology, applied in chemiluminescence/bioluminescence, introduction of foreign genetic material using vectors, material analysis by optical means, etc. There are no problems such as the mechanism of action of antibiotics, time-consuming and labor-intensive, etc., to achieve the effect of improving the speed of identification, huge social and economic benefits, and improving the speed of discovery

Active Publication Date: 2021-06-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, the existing methods for identifying the mechanism of action of antibiotics rely heavily on complex biochemistry, molecular biology, and genetic studies, which are time-consuming and labor-intensive, and rely on high-level scientific research. There is still no method that can quickly determine the action of antibiotics. Mechanism method
This situation has seriously affected the rapid screening, discovery, identification and research of the mechanism of action of antibiotics.
After searching, there is no report on the use of a luminescent reporter system constructed from the promoter fragments of the chaperone genes clpX, groE, dnaJ, dnaK and the luxABCDE gene fragment connected to the promoter of the molecular chaperone gene to determine the mechanism of action of antibiotics.

Method used

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  • A method for determining the mechanism of action of antibiotics using a bioluminescent reporter system
  • A method for determining the mechanism of action of antibiotics using a bioluminescent reporter system
  • A method for determining the mechanism of action of antibiotics using a bioluminescent reporter system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Experimental strains and plasmids:

[0036]Escherichia coli DH5α: used for competent preparation, obtained from China General Microorganism Culture Collection Center (CGMCC1.12873).

[0037] Escherichia coli DH10B: used for competent preparation, obtained from ThermoFisher Scientific (Cat. No. 18297010)

[0038] Staphylococcus aureus RN4220: used for competent preparation and luminescence detection, acquired from BEI Resources.

[0039] pMD19-T: Purchased from Dalian Bao Biological Company (TaKaRa)

[0040] pAmilux: from the University of British Columbia, Canada (Document: Mesak LR, Yim G, Davies J, Improved lux reporters for use in Staphylococcus aureus, Plasmid, 2009, 61(3): 182-187.)

[0041] The medium and its preparation required for the experiment:

[0042] LB medium: For each liter of medium, tryptone (10g), sodium chloride (10g), and yeast extract (5g) should be added to 950ml of deionized water, and stirred with a magnetic stirrer until dissolved. Add 5 mo...

Embodiment 2

[0154] The drawing of embodiment 2 characteristic luminous curves

[0155] Experimental strains: The strains used in the experiment are four reporter strains of Staphylococcus aureus lux: S1, S2, S3, and S4.

[0156] Media required for experiment and its preparation

[0157] 1. NYE agar medium

[0158] 2. 0.7% agar: Weigh 0.7g of agar with a balance and add it to the Erlenmeyer flask, then add 100ml of deionized water, and sterilize under high pressure steam at 121°C for 20 minutes for later use.

[0159] Antibiotics required for experiments and their preparation

[0160] 1. Daptomycin (DAP) solution: Dissolve 30 mg of daptomycin in 1 ml of ultrapure water to make the final concentration 30 mg / ml, and filter through a 0.22 μm membrane to sterilize;

[0161] 2. Polymyxin B (PMB) solution: Dissolve 100mg of polymyxin B in 1ml of ultrapure water to a final concentration of 100mg / ml, and filter through a 0.22μm membrane to sterilize;

[0162] 3. Imipenem (IPM) solution: Dissol...

Embodiment 3

[0202] Cluster Analysis of Example 3 Characteristic Luminescence Curves

[0203] Experimental procedure

[0204] Step 1. Cluster analysis

[0205] 1. Record each characteristic luminous curve (from the edge of the inhibition zone to the edge of the plate) as a vector (luminous intensity characterization vector) represented by 300 luminous intensities, and each luminous intensity represents in the luminous picture of embodiment 2 The average luminous intensity in each circle from the edge of the inhibition zone to the edge of the plate (the span is 1 / 300 of the edge of the inhibition zone to the edge of the plate).

[0206] 2. Merge the characteristic luminescence curves of the four reporter strains corresponding to each antibiotic into a combined luminescence intensity characterization vector represented by 1200 luminescence intensities.

[0207] 3. Using the Euclidean distance calculation method, calculate the distance between the combined luminous intensity representation ...

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Abstract

The invention discloses a method for determining the action mechanism of antibiotics by using a bioluminescent reporter system. It is to use the bioluminescent reporter system to prepare the lux reporter strain of Staphylococcus aureus, arrange the filter paper sheets of the solution containing antibiotics to be tested on the agarose double-layer plate containing the strain, cultivate and obtain the luminescent picture, and convert the luminescent picture into luminescent curve, extract the characteristics of the luminescence curve and compare the characteristic luminescence curves of known antibiotics for hierarchical clustering. If the luminescence curves of the detected antibiotics and the luminescence curves of antibiotics with known mechanisms of action are clustered together, it is determined that the mechanism of action of the antibiotic to be tested is similar to that of antibiotics with known mechanisms of action. Antibiotics with known mechanisms of action are the same. The method of the invention can realize the determination of the action mechanism of antibiotics, reduce the cost of identifying the action mechanism of antibiotics, increase the speed of identification of antibiotic action mechanisms, improve the ability of antibiotic screening, and has broad application prospects.

Description

technical field [0001] The invention relates to a method for determining the action mechanism of antibiotics by using a bioluminescent reporter system. Belongs to the field of biotechnology. Background technique [0002] Antibiotics are humans' main weapon against bacterial infections. The widespread use of antibiotics has caused serious problems of antibiotic resistance, making existing antibiotics rapidly ineffective, and bacterial infections have once again become a major threat to human health. According to statistics, there are currently about 700,000 deaths worldwide due to antibiotic resistance. In 2050, it is estimated that 10 million people will die every year because of antibiotic resistance, becoming the number one cause of death for human beings. Because of the existence of this threat, the importance of discovering new antibiotics is increasing day by day, and the discovery of new antibiotics has also become the focus of research and work in the scientific an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76C12N15/74
CPCC12N15/74G01N21/763
Inventor 王明钰徐海
Owner SHANDONG UNIV
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