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Biosensors comprising allosteric transcription factor regulatory systems, kits and their use in small molecule detection

A technology of biosensors and transcription factors, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, and analysis through chemical reactions of materials, which can solve the problems of difficulty in lowering the detection limit and poor detection stability

Active Publication Date: 2020-10-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the production of fluorescent proteins is closely related to the density and physiological conditions of cells, the detection stability of the above method is poor
On the other hand, the detection limit of this type of detection system not only depends on the interaction between the inducer and the trans-acting factor, but also is limited by the transcriptional machinery of the cell. Problems such as the osmotic expression of inducible promoters often reduce the detection limit. more difficult

Method used

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  • Biosensors comprising allosteric transcription factor regulatory systems, kits and their use in small molecule detection
  • Biosensors comprising allosteric transcription factor regulatory systems, kits and their use in small molecule detection
  • Biosensors comprising allosteric transcription factor regulatory systems, kits and their use in small molecule detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0155] Example 1: Uric acid biosensor

[0156] In this example, the allosteric transcription factor HucR and the DNA fragment hucO interacting with it are used as the recognition element, and the ALPHA technology microbeads are used as the transducer element to construct a biosensor platform capable of sensing uric acid, and use a mathematical model to analyze the sensitivity of the biosensor It provides a general, systematic and effective strategy to improve the sensitivity and detection range of biosensors.

[0157] The HucR protein is a repressor protein derived from Deinococcus radiodurans. In the absence of uric acid (UA), HucR can bind to its operator site hucO, preventing the transcription of downstream target genes. In the presence of uric acid, uric acid specifically binds to HucR, releasing HucR from hucO and turning on the transcription of target genes. Therefore, the concentration of uric acid in the sample can be determined from the degree of binding of HucR to ...

Embodiment 2

[0273] Embodiment 2: oxytetracycline biosensor

[0274] In this example, the allosteric transcription factor OtrR and the DNA fragment containing its action site otrO are used as the recognition element, the donor beads and acceptor beads of ALPHA technology are used as the first transduction element, and HucR and its interaction site hucO are used as the second transducing element. The second transducer element is used to construct a biosensor platform capable of sensing oxytetracycline. The DNA fragment has both otrO and hucO fragments ( image 3 a).

[0275] OtrR is a repressor protein derived from Streptomyces chamotis. In the natural state, in the absence of oxytetracycline, OtrR can bind to its operator site otrO, preventing the transcription of downstream target genes. In the presence of oxytetracycline, oxytetracycline specifically binds OtrR, releasing OtrR from otrO and turning on the transcription of target genes. Therefore, the concentration of oxytetracycline ...

Embodiment 3

[0305] Embodiment 3: prunamycin biosensor

[0306]In this example, two kinds of prunamycin biosensors using nuclease as the second transduction element were constructed. The recognition elements of the sensor are all allosteric transcription factors Pip and ptr sites, and the first transducer elements are all gold nanoparticle pairs. In an embodiment utilizing an exonuclease, exonuclease III and a DNA fragment with a 3' overhang are used as the second transducing element. In embodiments utilizing an endonuclease, a HindIII endonuclease and a HindIII restriction site are used as the second transducing element.

[0307] Pip is a repressor protein derived from Streptomyces coelicolor. In the natural state, in the absence of prunamycin, Pip can bind to its operator site ptr, preventing the transcription of downstream target genes. Prinamycin specifically binds Pip when it is present, releasing Pip from ptr and turning on the transcription of the target gene. Therefore, the con...

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Abstract

The invention relates to a biosensor containing an allosteric transcription factor, and a kit and application of the same to micromolecular detection.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to an invitro method, a kit and a device for detecting whether a specific type of small molecule exists in a sample by means of the interaction between an allosteric transcription factor (aTF) and DNA. Background technique [0002] Effective detection of small molecules in samples is extremely important for environmental pollution monitoring, food quality control, and disease diagnosis. Small molecule detection methods in the prior art are mainly divided into two categories, physical and chemical analysis methods and biological analysis methods. The physical and chemical analysis methods mainly use spectroscopy, chromatography and their combined techniques. These techniques have high separation efficiency, good selectivity, and strong qualitative and quantitative capabilities. However, the sample preparation process is complicated, the equipment is expensive and time-consuming. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6818G01N21/31G01N21/64G01N21/78
CPCG01N21/314G01N21/6486G01N21/78G01N2021/3185
Inventor 杨克迁王为善李珊珊范可强李子龙
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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