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Trc promoter mutation library and application thereof

A promoter and library technology, applied in the biological field, can solve problems such as inability to meet production requirements, weak strength, and limited use

Inactive Publication Date: 2020-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the expression intensity of an inducible promoter is closely related to the inducer, it is necessary to add a variety of different inducers to regulate the expression levels of different genes in the precise regulation of multi-gene metabolic pathways, which limits its role in metabolic regulation. usage of
In addition, most inducible promoters have leaky expression, and the addition of inducers also greatly increases the cost of fermentation
Constitutive promoters have the advantage of stable expression levels under different growth conditions. However, the existing constitutive promoters in nature are generally weak in strength and cannot meet actual production needs.

Method used

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  • Trc promoter mutation library and application thereof
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  • Trc promoter mutation library and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Error-prone mutations of the Trc promoter

[0053] Trc promoter is a constitutive promoter, which is a spliced ​​hybrid promoter of Trp and Lac. It has higher transcription efficiency than Trp and strong promoter characteristics regulated by LacI repressor protein.

[0054] Select the Trc promoter (SEQ ID NO.42) as a template, adjust the magnesium ion concentration (1mM, 2mM, 0.5mM) and manganese ion concentration (3.0mM) in the PCR reaction system through a concentration gradient, and add non-fidelity Taq DNA polymerase Increase the mutation rate of the amplification to obtain promoters of different strengths.

[0055] Green fluorescent protein (sfGFP) was used as a reporter gene to characterize the Trc promoter strength regulating its expression. In order to construct a reporter gene expression vector and facilitate the insertion of a mutant promoter, the original unmutated Trc promoter and other promoters in the promoter library were used to regulate the ...

Embodiment 2

[0056] The construction of embodiment 2 recombinant plasmids pTrc-sfgfp and mpTrc-sfgfp

[0057] Such as figure 1 As shown in (A), using pUC57-sfgfp as a template (the construction method is disclosed in the paper "Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi-and Pan-Drug-Resistant Strains of Acinetobacter baumannii"), with primers pTrc1 F (SEQ ID NO.43) and pTrc1 R (SEQ ID NO.44)) were amplified to obtain the PCR product of sfGFP. Plasmid pTrc99a and the above PCR product were digested with EcoR I and Hind III at the same time, and recovered using a gel recovery kit. The target gene and pTrc99a were ligated with the target sfGFP and pTrc99a by T4 DNA ligase overnight at 16°C to obtain the recombinant plasmid pTrc- sfgfp. Add 100 μL of Escherichia coli JM109 to the above ligation reaction solution, place it on ice for 30 min, heat shock at 42 °C for 90 s, add 1 mL of LB liquid medium, incubate at 37 °C for 1 h, and coat the Kana-resistant plat...

Embodiment 3

[0058] The construction of embodiment 3 genetically engineered bacteria ECTrc-sfgfp and ECmpTrc-sfgfp

[0059] Thaw Escherichia coli JM109 competent cells on ice; add 5 μL of the recombinant plasmids pTrc-sfgfp and mpTrc-sfgfp constructed in Example 2, mix well and then ice-bath for 30 min; heat shock the above mixture at 42°C for 90 seconds, then add 1 mL LB medium, and only incubate it at 37°C and 250rpm for 1h, centrifuge at 5000rpm for 3min, then pour out the supernatant, and spread the bacterial solution on the corresponding antibiotic plate; culture it upside down for 12-16 hours until a single clone appears. The transformants were picked and verified by colony PCR to obtain positive recombinant strains. Finally, it was verified by Shanghai Sangon sequencing.

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Abstract

The invention relates to a Trc promoter mutation library and application thereof, and belongs to the technical field of biology. Promoters shown in SEQ ID NO.1-41 are obtained through a large number of screening, and the range of regulating and controlling the intensity of a target gene table is wide so that a Trc promoter mutation library is established. According to the invention, the promoter library is constructed, and the opening amount type fine adjustment gene expression becomes possible; in metabolic analysis and transformation, the disturbance on the cell genotype can be realized onlyby two non-'on ', namely'off' means of gene knockout and overexpression in the past, namely, the influence of the target gene expression on phenotype and metabolic flux distribution can be analyzed and controlled by performing gradient type accurate fine adjustment on the target gene expression.

Description

technical field [0001] The invention relates to a Trc promoter mutation library and an application thereof, belonging to the field of biotechnology. Background technique [0002] Promoter is an important regulatory element of gene expression, which determines the intensity and timing of gene expression. With the development of synthetic biology and metabolic engineering, people have higher and higher requirements for the scope and strength of promoter regulation in the process of regulating cell metabolism. Promoters with a wider regulatory range can provide finer regulation of the expression of different genes in metabolic pathways, while higher-strength promoters provide sufficient power to achieve high-efficiency expression of target proteins. Functionally, promoters can be divided into two types: inducible and constitutive. Inducible promoters regulate the transcription level of target genes under the action of corresponding regulatory proteins and inducers. Their bigge...

Claims

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Application Information

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IPC IPC(8): C40B40/06C12N9/38C07K14/435C12N15/113C12N15/70C12N1/21C12R1/19
CPCC40B40/06C12N9/2471C07K14/43595C12Y302/01023C12N15/70C12N2830/001
Inventor 邓禹赵梅张佳南娜荷芽周宇辰汪敏叶乘锋
Owner JIANGNAN UNIV
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