Unlock instant, AI-driven research and patent intelligence for your innovation.

Efficient shuttle plasmid for Escherichia coli-Riemerella anatipestifer

A technique of Riemer's anatipestifer and shuttle plasmid, which is applied in the direction of introducing foreign genetic material, viruses/phages, microorganisms, etc. using vectors, can solve the problems of few enzyme cutting sites, disadvantage, and limited number of shuttle plasmids, and achieve transfer efficiency. High, easy-to-operate effects

Inactive Publication Date: 2017-03-15
SICHUAN AGRI UNIV
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the number of shuttle plasmids that can be used for gene complementation of Riemerella anatipestifer is limited, and there are few restriction sites, and the efficiency of conjugative transfer is low, which is very unfavorable for gene complementation experiments

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
  • Efficient shuttle plasmid for Escherichia coli-Riemerella anatipestifer
  • Efficient shuttle plasmid for Escherichia coli-Riemerella anatipestifer
  • Efficient shuttle plasmid for Escherichia coli-Riemerella anatipestifer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Construction of recombinant plasmid pPM5+oriT

[0052] The shuttle plasmid of the present invention is modified on the basis of plasmid pPM5. In order to improve its combined transfer efficiency, the applicant cloned the transfer site oriT sequence (the sequence number in NCBI is AF047518.1) into this plasmid.

[0053] Design two PCR primers according to the oriT sequence:

[0054] Upstream primer: 5'-CCCAAGCTTCGCCTGATGCGGTATTTTTCTCC-3', as shown in SEQ ID NO.2;

[0055] Downstream primer: 5'-ACATGCATGCCTAGAGTCGATCTTCGCCAGC-3', as shown in SEQ ID NO.3.

[0056] Using the plasmid pEX18GM as a template, the primers shown in SEQ ID NO.2 and SEQ ID NO.3 were used to amplify the oriT sequence by PCR. The amplification program was: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, 72°C ℃ extension 30s, 30 cycles, 72 ℃ extension 7min, 22 ℃, ∞. The PCR product oriT sequence was recovered with a DNA purification and recovery kit, the p...

Embodiment 2

[0058] Construction of recombinant plasmid pFY01

[0059] Since the replication initiation site in this plasmid is different from that of Riemerella anatipestifer, in order to enable it to exist stably in Riemerella anatipestifer, the applicant introduced Riemerella anatipestifer The replication initiation gene pRA0726ori (sequence number JF268689.1 in NCBI) was cloned into this plasmid.

[0060] Design two PCR primers based on the replication initiation gene pRA0726 ori:

[0061] Upstream primer: 5'-ACATGCATGCCTATTTAGGCATTAGCCCTC-3', as shown in SEQ ID NO.4;

[0062] Downstream primer: 5'-AAAACTGCAGCCAATGCATTGGAACAGATCTCGTATAGAGCTCG-3', as shown in SEQ ID NO.5.

[0063] Using pLMF01 (Genbank: KU963002) as a template, the primers shown in SEQ ID NO.4 and SEQ ID NO.5 were used to amplify the pRA0726 ori gene sequence by PCR. The amplification program was: 98°C pre-denaturation for 30s; 98°C denaturation for 10s, Anneal at 55°C for 30s, extend at 72°C for 1min, 30 cycles, ext...

Embodiment 3

[0065] Construction of recombinant plasmid pFY02

[0066] In order to facilitate the insertion of gene fragments into the shuttle plasmid and increase the expression of the gene, the applicant cloned a high expression promoter sequence High EXP with multiple endonuclease sites on the shuttle plasmid (the sequence number in NCBI is CP003787.1) .

[0067] Design two PCR primers according to the high expression promoter High EXP sequence:

[0068] Upstream primer: 5'-ACGCGTCGACGTCGGCCATATTTCAAAAATTTAACTTAAACC-3', as shown in SEQ ID NO.6;

[0069] Downstream primers:

[0070] 5'-TGCTCTAGAGCAGCGCGCAGGCCTGCTAGCCCGCGGAATTTTAAATAATTTTTTTAAATTTG-3', as shown in SEQ ID NO.7.

[0071] Wherein the downstream primer shown in SEQ ID NO.7 has added restriction sites SacII, NheI, StuI, BsshII.

[0072] Use the RA CH-1 strain as a template, and use the primers shown in SEQ ID NO.6 and SEQ ID NO.7 as primers to amplify the High EXP gene sequence by PCR. The amplification program is: pre-den...

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 an efficient shuttle plasmid for Escherichia coli-Riemerella anatipestifer, and a preparation method and application thereof; a gene sequence of the shuttle plasmid is shown as in SEQ ID NO. 1 and is modified based on plasmid pPM5; Riemerella anatipestifer start replicator gene (pRA0726 ori) is cloned to the plasmid firstly, transfer site (oriT) is then cloned to the plasmid, and therefore, the shuttle plasmid can be introduced to Riemerella anatipestifer through efficient conjugal transfer and can be replicated stably; in order to facilitate the insertion of gene fragments into the shuttle plasmid and increase gene expression quantity, high-expression starter sequence (High EXP) with multiple restriction enzyme sites is cloned to the shuttle plasmid. The shuttle plasmid acquired herein may exist stably in Riemerella anatipestifer to act for the retro-complementation of gene knock-out mutants, has small size and multiple polyclonal sites, is easy to operate, and is very high in conjugal transfer efficiency.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a shuttle plasmid, in particular to a high-efficiency shuttle plasmid for Escherichia coli-Riemerella anatipestifer. Background technique [0002] Gene knockout technology is a common method for studying gene function. The deletion of a certain gene in bacteria often causes its phenotype to change, but the change in phenotype may be caused by the gene being knocked out and affecting the expression of downstream genes. Therefore, it is necessary to clone the deficient gene into a shuttle plasmid and conduct functional complementation research. [0003] At present, the number of shuttle plasmids that can be used for gene complementation of Riemerella anatipestifer is limited, and there are few restriction sites, and the efficiency of conjugative transfer is low, which is very unfavorable for gene complementation experiments. Contents of the invention [0004] In view of this, the objec...

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): C12N15/74
CPCC12N15/74C12N2800/101C12N2820/55C12N2830/34
Inventor 刘马峰冯言王梦怡朱德康程安春汪铭书贾仁勇陈舜孙昆峰杨乔吴英
Owner SICHUAN AGRI UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com