Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Escherichia coli-clostridium shuttle expression vector, and construction and expression thereof

A shuttle expression vector, Escherichia coli technology, applied in the field of bioengineering, can solve problems such as not very ideal application prospects

Inactive Publication Date: 2014-07-02
SHANGHAI ACAD OF AGRI SCI
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clostridium perfringens chloramphenicol acetyltransferase, which is very suitable as a reporter gene for Clostridium, is promising because Clostridium contains many high levels of non-specific acetyl-CoA that may interfere with the expression of chloramphenicol acetyltransferase. not ideal

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Escherichia coli-clostridium shuttle expression vector, and construction and expression thereof
  • Escherichia coli-clostridium shuttle expression vector, and construction and expression thereof
  • Escherichia coli-clostridium shuttle expression vector, and construction and expression thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Synthesis of embodiment 1 Escherichia coli lacZ gene

[0023] According to Clostridium (GenBank: AE001437.1) preferred codon synthetic biology method [Ai-Sheng Xiong, Quan-Hhong Yao, Ri-He Peng, Xian Li, Huiqin Fan, Zong-ming Cheng and Yi Li, Nucleic Acid Research, 2004, 32(12), e98:1-10] Design the following primers, wherein the primers are all from 5' end to 3' end, and further optimize the synthesis of Escherichia coli lacZ gene.

[0024] 1.EMPII-1:Tm=54,60mer

[0025] GGA,TCC,ATG,ACT,ATG,ATT,ACT,GAT,TCT,CTT,GCT,GTT,GTT,TTA,CAA,AGA,AGA,GAT,TGG,GAA

[0026] 2.EMPII-2:Tm=54,60mer

[0027] GAT, GTG, CAG, CAA, GTC, TAT, TAA, GTT, GAG, TAA, CAC, CAG, GAT, TTT, CCC, AAT, CTC, TTC, TTT, GTA

[0028] 3.EMPII-3:Tm=54,60mer

[0029] TAA,TAG,ACT,TGC,TGC,ACA,TCC,TCC,TTT,TGC,TTC,TTG,GAG,AAA,TTC,TGA,AGA,AGC,TAG,AAC

[0030] 4.EMPII-4:Tm=54,60mer

[0031] TTC,ACC,ATT,AAG,TGA,TCT,AAG,TTG,TTG,AGA,TGG,TCT,ATC,AGT,TCT,AGC,TTC,TTC,AGA,ATT

[0032] 5.EMPII-5:Tm=54,60mer

[0033] ...

Embodiment 2

[0167] Example 2 Cloning of Clostridium CRO repressor-like DNA-binding gene (OABC) promoter and construction of plasmid pYN7443

[0168] Use R27320 (5'-TCT, AGA, AAA, GGA, AAA, TAT, GAT, AAA, AAA, TTT, CA-3') and R27321 (5'-GGA, TCC, CAT, TAA, TAT, CGA, AAA ,TAG, CTT, AAA, CCC, AAC-3') as primers, and Clostridium genomic DNA (GenBank: AE001437.1) as a template, and the high-fidelity and high-temperature resistant DNA polymerase Pyrobest from Japan TAKARA Company was used to carry out Clostridium OABC Amplification of gene promoters. The PCR amplification program was: hot start at 94°C for 5 min; denaturation at 94°C for 20S, annealing at 53°C for 20S, extension at 72°C for 15S, a total of 35 cycles; final extension at 94°C for 10 min; Fragments around 300bp were recovered by electrophoresis at a concentration of Agarose. Purify the PCR amplified product with the UNIQ-10 Column DNA Gel Recovery Kit produced by Shanghai Sangon to obtain the Clostridium OABC gene promoter, the ...

Embodiment 3

[0169] Example 3 Construction of Escherichia coli-clostridium shuttle expression vector pYN7443-EclacZS

[0170] see figure 1 , with BamHI and SacI (purchased from Treasure Bioengineering (Dalian) Co., Ltd.) to carry out double enzyme digestion respectively, recover the DNA fragments, connect the EclacZS gene of Example 1 to the pYN7443 plasmid of Example 2 by T4 DNA ligase-purify, that is The Escherichia coli-Clostridium shuttle vector pYN7443-EclacZS containing the Escherichia coli lacZ gene was obtained.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an escherichia coli-clostridium shuttle expression vector, and construction and expression thereof. The escherichia coli-clostridium shuttle expression vector is constructed by inserting escherichia coli lacZ gene (EclacZS) coding sequence between BamHI and SacI restriction enzyme cutting sites of pYN7443 plasmid, wherein the escherichia coli lacZ gene (EclacZS) coding sequence is obtained via clostridium preferred codon chemical synthesis. It is confirmed by escherichia coli lacZ gene activity determination on clostridium transformed from the escherichia coli-clostridium shuttle expression vector that the lacZ gene, which comes from escherichia coli and is obtained via clostridium preferred codon chemical synthesis, is a report gene suitable for expresion in clostridium. Construction of high butanol yield engineered strains via determination of optimal promoter-terminator combination possesses important theoretical significance, wherein determination of the optimal promoter-terminator combination is realized by comparing different promoters and terminators.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to an Escherichia coli-clostridium shuttle expression vector and its construction and expression, in particular to an Escherichia coli-clostridium shuttle expression vector chemically synthesized according to the codon preference of Clostridia and its construction, And the expression vector can successfully see the application of Escherichia coli lacZ gene expressed in Clostridium when transformed into Clostridium. Background technique [0002] Although considerable achievements have been made in the metabolic engineering of Clostridium in recent years, poor research on several necessary genetic tools has limited the further development of acetone-butanol fermentation, and one of the most important genetic tools is the control of exogenous gene expression the reporter gene. A good reporter gene can help researchers to study different homologous or heterologous promoters in Clo...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/113C12N15/74C12Q1/34
Inventor 姚泉洪田永生彭日荷许晶王波王丽娟韩静
Owner SHANGHAI ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com