Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

DNA assembly method and application thereof

A purpose, recombinant bacteria technology, applied in the field of DNA assembly, can solve problems such as increasing the difficulty of DNA sequence, affecting DNA sequence, and restricting applications

Pending Publication Date: 2020-07-07
NANJING ZHONGKEYOUZI INST OF BIOTECHNOLOGY CO LTD
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The scar sequence between the spliced ​​DNA fragments will affect the integrity of the DNA sequence, the secondary structure of the mRNA and the correct expression of the protein, which increases the difficulty of DNA sequence design and limits its application in the need for scarless assembly and precise assembly

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
  • DNA assembly method and application thereof
  • DNA assembly method and application thereof
  • DNA assembly method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] (1) Construction of PS-Brick original vector pOB plasmid and pOM plasmid

[0056] Plasmid pUC19 (SEQ ID NO.1) is used as the basic vector for verifying the PS-Brick assembly method, and a BmrI site and three MlyI sites in the vector are detected by overlap extension PCR (overlap extension polymerase chain reaction, overlap PCR) method (Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. (1989) Site-Directed Mutagenesis by Overlap Extension Using the Polymerase Chain-Reaction, Gene 77, 51-59.) removed. Specifically, first use the primer pair UC709-F / UC1179-R and UC1179-F / UC1695-R to amplify two DNA fragments, and then use the primer UC709-F / UC1746-R to splice the two DNA fragments by overlapping PCR Fragments, the spliced ​​product was used as a large primer, and the plasmid pUC19 was used as a template to mutate one of the BmrI sites and three MlyI sites. The other two BmrI sites and MlyI sites located in the multiple cloning site sequence on the plasmid ...

Embodiment 2

[0065] The reaction condition optimization of embodiment 2PS-Brick assembly

[0066] Use IIS-type restriction endonucleases BmrI and MlyI to single-enzyme pOB and pOM vectors respectively. The enzyme digestion reaction system is as described in Example 1. According to the reaction conditions of Time-Saver products, the reaction time is from 15 minutes to 180 minutes, and then electrophoresis Check cutting efficiency. Such as figure 2 As shown in BC, almost all vectors were completely cleaved within 15 min, yielding a single band of linearized size.

[0067] Cut the gel to recover the bands of pOB cut by BmrI and pOM cut by MlyI, and then digested with SphI for 15 to 180 minutes, inactivated at 65°C, recovered, ligated with the PCR products FB and FM cut by SphSphI respectively, and transformed into E. coli DH5α competent cells. The number of transformant units (colony-forming units (CFUs)) represents the DNA assembly efficiency. 20 clones were picked for sequencing, and t...

Embodiment 3

[0070] Example 3 Engineering Transformation of Threonine Metabolic Pathway Using PS-Brick Method

[0071] The PS-Brick assembly technology was used to carry out multiple rounds of iterative "design-build-test" cycles, and a threonine-producing engineered bacterium was constructed. Due to the complexity of the regulation and interaction of gene expression and signaling networks in organisms, and the lack of prior knowledge to predict how a DNA assembly introduced into a cell will function, it is necessary to test multiple versions of the construct to obtain optimal results. Assembly scheme. Multiple rounds of step-by-step "design-build-check" experiments require an iterative nature of the DNA assembly approach.

[0072] The metabolic engineering strategy for constructing threonine engineered bacteria usually includes the following steps: release the feedback inhibition of the threonine operon, enhance the threonine terminal synthesis pathway, remove the metabolic bottleneck, b...

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 relates to a plasmid, a DNA assembly method and application of the method in recombinant bacteria. The plasmid comprises single contiguous IIP and IIS type restriction endonuclease recognition sites. The plasmid comprehensively uses the characteristics of IIP and IIS type restriction endonucleases to realize recursive cycle assembly, traceless assembly and repetitive sequence assembly.

Description

technical field [0001] The present invention generally relates to the field of biotechnology, in particular to a DNA assembly method and its application. Background technique [0002] DNA assembly is a fundamental enabling technology for synthetic biology and bioengineering. Currently, DNA assembly methods are mainly divided into two categories: restriction endonuclease (Restriction endonucleases, RE)-based assembly strategies and homologous fragment-mediated assembly strategies. Among them, the RE-based DNA assembly method is the most widely used assembly method. [0003] During the development of RE-based DNA assembly methods, BioBrick TM The method (Shetty, R.P., Endy, D., and Knight, T.F., Jr. (2008) Engineering BioBrick vectors from BioBrick parts, JBiol Eng 2, 5.) was the first to be developed and practically applied. This method utilizes four type IIP restriction enzymes (e.g. EcoRI, XbaI, SpeI, PstI) for recyclable DNA fragment assembly, two of which are isologous...

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): C12N15/70C12N15/66C12N1/21C12P13/08C12R1/19
CPCC12N15/70C12N15/66C12N9/1217C12N9/1205C12N9/88C12N9/0008C12N9/0006C07K14/245C12P13/08C12Y207/02004C12Y207/01039C12Y402/03001C12Y102/01011C12Y101/01103C12Y403/01019C12N15/11C12P19/34
Inventor 刘树文温廷益张芸邓爱华
Owner NANJING ZHONGKEYOUZI INST OF BIOTECHNOLOGY CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
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