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

Integrative plasmid pOPHI and resistance screening marker-free self-luminescent mycobacterium

A technology for integrating plasmids and mycobacteria, applied in the field of genetic engineering, can solve problems such as inconvenient genetic manipulation, and achieve the effects of high sensitivity, less compound amount, and strong repeatability

Active Publication Date: 2012-10-10
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
View PDF3 Cites 16 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, when using autonomously luminescent mycobacteria for research, if the strain used has a resistance gene, it will bring inconvenience to the genetic manipulation in the future.

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
  • Integrative plasmid pOPHI and resistance screening marker-free self-luminescent mycobacterium
  • Integrative plasmid pOPHI and resistance screening marker-free self-luminescent mycobacterium
  • Integrative plasmid pOPHI and resistance screening marker-free self-luminescent mycobacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Construct the integrated plasmid pOPHI, see the construction procedure Figure 1~3 , for a map of plasmid pOPHI see Figure 5 . Such as Figure 5 As shown, the plasmid pOPHI contains a strong mycobacterial promoter ( Hsp60 ), the enzyme gene required for luminescence ( LuxCDABE ), the integrase gene ( Int ) and hygromycin resistance gene ( Hyg ) of the fusion gene, the direct repeat sequence located at both ends of the fusion gene DifR and Dif L , the phage integration site ( attP ), and the ampicillin resistance gene ( Ampr ) and replicators ( rep ). The functions of each component are as follows:

[0052] Hsp60 : A mycobacterial strong promoter, which can promote the strong expression of subsequent genes.

[0053] LuxCDABE : Enzyme gene required for luminescence, the expression of this gene allows the host bacteria to emit light autonomously [5] .

[0054] Integrase gene ( Int ): Integrase can be expressed to integrate the plasmid into the...

Embodiment 2

[0081] A preparation method of self-luminescent mycobacteria without resistance selection marker

[0082] The 7H9 medium, 7H11 medium, and Tween 80 involved in this example were all purchased from Guangzhou Huaqisheng Biological Company.

[0083] Biorad electroporation instrument (Biorad GenePulser Xcell) and electroporation cup were purchased from Biorad Company.

[0084] 1. Materials to be prepared:

[0085] 1) Mycobacterium smegmatis ( Mycobacterium.smegmatis ), which can be obtained from the China General Microorganism Culture Collection and Management Center, with the number 1.2621.

[0086] 2) 10% glycerin + 0.05% Tween 80 [20ml glycerin + 180ml water + 100ul Tween 80, and filter with a 0.2μm filter. It is required to be freshly prepared and not older than 1 week.

[0087] 3) Sterilized glass beads, 50ml centrifuge tube, Biorad 0.2cm electric cup, 25ml pipette.

[0088] 4) 50ml of well-grown bacteria solution in the logarithmic phase [the OD value of the bacteria ...

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 integrative plasmid pOPHI and a resistance screening marker-free self-luminescent mycobacterium. The integrative plasmid pOPHI comprises a promoter, an enzyme gene (LuxCDABE) needed for luminescence, a fusion gene of an integrase gene (Int) and a resistance screening gene, direct repeat sequences DifR and DifL positioned on two ends of the fusion gene, a phage integration site (attP) and a replicon. After the integrative plasmid pOPHI is transferred into the mycobacteria, a photobacterium of which the resistance gene and the integrase gene are lost is screened out by subculturing, and the photobacterium is the resistance screening marker-free self-luminescent mycobacterium. Whether the resistance screening marker-free self-luminescent mycobacterium obtained by construction is dead or not is judged according to whether the bacterium is luminescent or not, the mycobacterium can be used for a plurality of detections of experiments, and credible experimental data can be quickly obtained.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an integrated plasmid pOPHI and an autonomous luminous mycobacterium without a resistance screening marker. Background technique [0002] Mycobacterium (Mycobacterium) is a class of slender slightly curved microorganisms, sometimes branched or filamentous. At present, mycobacteria have been classified into actinomycetes in taxonomy. Actinomycetes that are pathogenic to humans can be divided into two categories: those containing and those not containing mycolic acids. Mycobacteria belong to the group containing mycolic acids. [0003] The main characteristic of this genus of bacteria is that the cell wall contains a large amount of lipids, mainly mycolic acids. This is closely related to its dyeability, growth characteristics, pathogenicity, and resistance. Generally, it is not easy to be colored, but it can resist the decolorization of strong decolorizing agent ...

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): C12N15/74C12N15/66C12N1/21C12R1/32
Inventor 张天宇
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH 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