New-type efficient biological energy regeneration system (BES) for in-vitro biological reaction system, kit and preparation method

A bio-energy and system technology, applied in the biological field, can solve the problems of unsustainable energy supply, high cost, and unfavorable industrial application of in vitro protein synthesis system efficiency.

Pending Publication Date: 2019-07-09
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these compounds can release energy to generate ATP through the corresponding kinase reaction, they often only provide a large amount of energy quickly and briefly at the initial stage, and these high-energy compounds have an inhibitory effect on in vitro cell synthesis[6,7], and cannot provide sustained high cost, which is not conducive to the improvement of the efficiency and industrial application of in vitro protein synthesis system[8,9,10]
[0007] At present, many biosynthetic systems use compounds containing high-energy phosphate bonds as sources of regenerative energy, which is costly, and at the same time, a large amount of instantaneous ATP can inhibit the synthesis reaction in vitro

Method used

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  • New-type efficient biological energy regeneration system (BES) for in-vitro biological reaction system, kit and preparation method
  • New-type efficient biological energy regeneration system (BES) for in-vitro biological reaction system, kit and preparation method
  • New-type efficient biological energy regeneration system (BES) for in-vitro biological reaction system, kit and preparation method

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Experimental program
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Effect test

preparation example Construction

[0147] Preparation of BES reaction system:

[0148] BES reaction system: final concentration of 22mM 4-hydroxyethylpiperazineethanesulfonic acid with a pH of 7-8, 30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM nucleoside triphosphate mixture (adenine nucleus glycoside triphosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate), 0.08-0.24mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine amino acid, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 25 mM potassium phosphate, 1.7 mM dithiothreitol, 40 mM glucose, and 50% volume of the above cell extract.

[0149] biological response

[0150] Biological reactions, that is, biochemical reactions, refer to chemical reactions carried out in living organisms. These reactions are all catalyzed by enzymes. Enzymes and reactants are dissolved in water i...

Embodiment 1

[0225] Embodiment 1: Cell-free in vitro protein Fluc synthesis system

[0226] 1.1 Preparation of storage solution for in vitro protein synthesis system: 1M 4-hydroxyethylpiperazineethanesulfonic acid at pH 7.4, 5M potassium acetate, 250mM magnesium acetate, 25mM mixture of four nucleoside triphosphates, including adenine nucleoside triphosphate Phosphoric acid, guanosine triphosphate, cytidine nucleoside triphosphate and uridine nucleoside triphosphate, 1mM mixture of twenty kinds of amino acids: glycine, alanine, valine, leucine, isoleucine, Phenylalanine, Proline, Tryptophan, Serine, Tyrosine, Cysteine, Methionine, Asparagine, Glutamine, Threonine, Aspartic Acid, Glutamic Acid, Lysine , arginine and histidine, the concentrations of twenty amino acids are 1.0mM, 1M potassium phosphate, 1M dithiothreitol, 1M glucose, 1.7mg / mL T7 RNA polymerase 20%-50% polyethylene glycol Alcohol (polyethylene glycol, PEG) 3350 or (polyethylene glycol, PEG) 8000, 20%-40% sucrose, 1-4mM spermi...

Embodiment 2

[0248] Embodiment 2: Cell-free in vitro protein eGFP synthesis system

[0249] Cell-free in vitro protein eGFP synthesis system: final concentration of 22mM 4-hydroxyethylpiperazineethanesulfonic acid at pH 7-8, 30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate), 0.08-0.24mM amino acid mixture (glycine, alanine, valine, leucine acid, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 25mM potassium phosphate, 1.7mM dithiothreitol, 40mM glucose, 8-20ng / μLeGFP DNA, 0.027-0.054mg / mL LT7 RNA polymerase, 1% - 4% polyethylene glycol, and finally 50% by volume of yeast cell extract;

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Abstract

The invention provides a new-type efficient biological energy regeneration system (BES) for an in-vitro biological reaction system, a kit and a preparation method. The biological energy regeneration system can be applied for energy supply of in-vitro biosynthesis, and applied to a cell-free in-vitro biological reaction system. Cost of the in-vitro biosynthesis is saved, and capacity of in-vitro cell-free biosynthesis is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a novel high-efficiency energy regeneration system (BES), a kit and a preparation method for an in vitro biological reaction system. Background technique [0002] Biochemical reactions Biochemical reactions refer to chemical reactions that take place in living organisms. The biochemical reactions in the body are all catalyzed by enzymes. The enzymes and reactants dissolve in the water in the internal environment to react, and the water provides the carrier and medium for the substances in the body. [0003] The biochemical reaction in the cell is called the metabolism of the cell, which is the basis of the life activity of the cell. Metabolism, referred to as metabolism, is one of the important characteristics of organisms to express their life activities. Metabolism in organisms is not completely spontaneous, but catalyzed by biocatalysts-enzymes. Metabolism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00
CPCC12P21/00
Inventor 郭敏柴智王海鹏徐开周子鉴于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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