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Light-operated cell-free protein synthesis method, plasmid used in method and product using method

A protein, cell-free technology, applied in animal/human proteins, medical preparations containing active ingredients, introduction of foreign genetic material using carriers, etc., can solve difficult to achieve precise positioning of targeted cells, adverse effects of chemical stability problems, to achieve rapid response, increased flexibility, and low cost

Active Publication Date: 2020-11-27
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the regulation rate of chemical substances will be limited by the process rate of entering and exiting the membrane and body binding, and the stability of the chemical substance itself and other side effects will have an adverse effect on the regulation. Traditional chemical small molecule ligands and antagonists are difficult to achieve. Precise positioning of targeted cells

Method used

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  • Light-operated cell-free protein synthesis method, plasmid used in method and product using method
  • Light-operated cell-free protein synthesis method, plasmid used in method and product using method
  • Light-operated cell-free protein synthesis method, plasmid used in method and product using method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1: Making pDark plasmid, culturing it in the cell-free extract of E.coli BL21, and expressing red fluorescent protein under dark and blue light irradiation conditions

[0133] - Make pDark plasmid

[0134] Use the PCR method to clone the red fluorescent protein gene mCherry from the plasmid pET23a-mCherry (the plasmid was synthesized by Jinweizhi Company), and then use the PCR technology to clone the plasmid pDusk (the product number of the plasmid is: Addgene #43795) from the promoter FixK2 To ensure that there are 25 bases of the same sequence at both ends of the mCherry gene and the linear vector. The mCherry gene was connected with the vector by Gibson's self-assembly method to form the pDark plasmid.

[0135] - Prepare E.coli BL21 cell extract and mix with pDark to form a cell-free reaction system

[0136] The cells were cultured to prepare cell extracts E.coli BL21(DE3) containing two light-regulated proteins, and the cell-free system was prepared accord...

Embodiment 2

[0141] Example 2: Making pLight plasmid, culturing it in the cell-free extract of E.coli BL21, and expressing red fluorescent protein under dark and blue light irradiation conditions

[0142] -The pLight plasmid was further constructed on the basis of the pDark plasmid structure. pLight was based on the pDark, and the cI-pR gene circuit was assembled behind the promoter FixK2. Open the empty vector pDawn (product number of the plasmid: Addgene #43796) from the promoter pR by using PCR technology to ensure that there are 25 bases of the same sequence at both ends of the mCherry gene and the linear vector. Use Gibson self-assembly method to connect mCherry gene and vector to form pLight plasmid. - Prepare E.coli BL21 cell extract and mix with pLight to form a cell-free reaction system

[0143] Prepare cell extracts E.coli BL21(DE3) containing two light-regulated proteins respectively. When preparing a cell-free system, mix the two cell extracts according to 0, 0.01, 0.04, 0.1, ...

Embodiment 3

[0148] Example 3: Select new pLight plasmids (replacing the red fluorescent protein with NbmCherry, NbRota3B2 and NbCEA5 three drug proteins), and study their expression in the cell-free extract of E.coli BL21.

[0149] - On the basis of the pLight plasmid structure in Example 2, pLight plasmids of three drug proteins were further constructed, namely pLight-NbmCherry, pLight-NbRota3B2 and pLight-NbCEA5. Use PCR technology to NbmCherry, the gene of NbRota3B2 and NbCEA5 three kinds of drug proteins from respective plasmid (can refer to following literature about NbmCherry: Fridy, P.C., Li, Y., Keegan, S., Thompson, M.K., Nudelman, I. , Scheid, J.F., Oeffinger, M., Nussenzweig, M.C., D., and Chait, B.T. (2014) A robust pipeline for rapid production of versatile nanobody repertoires, Nature methods 11, 1253. Regarding NbRota3B2, you can refer to the following literature: Vega, C.G., Bok, M., Vlasova, A.N., Chattha, K.S. ,Gómez-Sebastián,S., C., Alvarado, C., Lasa, R., Escriban...

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Abstract

The invention relates to a plasmid. The plasmid comprises a gene of a photosensitive protein, a promoter capable of responding to the photosensitive protein, and an exogenous gene connected with the promoter and used as an exogenous protein transcription template, or an inversion circuit connected with the promoter and comprising the exogenous gene used as the exogenous protein transcription template. In addition, the invention further relates to application of the plasmid, a product based on the application and a light-operated cell-free protein synthesis method.

Description

technical field [0001] The application belongs to the field of biological reaction systems, and specifically relates to a light-controlled cell-free protein synthesis method, a plasmid used in the method, and a product using the method. Background technique [0002] Bio-manufacturing is an industry that provides new products for social development through the large-scale material processing and material transformation of biological functions. Compared with the traditional manufacturing industry, bio-manufacturing has lower demand and consumption of resources, and at the same time causes less pollution and damage to the environment. It is an important driving force to promote the transformation and upgrading of traditional manufacturing industries and optimize the industrial structure. [0003] In recent years, cell-free protein synthesis (CFPS), as a new protein synthesis method, has attracted attention in the field of biomanufacturing, showing great potential and applicatio...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/70C12N15/12C12N15/13A61K48/00A61K41/00
CPCC12N15/63C12N15/70C07K14/43504C07K16/10C07K14/4748A61K48/0008A61K41/0028
Inventor 卢元杨俊祝
Owner TSINGHUA UNIV
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