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

Shuttling expression vector of bacillus coli-bacillus subtilis and use thereof

A shuttle expression vector, the technology of Bacillus subtilis, applied in the field of Escherichia coli-Bacillus subtilis shuttle expression vector, can solve the problems of limited expression vector, low conversion efficiency of exogenous DNA, difficulty in establishing protein mutant gene library, etc., to increase The effect of library capacity, convenient detection and screening, simple and flexible library capacity size

Inactive Publication Date: 2009-06-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
View PDF0 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the development of molecular biology and genetic engineering, Bacillus subtilis has attracted more and more attention as a genetic engineering expression system, but Bacillus subtilis is a Gram-positive bacterium, the transformation efficiency of foreign DNA is low, and the available expression Vectors are quite limited, so it is difficult to establish a gene library of protein mutants in the Bacillus subtilis operating system using traditional methods

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
  • Shuttling expression vector of bacillus coli-bacillus subtilis and use thereof
  • Shuttling expression vector of bacillus coli-bacillus subtilis and use thereof
  • Shuttling expression vector of bacillus coli-bacillus subtilis and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, construction of Escherichia coli-Bacillus subtilis shuttle expression vector

[0057] 1. Cloning of fructan sucrose transferase gene signal peptide sequence and P43 promoter sequence

[0058] Primer A carrying EcoR I and Sac II restriction sites and primer B carrying HindIII and Sph I restriction sites were selected, and Bacillus subtilis W168 (China General Microorganism Collection and Management Center, the preservation number is CGMCC1.1390) ( Kunst F , Ogasawara N , Moszer I , Albertini AM , Alloni G , Azevedo V , Bertero MG , Bessières P , Bolotin A , Borchert S , Borriss R , Boursier L , Brans A , Braun M , Brignell SC , Bron S , Brouillet S , Bruschi CV , Caldwell B. , Capuano V , Carter NM , Choi SK , Codani JJ , Connerton IF , Danchin A , et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 390 (6657): 249-56, 1997) (Institute of Microbiology, Chinese Academ...

Embodiment 2

[0071] Example 2, detection of protein expression level of Escherichia coli-Bacillus subtilis shuttle expression vector

[0072] One, Bacillus subtilis alkaline protease gene (apr) sequence cloning

[0073] Design primers containing Xho I and Kpn I restriction sites as follows:

[0074] Forward primer: 5'-ATCG CTCGAG TGCCGGAAAAAGCAGTACAGA-3'; reverse primer: 5'-CTAG GGTACC TTATTGTGCAGCTGCTTGT-3'.

[0075] Using the genomic DNA of Bacillus subtilis W168 as a template, the PCR reaction was carried out according to the following conditions.

[0076] The PCR amplification system (50 μl) is as follows: 5 μl 10×PCR buffer, 4 μl 2.5 mM dNTP mixture, 0.5 μl forward primer, 0.5 μl reverse primer, 0.5 μl template DNA, 0.5 μl ExTaq DNA polymerase, plus sterilized Water 39 μl to make a final volume of 50 μl.

[0077] PCR reaction conditions: 94°C pre-denaturation for 5 minutes; continue for 30 cycles: 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute and 30 seconds; final...

Embodiment 3

[0087] Embodiment 3, the establishment of alkaline protease mutant gene bank

[0088] 1. Random mutation and gene rearrangement (DNA shuffling) of the alkaline protease gene apr

[0089] (1) Error-prone PCR mutation of alkaline protease gene

[0090] Error-prone PCR reaction system:

[0091] 10×PCR buffer 5μl

[0092] 8mM MnSO 4 4μl

[0093] 2mM dGTP 5μl

[0094] 50×dNTP mix 1μl

[0095] Forward primer 0.75μl

[0096] Reverse primer 0.75μl

[0097] Template DNA (alkaline protease gene apr prepared in Example 2) 0.5 μl

[0098] Taq DNA polymerase 1μl

[0099] Add 32 μl of sterile water to make a final volume of 50 μl.

[0100] Forward primer: 5'-ATCG CTCGAG TGCCGGAAAAAGCAGTACAGA-3'; reverse primer: 5'-CTAG GGTACC TTATTGTGCAGCTGCTTGT-3'.

[0101] Error-prone PCR reaction conditions: 94°C pre-denaturation for 30 seconds; then 30 cycles of 94°C for 30 seconds, 55°C for 1 minute, and 72°C for 1 minute and 20 seconds; finall...

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-Bacillus subtilis shuttle expression vector and application thereof. The Escherichia coli-Bacillus subtilis shuttle expression vector comprises the following elements: a coding sequence of secretory signal peptide, a coding sequence of replication initiation protein of Bacillus subtilis, a promoter sequence of the Bacillus subtilis, and a replication initiation sequence of Escherichia coli. The vector can perform steady replication in the Escherichia coli, and can also replicate and express in the Bacillus subtilis. The vector can be applied to establishing a protein mutant gene library, mutant genes are connected to the vector first to ensure that various mutant genes are replicated in the Escherichia coli and then are transferred into the Bacillus subtilis to be expressed and screened, the efficiency of constructing the mutant library is equivalent to that of an Escherichia coli operation system, and the vector can simply and flexibly control the capacity of the library and can also fully utilize the external secretion expression ability of host cells of the Bacillus subtilis, thereby being convenient for the detection and screening of the performance of the mutant library.

Description

technical field [0001] The invention relates to an Escherichia coli-Bacillus subtilis shuttle expression vector and application thereof. Background technique [0002] Directed protein evolution refers to simulating the natural evolution mechanism, artificially creating special evolutionary conditions, starting from one or more existing parental proteins, changing the protein gene sequence and creating protein molecular diversity through gene mutation and recombination, so as to construct artificial The protein mutant gene library, further combined with sensitive and high-throughput screening technology can quickly obtain the ideal evolution protein, so it is also called protein in vitro molecular evolution (Kolkman JA, Stemmer W P C.Directed evolution of proteins by exon shuffling.Nature Biotechnology , 19: 423-428, 2001.). The directed evolution of proteins can cause various changes in the structure of the protein, and some subtle changes in its function, resulting in prot...

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
IPC IPC(8): C12N15/70C12N15/75C12N1/21C40B40/02C40B50/06C12R1/125
Inventor 温廷益邓爱华张芸于雷
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF 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