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Blue light regulation promoter, fusion gene of blue light regulation promoter, blue light-mediated regulation plasmid and construction method and application of blue light-mediated regulation plasmid

A technology that combines genes and construction methods, applied in the field of microorganisms, can solve problems such as unfavorable applications, non-specific toxicity transfer delays, and pleiotropic effects, and achieve the effects of simple operation, good reversibility, and rapid response

Active Publication Date: 2021-10-01
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing reports on the Yarrowia lipolytica promoter are still in the traditional exogenous chemical inducer regulation mode, but there are defects such as non-specificity, toxicity, transport delay and pleiotropy, which are not conducive to its industrial production and applications in social life

Method used

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  • Blue light regulation promoter, fusion gene of blue light regulation promoter, blue light-mediated regulation plasmid and construction method and application of blue light-mediated regulation plasmid
  • Blue light regulation promoter, fusion gene of blue light regulation promoter, blue light-mediated regulation plasmid and construction method and application of blue light-mediated regulation plasmid
  • Blue light regulation promoter, fusion gene of blue light regulation promoter, blue light-mediated regulation plasmid and construction method and application of blue light-mediated regulation plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Optimize the gene sequence

[0042] 1. The fusion gene is composed of the blue light-regulated promoter PC120-CYC, the green fluorescent protein GFP and the Yarrowia lipolytica xpr2 terminator, and the nucleic acid sequence is shown in SEQ ID NO.2.

[0043] 2. The VP16 and EL222 gene sequences and protein sequences retrieved from NCBI were optimized according to the codon preference of Yarrowia lipolytica, and the DNA sequences were re-synthesized.

[0044] The specific optimization method is: according to the gene synthesis company GenScript codon optimization software OptimumGene TM To optimize the gene, mainly refer to the codon preference of Yarrowia lipolytica, and then combine the filter element and balance the GC content of the gene to finally complete the optimization of the gene codon, and obtain the nucleic acid sequence of the fusion gene SV40-VP-EL, such as SEQ As shown in ID NO.4, in the fusion gene SV40-VP-EL, SV40 represents the nuclear localization sign...

Embodiment 2

[0046] Construction of blue light-mediated regulatory plasmid phSVExPCGx

[0047] Using the plasmid pINA1296 as the backbone vector, the blue light-mediated regulation plasmid phSVExPCGx was designed and constructed.

[0048] The specific construction method is:

[0049] The fusion gene SV40-VP-EL gene sequence optimized in Example 1 was fully synthesized, and then the sequence between the pINA1296 plasmid hp4d promoter and xpr2 terminator was replaced by the fusion gene SV40-VP-EL using Gibson assembly technology to obtain a plasmid phSVEx;

[0050] The gene sequence of the fusion gene optimized in Example 1 was fully synthesized, and then the fusion gene was inserted into the upstream of the hp4dpromoter in the plasmid phSVEx using Gibsonassembly technology to obtain the blue light-mediated regulatory plasmid phSVExPCGx. In this example, the synthesized Blue light-mediated regulation plasmid carrying the reporter gene GFP. The blue light-mediated regulatory plasmid phSVEx...

Embodiment 3

[0052] Transformation, induced expression and analysis of the blue light-mediated regulatory plasmid phSVExPCGx

[0053] (1) Plasmid extraction and quantitative detection

[0054] 1) Take 10 mL of the overnight bacterial culture, centrifuge at 13400 g for 1 min, discard the supernatant and collect the precipitate.

[0055] 2) Add 500 μL of solution Ⅰ, solution Ⅱ and solution Ⅲ in sequence, immediately flip up and down gently for 6 to 8 times, let stand for 5 minutes, and centrifuge at 13400 g for 10 minutes.

[0056] Wherein the composition of solution I is 25mM Tris-HCl (pH=8.0), 10mM EDTA, 50mM glucose;

[0057] The composition of solution II is 250mMNaOH, 1% (W / V) SDS;

[0058] The composition of solution III is 3M potassium acetate, 5M acetic acid.

[0059] 3) Add the supernatant collected in the previous step into the filter column, centrifuge at 13400 g for 1 min, add 450 μL of isopropanol and mix well. Then add to the adsorption column, centrifuge at 13400g for 1min...

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Abstract

The invention relates to the technical field of microorganisms, in particular to a blue light regulation promoter, a fusion gene of the blue light regulation promoter, a blue light-mediated regulation plasmid and a construction method and application of the blue light-mediated regulation plasmid. The invention provides a blue light regulation promoter PC120-CYC. The feasibility of a blue light-induced regulation mode in the gene expression and regulation process of the yarrowia lipolytica is verified by utilizing a fusion gene synthesized based on the blue light-induced regulation mode and a blue light-mediated regulation plasmid. The blue light-mediated regulation plasmid provided by the invention is very simple and compact, and can be induced by blue light on the single cell level of yarrowia lipolytica. In the application of the blue light-mediated regulation plasmid, the blue light-mediated regulation plasmid is non-toxic and harmless, quick in response, simple and convenient to operate and good in reversibility, and can effectively promote the wide application of the yarrowia lipolytica in the fields of food, medicine, agricultural product processing and the like.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a blue-light-regulated promoter, a fusion gene of the blue-light-regulated promoter, a blue-light-mediated regulation plasmid, a construction method, and an application. Background technique [0002] As an important factor in the external environment, light provides energy for all living matter on the earth, and in some photosensitivity reactions, light can be used as a regulatory signal to achieve structural and functional changes. Optogenetics is an emerging direction for the study of light-sensitive proteins and gene circuits, which is dedicated to the precise regulation and expression of genes through optical and genetic techniques. [0003] As a common light-sensing module in plants and bacteria, the Light-Oxygen-Voltage (LOV) domain has blue-light-dependent regulation, thus realizing the blue-light-regulated enzyme and DNA binding and other effectors. active. LOVs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/81C12N15/62C07K19/00C12R1/645
CPCC07K14/39C12N15/815C07K14/195C12N2830/002C12N2800/22C07K2319/09C07K2319/71
Inventor 张后今闫云君王志乾龚梦瑶韩娟张婷
Owner HUAZHONG UNIV OF SCI & TECH
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