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Membrane-free transcriptional regulator and application thereof in precise regulation of gene expression

A regulator and transcription factor technology, applied in the field of synthetic biology and genetic engineering, can solve problems such as limiting downstream gene expression and yield

Pending Publication Date: 2021-11-02
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the amount of transcription factors in the organism greatly limits the expression and production of downstream genes

Method used

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  • Membrane-free transcriptional regulator and application thereof in precise regulation of gene expression
  • Membrane-free transcriptional regulator and application thereof in precise regulation of gene expression
  • Membrane-free transcriptional regulator and application thereof in precise regulation of gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Constructing a Membrane-Free Transcription Regulator

[0055] Intracellular transcription factors, transcription-related elements, and enzymes important in cell metabolism can form a liquid-liquid phase separation. This phase separation occurs in cells in the form of liquid droplets, often referred to as intracellular membraneless organelles. The construction of "membraneless" organelles requires the formation of low polymers based on intrinsically disordered sequences or multivalent interactions. Binding protein-protein interactions for inducible 'membraneless' organelles. The "membraneless" phase separation system induced in the current life science field is mainly based on two methods: light induction and chemical small molecule induction. Compared with the light induction system, the chemical small molecule induction system has the characteristics of convenient operation, good controllability and high sensitivity, and is more suitable for generalized and...

Embodiment 2

[0071] Example 2 Membraneless Transcription Regulator Regulates Downstream Gene Transcription Burst

[0072] Using real-time fluorescence imaging technology and live cell RNA labeling technology, the transcription dynamics in the current state of the cell can be analyzed in real time.

[0073] PP7 is a nucleotide sequence near the transcription initiation site of the PP7 replicon of a type of bacteriophage, which can be specifically recognized by the phage capsid protein PCP. Taking advantage of this property, the researchers developed them as a tool for monitoring RNA transcription in living cells. In order to observe the expression kinetics of downstream genes after the aggregation of transcription factors, the PCP-PP7 live cell RNA labeling technology was used to integrate the lentivirus PCP-mCherry gene into the monoclonal cell line obtained in Example 1, and to observe the expression kinetics of downstream genes. Transcriptional burst at integration sites.

[0074] 1. T...

Embodiment 3

[0091] Example 3 "Membraneless" transcriptional regulators can achieve inducible nonlinear regulation of gene expression

[0092] In order to verify the performance of the "membrane-free" transcriptional regulator induced by this small molecule, the input (expression of transcriptional components, Here is the fluorescent signal of EGFP) and output (the protein level of the downstream gene, here is the fluorescent signal of iRFP) to measure, quantitatively analyze the input and output signals produced under different concentrations of small molecule induction conditions ( image 3 a).

[0093] In this example, the same monoclonal cell line is added with different concentrations of aggregation-inducing agent (Rapamycin) to induce transcription factors to form different degrees of aggregation. The concentration reference range for rapamycin is based on the results of chemically small molecule-induced assembly of "membrane-less" transcriptional regulators, within which rapamycin ...

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Abstract

The invention relates to a membrane-free transcriptional regulator and application thereof in precise regulation of gene expression. According to the invention, a transcription factor aggregation system capable of being induced by small molecules or light is constructed based on the interaction of inherent disordered sequences or multivalent peptide fragments to form a low polymer and the interaction between proteins, the transcription factor aggregation system is called as a membrane-free transcription regulator, and the regulator is applied to gene expression regulation; under induction of different small molecule concentrations or different light, different degrees of aggregation of transcription factors can be induced by changing the concentration or light intensity of chemical small molecules, the aggregation state of the transcription factors is changed, the activation ability of the transcription factors is improved, and expression of downstream genes is remarkably activated, so the efficient and accurate regulation and control of gene expression are realized.

Description

technical field [0001] The invention relates to the technical fields of synthetic biology and genetic engineering, in particular to a membraneless transcription regulator and its application in precise regulation of gene expression. Background technique [0002] Nonlinear regulation (hypersensitive regulation) has the regulation characteristics of filtering low stimulus signals (below the threshold) and sensitively responding to signals in the S-curve interval (within the threshold range), and plays an important role in life processes such as signal processing and decision making. For example, Bicoid, which is a transcription factor related to morphogenesis in Drosophila embryonic development, is distributed in a gradient in the Anterior-posterior axis (A-P axis) of early Drosophila embryos. The Bicoid distributed in a gradient on the A-P axis binds to downstream genes with multiple Bicoid binding sites in a coordinated manner, and non-linearly regulates the expression of do...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C07K14/00
CPCC12N15/113C07K14/00C12N2830/002
Inventor 吴结根林一瀚
Owner PEKING UNIV