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A method for identifying biological elements based on a dual fluorescent reporter gene system

A fluorescent reporter gene and reporter gene technology, which is used in biochemical equipment and methods, microbial determination/inspection, microbial library, etc. And other issues

Active Publication Date: 2021-09-14
武汉睿嘉康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) In the prior art, the expression of the reporter gene in the single reporter gene system will be affected by various factors from inside and outside the strain, which will affect the identification of the promoter by the system
[0007] (2) In the prior art, the cycle of obtaining the promoter to be tested is long and the experiment is complicated, and it is impossible to quickly and high-throughput the strength identification, which is not conducive to the efficient quantitative research of the promoter
[0008] (3) In addition, in the prior art, in vitro detection technology is not widely used because of its low accuracy

Method used

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  • A method for identifying biological elements based on a dual fluorescent reporter gene system
  • A method for identifying biological elements based on a dual fluorescent reporter gene system
  • A method for identifying biological elements based on a dual fluorescent reporter gene system

Examples

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Effect test

Embodiment 1

[0055] (1) Using the pEZ15Asp plasmid as the backbone, construct a single fluorescent reporter gene system for screening fluorescent proteins ( figure 2 A), including EGFP, mCherry, RFP, CFP, and codon-optimized EGFP (opEGFP), mCherry (opmCherry) and CFP (opCFP), fluorescent proteins are all activated by the promoter PlacUV5.

[0056] (2) Determination of fluorescent reporter genes: according to the expression results of different fluorescent gene proteins in Zymomonas mobilis ( figure 2 B), after screening the appropriate fluorescent reporter genes (EGFP and opmCherry), the pEZ15Asp plasmid was also used as the backbone to construct a dual fluorescent reporter gene system.

[0057] like figure 2 As shown, a schematic diagram of the single fluorescent reporter gene system and the results of screening fluorescent proteins provided by the embodiment of the present invention.

[0058] (A) Schematic diagram of a single fluorescent reporter gene system; (B) Fluorescent protein...

Embodiment 2

[0069] (1) Firstly, genes with strong downstream expression were screened out according to different omics data, and Venn analysis was performed to screen out the genes shared by each omics data. Among the different omics data, according to the average value of each gene under all conditions, the present invention defines strong promoters ranked above 90%, medium-strength promoters ranked 40-60%, and ranked Below 10% are weak promoters. The present invention screens 19 strong promoters, 9 medium-strength promoters and 10 weak promoters.

[0070] like Figure 4 As shown, the Venn analysis provided in the embodiment of the present invention screens promoters with different strengths.

[0071] (2) Search, sequence analysis and operon prediction for the promoter of the target gene (taking Pgap as an example), and determine its promoter sequence:

[0072] Pgap promoter sequence:

[0073] 5’-GTTCGATCAACAACCCGAATCCTATCGTAATGATGTTTTGCCCGATCAGCCTCAATCGACAATTTTACGCGTTTCGATCGAAGCAGGG...

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Abstract

The invention belongs to the technical field of biological identification, and discloses a method for identifying biological elements based on a dual fluorescent reporter gene system, using pEZ15Asp plasmid as a backbone to construct a single fluorescent reporter gene system for screening fluorescent proteins; determination of fluorescent reporter genes; pEZ15Asp backbone The acquisition of fluorescent genes; the assembly of fluorescent genes; the acquisition of bacterial strains; system verification; screening of promoters with different strengths; quantitative analysis of fluorescence intensity by flow cytometry. The present invention is convenient and fast, can batch screen and identify promoters of different strengths in a relatively short period of time, and can perform quantitative analysis; can quickly and high-throughput screen and quantify promoters of different strengths and other biological elements, and can quickly Expand the biological component library of Zymomonas mobilis for metabolic engineering of different needs.

Description

technical field [0001] The invention belongs to the technical field of biological identification, in particular to a method for identifying biological elements based on a double fluorescent reporter gene system. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] The traditional promoter screening method is to randomly cut the genome, and then identify whether the obtained sequence is a promoter sequence and its activation strength through the expression of a single downstream reporter gene. Most of the previous detection systems are single reporter gene systems, but the expression of the reporter gene in the single reporter gene system will be affected by various factors from inside and outside the strain, which will affect the identification of the promoter by the system. In addition, the speed of obtaining the promoter to be tested by the previous method is slow, and it is impossible to quickly and hi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12Q1/6851C40B40/02G01N33/68G01N33/569
Inventor 杨世辉沈威杨永富黄钜易犁马立新
Owner 武汉睿嘉康生物科技有限公司
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