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Biosensor for detecting S-adenosylmethionine and preparation method of biosensor

An adenosylmethionine, biosensor technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of inability to detect intracellular SAM concentration

Active Publication Date: 2020-07-10
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this electrode is only suitable for the detection of SAM in vitro, and cannot detect the concentration of SAM in cells.

Method used

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  • Biosensor for detecting S-adenosylmethionine and preparation method of biosensor

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Embodiment 1

[0038] In this embodiment, the biosensor for detecting S-adenosylmethionine and its preparation method are as follows:

[0039] 1) Plasmids for constructing artificial transcription factor-based biosensors

[0040] a. Amplify to obtain TEF2 promoter fragment

[0041] Genomic DNA was extracted from Saccharomyces cerevisiae using a fungal genome extraction kit purchased from Tiangen as a template, and the TEF2 promoter fragment was amplified by PCR with SalI-TEF2-overlap-F and TEF2(metJGS)-overlap-R as primers. The primer sequences are shown below (SEQ ID NO. 7-8):

[0042] SalI-TEF2-overlap-F: 5'-ACGCGTCGACGGGCGCCATAACCAAGGT-3';

[0043] TEF2(metJGS)-overlap-R: 5'-TCGCCGCTCCATTCAGCCATGTTTAGTTAATTATAGTTCGTTGACCGTATATTC-3'.

[0044] PCR amplification system (100μL): double distilled water 60.5μL, dNTP 8μL (200μmol / L), buffer 20μL (Mg 2+ 50 mmol / L), 5 μL of upstream and downstream primers (10 μmol / L), 0.5 μL of template, and 1 μL of Phusion High-Fidelity polymerase.

[0045] ...

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Abstract

The present invention discloses a biosensor for detecting S-adenosylmethionine (SAM) and a preparation method of the biosensor, and belongs to the field of biochemical industry. A gene recombination technology synthesizes the biosensor that responds to the S-adenosylmethionine based on a transcription factor module and a recombinant promoter module. The biosensor uses MetJ derived from escherichiacoli as a DNA binding domain, the DNA binding domain interacts with hER and an activation domain (VP16) to constitute a new type of ATF, at the same time, a natural met box with four conserved sequences derived from metA is selected as a specific recognition sequence of the MetJ, and a green fluorescent protein gene is connected to downstream of the sequence to construct the biosensor that responds to the SAM. Under actions of a beta-estradiol inducer and the intracellular SAM, the ATF enters the nucleus and binds to the specific recognition sequence met box of the MetJ to realize downstreamgene regulation. The biosensor can respond to concentration of the S-adenosylmethionine in living cells and achieve a high-throughput detection through an output of fluorescence intensity.

Description

technical field [0001] The invention relates to a biosensor for detecting S-adenosylmethionine and a preparation method thereof, belonging to the field of biochemical industry. Background technique [0002] S-adenosylmethionine (SAM) is a key small-molecule active substance in the body. It has various physiological functions such as transmethylation, transsulfurization and transaminopropyl, and is related to various detoxification processes in the body. It is an indispensable and important substance to maintain the normal function of cell membranes, normal metabolism and health of the human body. Clinically, S-adenosylmethionine is used for the treatment of liver disease, depression, arthritis and other diseases, and the effect is remarkable. [0003] The metabolic pathway of S-adenosylmethionine in microbial cells is long and complex, and it is not a terminal metabolite. Many researchers have tried to improve the ability of strains to produce S-adenosylmethionine through m...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/65C12Q1/6897C12R1/865
CPCC12N15/81C12N15/65C12Q1/6897
Inventor 陈雅维王书涵廖源孔维臻
Owner HENAN UNIV OF SCI & TECH
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