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GAP promoter library and application thereof

A technology of promoter and library, applied in the field of GAP promoter library of Pichia pastoris

Inactive Publication Date: 2010-12-22
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
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Problems solved by technology

When Alper et al. studied the synthesis of lycopene, they found that increasing the expression of dxs gene (deoxy-xylulose-p synthase, deoxy-xylulose phosphate synthase) in wild bacteria could only improve the synthesis of lycopene to a certain extent. However, in the recombinant bacteria overexpressing the two downstream enzymes of the synthetic pathway, the continuous enhancement of dxs gene expression can continuously increase the lycopene production, indicating that the activity of deoxy-phosphoxylulose synthase is the limitation of the synthetic pathway of recombinant bacteria factor

Method used

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  • GAP promoter library and application thereof
  • GAP promoter library and application thereof
  • GAP promoter library and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1, the construction of recombinant bacteria G / GHg

[0105] Green Fluorescent Protein (GFP) was used as a reporter gene to characterize the GAP 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 GAP promoter was first used to regulate the expression of the reporter gene gfp, and the GFP reporter gene expression vector pGHg and the corresponding recombinant strain G / GHg were constructed, and the The vector pGH containing the GFP gene and the recombinant strain G / GH were used as controls to investigate the expression of GFP.

[0106] 1. Construction and identification of expression vector pGHg

[0107] (1) Construction and identification of recombinant plasmid pGAPZAgfp

[0108] refer to figure 1 , using the chemically synthesized gfp gene (SEQ ID NO: 15) as a template, using the primer GFP F(AAT GCGGCCGC AAACCC AAGCTT ATGTCTAAAGGTGAAG...

Embodiment 2

[0123] Embodiment 2, the establishment of screening model

[0124] 1. Determination of screening medium

[0125] (1) Effects of different media on GFP fluorescence detection

[0126] PBS was used as a negative control to investigate the effects of different culture media on GFP fluorescence detection.

[0127] Sterile BMD, BMDY, YPD, BSM medium (see Invitrogen’s operating manual: A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris) and PBS were each added to a 96-well plate to measure the fluorescence intensity by a fluorescent microplate reader (excitation wavelength: 488nm, emission wavelength: 505nm). It was found that BSM medium had the least interference on the determination of fluorescence intensity (BSM: 537 (RFU); PBS: 460 (RFU)), followed by BMD: 616 (RFU). The fluorescence intensities of the sterile medium BMDY and YPD detected by a fluorescent microplate reader are 47581 (RFU) and 46499 (RFU) respectively, which are more than 100 times h...

Embodiment 3

[0165] Embodiment 3, the construction of GAP promoter mutation library

[0166] In order to construct a promoter library and obtain a mutant promoter with a wide range of promoter strength and a gradient change in promoter strength, it is necessary to obtain a promoter with abundant mutations and a method for quickly and efficiently screening mutants. Error-prone PCR (EP-PCR) is a simple and rapid method for making random mutations in a DNA sequence. The basic principle is that by changing the concentration of some components (such as dNTP and Mg 2+ Concentration), so that bases are randomly introduced to a certain extent to create sequence diversity. The key to error-prone PCR is to select an appropriate mutation frequency. Generally, the frequency of beneficial mutations is very low, and most mutations are harmful or neutral. When the mutation frequency is too high, it is almost impossible to screen beneficial mutations; when the mutation frequency is too low, the wild typ...

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Abstract

The invention relates to a GAP promoter library and application thereof. The invention discloses a nucleic acid sequence of each promoter in the GAP promoter library, a vector and a host cell which contain the nucleic acid sequence. The invention also discloses a method for screening a required mutation promoter. The invention establishes an effective screening model, obtains the mutation GAP promoter library with strength of graded distribution, and can realize continuous fine adjustment on expression of a target gene.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a GAP promoter library for Pichia pastoris. Background technique [0002] Metabolic Engineering, which optimizes cell metabolic network through genetic manipulation, is becoming a very important frontier research direction in the field of biochemical engineering. However, in the face of the complex dynamic network composed of genes, proteins, metabolites, various signaling molecules and their interactions in microbial cells, the analysis and regulation of metabolic processes are very complicated and difficult. Only by fine-tuning gene expression to study its effect on cell phenotype and metabolic flux can we resolve key metabolic nodes that can be modified. Therefore, the precise "open volume" gene fine-tuning is a very important advanced means to carry out metabolic engineering research, and its research has important scientific significance and broad app...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C12N15/63C12N1/19C12Q1/68C12R1/84
Inventor 钱江潮秦秀林储炬庄英萍张嗣良肖慈英
Owner EAST CHINA UNIV OF SCI & TECH
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