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Lactose induction type expression vector and viscidity Serratieae converted therefrom

A technology of Serratia marcescens and an expression vector, which is applied in the field of lactose-inducible expression vector, can solve problems such as difficulty in large-scale production of diol, and achieves the effect of wide practical value and huge application value

Inactive Publication Date: 2008-08-06
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Another object of the present invention is to provide a method for producing meso-2,3-butanediol to overcome the problem that meso-2,3-butanediol is difficult to produce on a large scale in the prior art

Method used

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  • Lactose induction type expression vector and viscidity Serratieae converted therefrom
  • Lactose induction type expression vector and viscidity Serratieae converted therefrom
  • Lactose induction type expression vector and viscidity Serratieae converted therefrom

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the construction of inducible expression vector pLHZH

[0041] expression plasmid pET28a (+) (Kan r ,lacI q , purchased from Invitrogen) as a template, using primer 1 and primer 2 as a primer pair for PCR amplification. Among them, primer 1: 5'-ACACCATCGAATGGCGCAA-3'; primer 2: 5'-TCACTGCCCGCTTTCCAGT-3'. In a sterilized 0.2mL PCR thin-walled tube, add 20μL of sterile water, 5μL of 10×PCR buffer, 1μL of dNTP (10mM, TaKaRa), 1μL of primer 1 (final concentration 50pmol), 1μL of primer 2 (final concentration 50pmol), 0.5 μL of plasmid pET28a (+) solution (concentration 50ng / μL), 0.5μL pfu DNA polymerase (5U / μL, TaKaRa), make up to a total volume of 50μL with sterile water, and place the PCR tube in a PCR cycler. In the PCR reaction, the temperature change process of the PCR reaction is: first raise the temperature to 94°C, keep it for 5 minutes, then cycle 29 times according to the following temperature change program: raise the temperature to 94°C, keep ...

Embodiment 2

[0042] Embodiment 2, meso-2, the cloning of 3-BDH gene

[0043] Klebsiella pneumoniae (Klebsiella pneumoniae) genomic DNA was used as a template and primer 3 and primer 4 were used for PCR amplification. Among them, primer 3: 5'-CGC GGATCC ATGAAAAAAGTCGCACTTGTTACCG-3' (underlined base is BamH I recognition site); Primer 4: 5'-ACGC GTC GAC TTAGTTAAATACCATCCCGCCGTC-3' (bases underlined are Sal I recognition sites). In a sterilized 0.2mL PCR thin-walled tube, add 5μL sterile water, 2.5μL 10×PCR buffer, 1μL dNTP (10mM, TaKaRa), 1μL primer 1 (final concentration 50pmol), 1μL Primer 2 (final concentration 50 pmol), 0.5 μL genomic DNA solution (concentration 50 ng / μL), 0.5 μL pfu DNA polymerase (5 U / μL, Qiagen), make up to a total volume of 25 μL with sterile water, and place the PCR tube in in a PCR cycler. In the PCR reaction, the temperature change process of the PCR reaction is: first raise the temperature to 94°C, keep it for 5 minutes, then cycle 29 times according to the...

Embodiment 3

[0044] Embodiment 3, meso-2, the expression of 3-BDH gene

[0045] The recombinant plasmid pLHZH-Bdh was transformed into the host cell Serratia marcescens B17 by the improved calcium chloride method to obtain the genetically engineered bacteria Serratia marcescens B17 / pLHZH-Bdh. The genetically engineered bacteria were inoculated in LB liquid medium (LB medium composed of: yeast powder 0.5%, tryptone 1%, NaCl 1%, pH 7.0) added with appropriate antibiotics, cultured on a shaker at 30°C overnight; % of the inoculum was transferred to a 250mL shake flask containing 30mL LB medium, cultured on a shaker at 30°C for 5 hours, added 0.5mL120% lactose solution (final concentration 2%), and continued to cultivate for 6 hours, then centrifuged to collect bacteria body. The collected bacteria were subjected to SDS-PAGE electrophoresis of the whole bacteria, and the results were as follows: Figure 4 shown. In the figure, lane 1 is the standard protein molecular weight (97.2, 66.4, 44....

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Abstract

The invention discloses a lactose induction type expression vector, which can utilize cheap lactose to replace expensive isopropyl thioamide-beta-D- galactoside (IPTG) to be exutory. The lactose induction type expression vector can be widely used on the expression of exogenous gene and the reconstruction of serratia marcescens metabolic pathway, for example, the lactose induction type expression vector is used to lead meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) in serratia marcescens. The invention also discloses a serratia marcescens which utilizes gene recombination technique to reconstruct, which changes the metabolic pathway of natural meso-2,3-butanediol in bacteria through leading in exogenesis meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) gene. The serratia marcescens which is reconstructed by gene recombination technique which is provided by the invention expresses meso-2,3-butanediol dehydrogenase in an expected level, which improves production and accumulation of meso-2,3-butanediol in host strain, and improves the productivity of meso-2,3-butanediol in serratia marcescens.

Description

technical field [0001] The invention relates to a lactose-inducible expression vector, Serratia marcescens transformed by the vector and its application. Background technique [0002] 2,3-Butanediol is an important chemical raw material for the preparation of resins, cosmetics, inks, spices, explosives and pharmaceutical intermediates, and is widely used in many fields such as chemical industry, food, fuel and aerospace. For example, 2,3-butanediol can be used in the liquor industry as a flavor additive and for the preparation of perfume 3-hydroxy-2-butanone. At present, the domestic market price of 2,3-butanediol is 100,000 yuan / ton, while the price of meso-2,3-butanediol is about twice that price. Due to the difficulty in synthesizing 2,3-butanediol by chemical methods, industrial production has not yet been realized, so the production of 2,3-butanediol by biological fermentation is the future development direction. [0003] The production of 2,3-butanediol by biological...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N1/21C12N15/53C12P7/18C12R1/43
Inventor 魏东芝沈亚领朱虎周文瑜曹学
Owner EAST CHINA UNIV OF SCI & TECH
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