Construction method and application of promoter library

A construction method and promoter technology are applied in the field of promoter library construction to achieve the effect of small screening workload

Inactive Publication Date: 2016-05-25
NANJING UNIV OF TECH
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Problems solved by technology

Secondly, when Alper et al. were studying the synthesis of lycopene, they found that increasing the expression of dxs gene (deoxy-xylulose-psynthase, deoxy-xylulose phosphate synthase) in wild bacteria could only increase the expression of lycopene to a certain extent. However, in the recombinant bacteria ov

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  • Construction method and application of promoter library
  • Construction method and application of promoter library
  • Construction method and application of promoter library

Examples

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Embodiment 1

[0033] Example 1. Construction and Identification of Recombinant Bacteria pMD19-T-RBS-GFP

[0034] The present invention uses green fluorescent protein (GFP) as a reporter gene to describe and characterize the strength of the artificially synthesized promoter regulating its expression. In order to construct a reporter gene expression vector to facilitate the insertion of a mutant promoter, it is first necessary to construct an expression vector with a reporter gene. In the present invention, it is special that when constructing the expression vector of the reporter gene, it is necessary to artificially add a section of RBS ribosome binding site in front of the GFP gene when designing primers, so that the downstream reporter gene can be smoothly translated. Protein (RBS is the ribosome binding site, which plays an important role in protein translation), thereby constructing the vector pMD19-T-RBS-GFP recombinant bacteria with the RBS sequence in front of the reporter gene GFP. ...

Embodiment 2

[0036] Embodiment 2. Design and recombinant plasmid with the oligonucleotide sequence of biological promoter function

[0037] pMD19-T-P spl - Construction and identification of RBS-GFP

[0038] In this example, for Escherichia coli organisms, the conserved type (-35 region: TTGACA; -10 region: TATAAT), semi-conserved region (transcription initiation site; upstream regulatory region) and random region of the constitutive promoter in Escherichia coli organisms (the spacer region of the -35 region and the -10 region), an oligonucleotide sequence with a biological promoter function is designed. Secondly, in order to facilitate the connection to the reporter gene expression vector, two restriction enzyme cutting sites were introduced in the upstream and downstream respectively: EcoRI and XbaI, which are convenient for subsequent enzyme digestion identification. Finally, a NcoI restriction site was introduced behind the transcription start site, its role is to facilitate the iden...

Embodiment 3

[0043] Example 3. Obtaining of Mutant Bacteria with Artificial Promoter Library and Establishment of Screening Method and Screening Results

[0044] The obtained recombinant plasmid pMD19-T-P spl -The RBS-GFP connection solution is transformed into E.coliDH5a strain, spread on the LB plate containing ampicillin (ampicillin: the concentration of the mother solution is 100mg / mL, weigh 3g of Aladdin's ampicillin and dilute to 30ml of sterile water , use a 0.22um sterile filter membrane to filter and sterilize, the dosage is 1 / 1000 (v / v); LB medium: peptone 10g / L, yeast powder 5g / L, NaCl 5g / L, pH7.0, the plate needs to be added 15-20g / L agar powder. The above reagents were all purchased from Nanjing Wanqing Company), and after culturing overnight at 37°C, a transformed single colony was obtained.

[0045] Screening method: Inoculate the single colony obtained after transformation into 5 mL of LB medium containing ampicillin, cultivate at 37°C and 200 rpm / min for 12 hours, centrif...

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Abstract

The invention discloses a construction method and application of a promoter library. The construction method includes the following steps that firstly, an oligonucleotide sequence with promoter features is designed according to a conserved region, a semi-conserved region, a random sequence and a transcription initiation locus of a prokaryote promoter; secondly, the oligonucleotide sequence designed in the first step is subjected to annealing and extension to form double strands, the double strands are connected to a carrier with a report gene, and a mutation library of the promoter is obtained; thirdly, the mutation library, obtained in the second step, of the promoter is migrated into a host cell, a flat plate with corresponding resistance is coated with the mutation library, and the promoter library is obtained. By the adoption of the oligonucleotide sequence which is artificially designed and has the promoter features and the technologies of molecule aggregation and extension, the promoter library with ideal promoter intensity span is obtained. The method is quick, efficient, cheap and small in screening workload.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a promoter library and its application. Background technique [0002] The optimization and fine research of cell metabolic network is becoming a very important frontier research direction in the field of biochemical industry. However, microbial cells are a complex dynamic network composed of genes, proteins, metabolites, various signaling molecules and their interactions. The analysis and regulation of metabolic processes are very complex and difficult. In order to be able to analyze the key nodes for fine regulation, it is necessary to study the role of each pathway through fine regulation, so as to realize the artificial transformation of cell phenotype and metabolic flow. 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 scientif...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06G01N21/31G01N21/64
CPCC40B50/06C40B40/06G01N21/31G01N21/6428
Inventor 姜岷朱兴贵马江锋吴明科高有军
Owner NANJING UNIV OF TECH
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