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A class of tunable putrescine bioluminescence sensor and its application

A fluorescent sensor, putrescine technology, applied in the fields of molecular biology and synthetic biology, can solve the problems of inability to monitor the dynamic process of metabolism in real time, slow analysis speed, and hidden health risks.

Active Publication Date: 2021-06-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, excessive putrescine itself has a certain degree of toxicity, and is widely present in fermented foods. Foods containing a large amount of putrescine (cheese, sausage, bacon, red wine, etc.) will also cause serious damage to the body of consumers. major health hazard
[0004] At present, traditional methods for the determination of putrescine, such as pre-column derivatized high-performance chromatography, fluorescent probe method, thin-layer chromatography and electrophoresis, are relatively complicated and cumbersome, require additional sample preparation, and their analysis speed is slow. However, real-time monitoring of metabolic dynamic processes cannot be realized, and all belong to the category of offline detection

Method used

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  • A class of tunable putrescine bioluminescence sensor and its application
  • A class of tunable putrescine bioluminescence sensor and its application
  • A class of tunable putrescine bioluminescence sensor and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] In this example, the artificially designed promoter was constructed.

[0055] The specific operation of this example includes: two steps of direct denaturation and annealing of the two single-stranded nucleotides of the artificially designed promoter, and the construction of an artificially designed promoter fragment containing sticky ends of XhoI and EcoRI.

[0056] Single-stranded nucleotides are:

[0057] pLR2: single-stranded nucleotide 1 as shown in Seq ID No.11, namely:

[0058] 5'-TCGAGATAAATTATCTCTGGCGGTGTTGACAGTGGTCATTATATTTTACGCGATACTGAGCACAGTGGTCATTATATTTTACGCG-3'

[0059] Single-stranded nucleotide 2 is shown in Seq ID No.12, namely:

[0060] 5'-AATTCGCGTAAAATATAATGACCACTGTGCTCAGTATCGCGTAAAATATAATGACCACTGTCAACACCGCCAGAGATAATTTATC-3'

[0061] pAR2: single-stranded nucleotide 1 as shown in Seq ID No.13, namely:

[0062] 5'-TCGAGAAAATTTATCAAAAAGAGTGTTGACTGTGGTCATTATATTTTACGCGATACTTAGATTCAGTGGTCATTATATTTTACGCG-3'

[0063] Single-stranded nucleotide 2 is sho...

Embodiment 2

[0085] In this embodiment, a putrescine bioluminescence sensor is built through the following operations:

[0086] 1) Mix the artificially designed promoter fragment containing sticky ends after digestion with XhoI and EcoRI in Example 1 with the linear fragment of plasmid pZA27gfp after digestion with XhoI and EcoRI at 37° C. for 4 hours, and then in T4 ligase Under the action, connect overnight at 16°C, and transfer into Escherichia coli Top10 competent cells. In order to construct the correct pLR2gfp, pAR2gfp, pLR3gfp, pAR3gfp, pTacR2gfp, pTacR3gfp plasmids, spread the transformants on the card containing a final concentration of 50 μg / mL On the LB solid plate of namycin, after culturing overnight at 37°C, pick positive single clones and inoculate them into LB medium, at 37°C, 220rpm, 12-14 hours, and extract the plasmid for sequencing.

[0087] 2) Transform the correct pLR2gfp, pAR2gfp, pLR3gfp, pAR3gfp, pTacR2gfp, pTacR3gfp plasmids into Escherichia coli MG1655, spread on...

Embodiment 3

[0092] In this implementation, putrescine biosensors with different PuuR expression levels were established through the following operations:

[0093] 1) Using MG1655 genomic DNA as a template, PCR was performed with primer sequence numbers: 23 and 24, and the puuR gene containing KpnI and XmaI restriction sites was amplified, and then ligated with the linearized vector pZA27gfp after double digestion to construct the The correct pZA27puuR plasmid.

[0094] Seq ID NO.23: 5'-CGG GGTACC ATGAGTGATGAGGGACTGGC-3'

[0095] Seq ID NO.24: 5'-TCCC CCCGGG TTAAAACGTGGTGGGCGTAT-3'

[0096] The reaction system is: template 1 μL, primers 1 and 2 each 1 μL, DMSO 5 μL, 2×Primer Max mix 25 μL, sterilized water 17 μL;

[0097] The reaction conditions are: denaturation at 98°C for 5min; denaturation at 98°C for 30s; annealing at 58°C for 30s; extension at 72°C for 1min; 30cycles; 5min at 72°C; 10min at 12°C;

[0098] 2) Using the pZA27puuR plasmid as a template and primer sequence numbers...

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Abstract

A class of adjustable putrescine bioluminescence sensor and its application, the recombinant plasmids pAR2gfp, pLR2gfp, pAR3gfp, pLR3gfp, pTacR2gfp, pTacR3gfp containing artificially designed promoters responsive to putrescine were transferred into Escherichia coli MG1655, and a A green fluorescent protein-labeled bioluminescent detection system that induces GFP when putrescine exists in the environment mut3 Protein expression, at this time to detect the fluorescence intensity. The present invention uses fluorescent labeling technology to construct a putrescine bioluminescent sensor, conducts qualitative and quantitative analysis of putrescine by detecting the fluorescence intensity, and provides technology and methods for putrescine detection. At the same time, by changing the expression level of putrescine recognition element PuuR and the culture medium Adjusting the response range of the sensor has important applications in the screening of production strains, real-time monitoring of putrescine synthesis process and environmental detection.

Description

technical field [0001] The invention relates to a technology in the field of molecular biology and synthetic biology, in particular to a genetically encoded putrescine bioluminescent sensor, which is used for qualitative and quantitative detection of putrescine concentration in the external environment, production capacity of putrescine production strains and Real-time monitoring of putrescine synthesis process. Background technique [0002] Putrescine, also known as 1,4-diaminobutane or 1,4-butanediamine, contains two amino functional groups, making it widely used and marketed in the chemical industry. For example, putrescine can be used as a component of polymers, medicines, agricultural products and other additives, especially, putrescine and adipic acid synthesize nylon-4,6 materials (Yamanobe et al., J.Mol.Struct. , 829:80-87, 2007). Traditional chemical synthesis adopts succinonitrile hydrogenation method (Sanders et al., Macromol.Biosci., 7: 105-117, 2007) has certa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N15/70G01N21/64
CPCC12N15/66C12N15/70C12N2800/101G01N21/6428
Inventor 钱志刚陈雪枫夏小霞
Owner SHANGHAI JIAO TONG UNIV
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