Gluconobacter oxydans promoter and its application

A technology for oxidizing glucose and acid bacteria, which is applied in the field of promoters, can solve the problems of lack of constraints on the genetic engineering improvement of Gluconobacter oxidans, and achieve the effect of high activation activity

Active Publication Date: 2014-04-23
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a promoter of Gluconobacter oxidans and its application, thereby solving the deficiency that the

Method used

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  • Gluconobacter oxydans promoter and its application
  • Gluconobacter oxydans promoter and its application
  • Gluconobacter oxydans promoter and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Cloning of the promoter gHp0169

[0039] According to the G.oxydans621H genome sequence, the gHp0169 sequence is located between GOX0168 and GOX0169 (eg figure 2 shown), design and synthesize primers (Shanghai Jierui Bioengineering Co., Ltd.), its sequence is as follows:

[0040] gHp0169-F: 5'-ATA GAGCTC GCCAAGAAAGCCGCAGAACTC-3';

[0041] gHp0169-R: 5'-GCA TCTAGA GCGGAAGGCGTTATACCCTGA-3';

[0042] Using the genome of G.oxydans as a template, using gHp0169-F and gHp0169-R as primers, PCR amplifies the target gene, and introduces Sac I and Xba I restriction sites (underlined) at both ends, respectively. The specific conditions are as follows :

[0043] Reaction system: 2×PCR mix 25μl, primer (10μmol / L) 1μl each, template 1μl, ddH 2 O22μl;

[0044] Reaction process: 94°C for 5min, 94°C for 30s, 60°C for 30s, 72°C for 30s, 30 cycles, 72°C extension for 10min, 4°C storage.

[0045] The result is as figure 1 As shown, primers gHp0169-F and gHp0169-R wer...

Embodiment 2

[0047] Example 2: Verification of the functional activity of the promoter gHp0169

[0048] 2.1. Construction of plasmid pBBR1pgHp0169-GFP

[0049] According to the enzyme cutting sites designed at both ends of the primers gHp0169-F and gHp0169-R, the gHp0169 fragment was excised from the T-gHp0169 recombinant vector constructed in Example 1 with Sac I and XbaI, and the recovered gHp0169 fragment was Ligated with pBBR1MCS5 vector fragment digested with Sac I and Xba I. The ligated product was transformed into Escherichia coli DH5α strain, the plasmid was extracted, and identified by digestion with Sac I and Xba I. Sequencing was performed on the initially identified correct recombinant plasmid, and the recombinant vector containing 142 bp shown in SEQ ID NO: 1 was named pBBR1pgHp0169.

[0050] Using PET28a-GFP (purchased from Luyang Biopharmaceutical Company, USA) as a template, GFP primers (Shanghai Jierui Bioengineering Co., Ltd.) were designed and synthesized. The sequence...

Embodiment 3

[0083] Embodiment 3: Utilize promoter gHp0169 to overexpress ga2dh gene to improve 2-KGA production

[0084] 3.1. Construction of plasmids pBBR1pgHp0169-ga2dh and pBBR1ptufB-ga2dh

[0085] According to the nucleotide sequence of ga2dh of Gluconobacter oxydans G.oxydans, design primers (Shanghai Jierui Biological Engineering Co., Ltd.), the sequence is as follows:

[0086] ga2dh-F: 5'-CTA TCTAGA GGAGAAACCTGTGCCCCCCATG-3';

[0087] ga2h-R: 5'-GAG GGATCC TTCAGTTCAGTGAGACCGCATCATC-3';

[0088] Using the genome of G.oxydans as a template, using ga2dh-F and ga2dh-R as primers for PCR amplification, respectively introducing Xba I and BamH I restriction sites at both ends of the promoter sequence, the amplification conditions are as follows:

[0089] Reaction system: 2×PCR mix 25μl, primer (10μmol / L) 1μl each, template 1μl, ddH 2 O22μl;

[0090] Reaction process: 30 cycles of 94°C for 5min, 94°C for 30s, 59°C for 30s, 72°C for 4min, extension at 72°C for 10min, and storage at ...

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Abstract

The invention provides a Gluconobacter oxydans (G.oxydans) promoter and its application. The promoter has the following nucleotide sequence: 1) a nucleotide sequence shown as SEQ ID NO:1 in a sequence table; or 2) a nucleotide sequence hybridizing with the nucleotide sequence shown as SEQ ID NO:1 under rigorous conditions and having promoter functions; or 3) a nucleotide sequence which is obtained by subjecting the nucleotide sequence defined by 1) or 2) to substitution, deletion, insertion or adding by one or more basic groups, and has over 90% homology with the defined nucleotide sequence and promoter functions. The promoter provided by the invention has the function of effective expression of the endogenous or exogenous gene in Gluconobacter oxydans, and has higher promoter activity compared with common promoters. The Gluconobacter oxydans promoter can realize application to gene expression and functional gene screening research in G.oxydans, and also can be applied to construction of 2-KGA high-yield gene engineering bacteria.

Description

technical field [0001] The invention relates to a promoter, more specifically to a promoter of Gluconobacter oxydans and its application. Background technique [0002] The final products of gene expression are RNA and protein. Any defect or disorder of gene expression regulation program will cause serious consequences to the organism. Therefore, the research on the mechanism of gene expression regulation is the focus and frontier of molecular biology. The expression of protein-coding genes requires transcription and translation, each of which has different regulatory sites for gene expression. Promoters have been studied in detail as an important element to start genes. Promoter refers to the DNA sequence region that can bind to RNA polymerase to initiate transcription, usually located upstream of the coding gene. It is a component of genes that controls when and how much gene expression (transcription) starts. Therefore, the screening and functional analysis of promoter...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/74C12N1/21C12R1/01
Inventor 林金萍魏东芝施露露
Owner EAST CHINA UNIV OF SCI & TECH
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