Cell-free protein synthesis method based on sigma factor as transcription element

A cell-free protein and protein synthesis technology, applied in the field of cell-free protein synthesis and expression components, can solve the problems of lack of genetic components and restricted gene circuits, etc., and achieve good orthogonality, low endogenous background, and less mutual interference

Inactive Publication Date: 2021-06-29
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the cell-free protein synthesis system has been developed for more than 60 years, the available genetic elements are still very scarce, mainly relying on bacteriophage T7 and T7 RNA polymerase for transcription and translation in the system
Limited phage transcriptional elements limit the development of genetic circuits in cell-free systems and the application of complex systems

Method used

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  • Cell-free protein synthesis method based on sigma factor as transcription element
  • Cell-free protein synthesis method based on sigma factor as transcription element
  • Cell-free protein synthesis method based on sigma factor as transcription element

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Experimental program
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Effect test

Embodiment 1

[0044] A variety of σ factors characterized by this method and their corresponding promoters are used to design gene circuits and perform protein production and expression. P 70 Ability to utilize σ in cell extracts 70 For subsequent protein expression, the target protein such as green fluorescent protein (sfGFP) is designed behind P70, and a protein yield of 0.2 mg / ml can be achieved in cell-free. At the same time, the gene circuit of secondary regulation can be designed, and the constitutive promoter (PT7 or P70) can be used to express σ factors such as σ 24 , σ 24 By regulating the expression of P24 subsequent genes, the expression level of fluorescent protein (sfGFP) of 0.01mg / ml can be achieved in cell-free, and the expression level in cell-free can be designed by using this secondary regulatory circuit. Utilize constitutive T7 to express σ factor, and add fluorescent protein particles expressed by promoters corresponding to different σ factors (see attached Figure 1A...

Embodiment 2

[0050] This method can take advantage of the fact that the σ factor itself needs to be combined with the core enzyme to exert a complete function and that different σ factors have different competitiveness for the core enzyme to design an oscillating gene circuit.

[0051] Designed and obtained by overlapping PCR H1 Express σ for the promoter 32 Factor DNA template. sigH constitutively expressed (P 70 ). with P 32 Expression of fluorescent protein (sfGFP) for the promoter as a reporter gene to represent σ 32 level of expression. Add the above gene circuit into the cell-free body system for 30°C reaction, during the reaction process sigH regulates σ 32 expression. express σ 32 After that, the expression of the fluorescent protein is started and the signal is output. But due to sigH and σ 32Both need to be combined with the core enzyme to function, so the two σ factors have a competitive relationship. in σ 32 When the expression reaches a certain level, the core enzy...

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Abstract

The invention provides a cell-free protein synthesis method based on a cell endogenous sigma factor for escherichia coli. The method is characterized in that protein expression is carried out in a cell-free protein synthesis system by using a protein expression gene element composed of the endogenous sigma factor of escherichia coli or bacillus subtilis and a corresponding promoter.

Description

technical field [0001] The invention belongs to the field of synthetic biological protein synthesis, and in particular relates to a method for synthesizing expression elements of cell-free protein. Background technique [0002] In the past few decades, with the development of metabolic engineering and synthetic biology, microorganisms have shown great potential and application prospects as production factories for the production of diverse (bio)chemicals. Cell-free protein synthesis (CFPS) is a new type of protein synthesis technology, which has advantages over whole-cell factories in the production of biological products. This technology mainly utilizes transcription-translation-related catalytic enzyme components in cell extracts combined with externally added energy regeneration systems, amino acid substrates for protein synthesis, inorganic salts and cofactors for in vitro protein synthesis. Cell-free synthesis can complete the protein synthesis process in vitro without...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/435
CPCC12N15/70C07K14/43595
Inventor 卢元林晓媚
Owner TSINGHUA UNIV
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