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Micro-droplets for cell-free protein synthesis and preparation method thereof

A protein synthesis and microdroplet technology, which is applied to the field of microdroplets for cell-free protein synthesis and its preparation, can solve the problems of complicated operation, difficult to realize the simple and efficient synthesis of cell-free protein, difficult separation and purification, etc. Simple and fast method

Active Publication Date: 2017-11-17
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional biosynthesis method is limited by the organism itself. It is necessary to consider the culture and survival of cells, adjust various environmental factors, and separation and purification are also difficult. At present, high-throughput, big data, fast and efficient research has become a major goal of protein synthesis. The trend of research, the traditional method can no longer meet the research requirements, and the cell-free protein synthesis method is superior to the traditional method in these aspects, can bypass the cumbersome process of transformed cells expressed in vivo, not limited to the cell itself, can incorporate non- Natural and isotope-labeled amino acids, enabling the production of proteins that are insoluble or toxic in vivo, and in addition, facilitate proteome screening projects
[0005] However, the current research on cell-free protein synthesis systems is mostly focused on protein synthesis at the macro scale, trying to achieve long-term and high-efficiency protein synthesis by various means, but the operation is too complicated to reflect the simplicity and high efficiency of cell-free protein synthesis characteristics

Method used

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  • Micro-droplets for cell-free protein synthesis and preparation method thereof
  • Micro-droplets for cell-free protein synthesis and preparation method thereof
  • Micro-droplets for cell-free protein synthesis and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 7

[0047] Preparation of cell disruption products:

[0048] The cultured Escherichia coli (Escherichia coli BL21(DE3) CICC 23796, purchased from CICC) was collected, suspended, centrifuged, mechanically broken, and stored.

[0049] In addition to mechanical methods, cell disruption methods can also be repeated freezing and thawing methods, ultrasonic treatment methods, enzymatic methods, alkaline lysis methods or chemical permeation methods.

[0050] Eukaryotic cells can also be used for disruption.

[0051] Examples 8-10 are composition 1, see Table 3.

[0052] table 3

[0053]

Example 8

Example 9

Example 10

Cell disruption products

Example 7 Preparation of 1g

Example 7 Preparation of 1g

Example 7 Preparation of 1g

Buffer solution 1

Example 1 Preparation of 0.1ml

Example 2 Preparation of 0.05ml

Example 3 Preparation of 0.01ml

Buffer solution 2

Example 4 Preparation of 3ml

Example 5 Preparation of 2ml

Example 6 Preparation of 1ml

[0054] Examples 11-13 are amino acid m...

Embodiment 20-22

[0063] Examples 20-22 are composition 2 (each component is volume ratio), see Table 7

[0064] Table 7

[0065]

Example 20

Example 21

Example 22

Amino acid mixture

8mL prepared in Example 11

5mL prepared in Example 12

2mL prepared in Example 13

Reaction buffer

30mL prepared in Example 14

25mL Example 15 preparation

20mL prepared in Example 16

Energy supplement

5mL prepared in Example 17

3mL prepared in Example 18

2mL prepared in Example 19

Ribonucleic acid polymerase aqueous solution

1mL 150μg / ml

1mL 70μg / ml

1mL 5μg / ml

[0066] Gene plasmids can be purchased or obtained through genetic modification.

[0067] pRset-CFP (purchased from: Promega Corporation)

[0068] pRset-YFP (purchased from: Promega Corporation)

[0069] pRset-eGFP (purchased from: Promega Corporation)

[0070] The above examples of plasmids are intended to enable those skilled in the art to better understand the present invention, but they are not limited, and other plasmids can also be used in ...

Embodiment 23-25

[0071] Examples 23-25 ​​are oil phase

[0072] Table 8

[0073]

[0074] EM 90 (Degussa)

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Abstract

The invention discloses micro-droplets for cell-free protein synthesis and a preparation method thereof. The method includes the steps that firstly, a micro-fluidic chip device is used; 2, raw materials for protein synthesis are mixed to serve as a water phase, and the water phase is injected from a water phase inlet 6; an oil phase is injected from an oil phase inlet 4; in a cross channel 13, the water phase disperses under the shear force of the oil phase to form water-in-oil micro-droplets, and the micro-droplets are collected from a micro-droplet outlet 7 and then put into a thermostatic reaction vessel. According to the method, the micro-droplets can be rapidly prepared through the micro-fluidic chip device, and the obtained micro-droplets are uniform in size and stable in structure, and can provide a site for in-vitro protein synthesis; due to the unique microscale of the micro-droplets, part of the micro-droplets are used for simulating a cell environment so that a protein synthesis system working in the cell environment originally can obtain a similar environment, which facilitates the work of the protein synthesis system. Protein synthesis can be quantified, and the micro-droplets can be applied to detection and diagnosis.

Description

Technical field [0001] The invention relates to a micro-droplet for cell-free protein synthesis and a preparation method and application thereof. Background technique [0002] Since the 20th century, with the deconstruction of the structure and function of various biological macromolecules, life sciences have gradually cracked the secrets of biological genetic variation and fully transferred to the molecular level to decrypt life processes. At the end of the 20th century, with the development of a large number of genome sequencing projects, the genetic information of various organisms was deciphered, and molecular biology emerged. It was possible to reconstruct genetic information using molecular manipulation to obtain various biological products that could meet human needs. [0003] Protein is the main undertaker of life activities. Various life activities require the participation of proteins, and proteins are the direct products of the expression of genetic information. Therefor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J19/00C12P21/02
CPCB01J19/0046B01J2219/00527B01J2219/00725C12P21/02
Inventor 仰大勇焦毅刘阳
Owner TIANJIN UNIV
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