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

High-conversion-ratio zymomonas mobilis as well as construction method and application thereof

A technology of Zymomonas and its construction method, which is applied in the field of Zymomonas mobilis with high transformation rate and its construction, which can solve the problems of limitations of genetic engineering transformation, difficult operation of large plasmids, and inability to introduce foreign genes.

Inactive Publication Date: 2018-07-20
SHANGHAI JIAO TONG UNIV
View PDF5 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above-mentioned defects in the prior art, the technical problem to be solved by the present invention is that the Zymomonas mobilis genetic modification operation system has low efficiency, large plasmid operation is difficult, and genetic engineering is greatly restricted. The source gene cannot be imported into it

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
  • High-conversion-ratio zymomonas mobilis as well as construction method and application thereof
  • High-conversion-ratio zymomonas mobilis as well as construction method and application thereof
  • High-conversion-ratio zymomonas mobilis as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] A construction of Zymomonas mobilis ZM4m and ZM401m bacterial strains with high transformation efficiency:

[0063] 1. Restriction-modification system-related gene prediction

[0064] By combining the references and the database KEGG, the genome-wide restriction-modification system (R-M system) related genes of Z. mobilis ZM4 and ZM401 were predicted, as shown in Table 1. The NCBI-ProteinIDs of the two genes of ZMO0028 and ZMO1933 are AAV88652 respectively and AAV90557.

[0065] Table 1 Predicted restriction modification systems in Zymomonas mobilis

[0066]

[0067] 2. Design of knockout primers

[0068] Gene knockout primers were designed for the two predicted genes related to the restriction modification system and used in subsequent screening and identification steps. The primers related to gene knockout and transformant identification are shown in Table 2.

[0069] Table 2 Gene knockout and transformant identification related primers

[0070]

[0071] Not...

Embodiment 2

[0108] Transform recombinant plasmids into Zymomonas mobilis ZM4 and ZM401 using electroporation by the following steps:

[0109] ●Transform the constructed plasmid into E.coli JM110, and extract the demethylated plasmid from it;

[0110] ●Take out Z.mobilis glycerolbacteria from the -80℃ refrigerator, and produce single colonies on the RM agar plate by dividing and marking;

[0111] Pick a single colony of Z.mobilis and culture it in RM liquid medium overnight to the end of logarithm;

[0112] ●According to 2%-3% transfer, static culture to OD 600 =0.4, place on ice for 15-20min, centrifuge at 4,500rpm for 5min at 4°C, discard the upper layer;

[0113] Use pre-cooled ddH containing 10% glycerol 2 O washed twice and washed with an appropriate volume of ddHO containing 10% glycerol 2 O resuspended bacteria, aliquoted into 80 μL per tube to make Z.mobilis competent;

[0114] ●Add 1 μg of demethylated plasmid to the competent cells, mix gently, let stand on ice for 5 minutes...

Embodiment 3

[0118] Transformer Single Exchange Verification:

[0119] Such as figure 1 As shown, the transformants formed by transforming the recombinant plasmids into Zymomonas mobilis ZM4 and ZM401 by electroporation were verified by single exchange colony PCR. One length is the sum of the upstream and downstream homology arms and the target gene. The upstream primers used for identification are shown in SEQ ID No.1 and SEQ ID No.5, and the downstream primers are shown in SEQ ID No.4 and SEQ ID No.8. .

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 a construction method of high-conversion-ratio zymomonas mobilis. Meanwhile, the invention further relates to the high-conversion-ratio zymomonas mobilis. Thirdly, the invention also relates to application of the high-conversion-ratio zymomonas mobilis to a gene engineering field. The construction method comprises the following steps: combining reference documents and a database to predicate related genes of a zymomonas mobilis ZM4 whole-genome limitation-modification system; designing gene knockout primers for the predicated two genes related to the limitation-modification system respectively; taking ZM4 and ZM401 as starting strains, and knocking out the related genes through a plasmid pEX18Tc, so as to finally obtain ZM4m and ZM401m which are used as knockout strains of the related genes of the limitation-modification system. According to the construction method disclosed by the invention, the conversion efficiency of the obtained strains is remarkably improved, and the quantity of obtained transformants is great; the strains have a high positive rate, and the growth performance and the fermentation performance of the strains are the same as those of wildtype strains. The double knockout strains are used as the starting strains, so that molecular modification of the zymomonas mobilis and related mechanism researches are facilitated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Zymomonas mobilis with high conversion rate and its construction method and application. Background technique [0002] Zymononas mobilis is a Gram-negative facultative anaerobic bacterium. Due to its unique ED metabolic pathway and efficient ethanol fermentation performance, this strain is considered to have broad application prospects in the field of biorefinery. In addition to the above-mentioned advantages, Zymomonas mobilis also has some disadvantages such as: narrow substrate utilization range, only glucose, fructose and sucrose; poor tolerance to inhibitors; too simple metabolic map, and few types of products. In order to expand the industrial application of Zymomonas mobilis, it is a better way to transform it using genetic engineering technology, and it is also the focus of current research in the field of biotechnology. [0003] Since the current genetic engineering of Z...

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): C12N15/74C12N15/54C12N1/21C12R1/01
CPCC12N9/1007C12N15/74
Inventor 刘晨光夏娟闻远曹莲莹李凯白凤武
Owner SHANGHAI JIAO TONG UNIV
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