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

A technology integrating plasmids and resistance screening, applied in the field of genetic engineering, can solve problems such as inconvenient genetic manipulation, and achieve the effects of high sensitivity, strong repeatability, and low amount of compounds

Active Publication Date: 2013-10-30
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • 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

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  • 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
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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 ...

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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 present invention is a genetic engineering field, which involves an autonomous luminescence bangs that integrate the plasmid POPHI, and a kind of non -resistant screening label. Background technique [0002] The mycobacterium is a type of slender and slightly curved microorganisms, sometimes branches or silk.At present, the belonging to the branch of branches has been summarized in the mobiliae.Plasses that are pathogenic can be divided into two categories: and without branch bacteria.Bacteria is contained in branches. [0003] The main characteristic of this genus bacteria is that the cell wall contains a large amount of lipids, mainly branched bacteria.This is closely related to its dyeing, growth characteristics, pathogenicity, and resistance.Generally, it is not easy to color. If it is heated or extended the dyeing time, it can resist the decolorization of ethanol hydrochloride hydrochloride hydrochloride, so it is also known as Acid-Fast Bacilli.Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N15/66C12N1/21C12R1/32
Inventor 张天宇
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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