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

Biological synthesis gene cluster of tetrokacin A and use thereof

A technology for biosynthesis and tetrikacin, which is applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., and can solve the problems of long chemical semi-synthetic route and low yield.

Active Publication Date: 2009-02-11
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
View PDF0 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complexity of the molecular structure of tetrocacin A, its chemical semi-synthetic route is long and the yield is low, and there is no report on the total synthesis of this molecule so far

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
  • Biological synthesis gene cluster of tetrokacin A and use thereof
  • Biological synthesis gene cluster of tetrokacin A and use thereof
  • Biological synthesis gene cluster of tetrokacin A and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Extraction of total DNA of Tetrocacin A-producing bacteria Micromonospora cyanococcus NRRL11289:

[0097] Draw 100 μl of -80℃ frozen Micromonospora mycelium suspension and inoculate it into a medium containing 3ml TSB (30g / L T krypton S oya B roth) culture medium, 30°C, shaking culture for about 36 hours, take 2.5ml of bacterial liquid and inoculate into 50ml TSB (containing 0.5% glycine, 25mM magnesium chloride) medium, 30°C, shaking culture for about 36 hours until the bacteria The liquid was fresh orange, and the cells were collected by centrifugation at 3500 rpm, washed twice with lysate (150 g / L sucrose, 25 mM Tris-Cl, 25 mM EDTA), and stored at -20°C for extraction of genomic DNA. Add 10ml of lysate (containing lysozyme 5mg / ml, achromogenic peptidase 1mg / ml) to the collected hyphae, vortex until uniform, incubate at 37°C for 30 minutes, add 0.1ml proteinase K (4mg / ml), 1ml 10% SDS, mix well and quickly place in a 70°C water bath for 15 minutes until the solutio...

Embodiment 2

[0099] Construction of Genome Cosmid Library of Tetrocacin A-producing Micromonospora cyanococcus NRRL11289:

[0100] First, through a series of dilution experiments to determine the amount of Sau3AI, on this basis, a large number of enzyme-digested DNA fragments slightly larger than 40kb, dephosphorylated. pOJ446 was first cut and dephosphorylated from the middle of the two cos sequences with BglII, and then cut with BamHI from the multiple cloning site to obtain two arms, which were ligated with the prepared 40kb DNA fragment overnight. Take out the packaging mixture from the -80°C refrigerator and place it in a dry ice bucket. Melt the packaging mixture quickly between your fingers. Add 4 μL of the ligation product when it just starts to melt, mix well with a gun, and place in a 22°C water bath for 2 hours. Add 500 μL SM buffer [(L -1 ): 5.8g NaCl, 2.0g MgSO 4 , 50.0ml of 1M Tris.HCl (pH=7.5), 5.0ml of 2% (w / v) agar] and 50 μL of chloroform, mixed gently, and centrifuged ...

Embodiment 3

[0105] Nucleic acid molecular hybridization:

[0106] 1) DIG DNA labeling: Dilute the DNA to be labeled with sterile water to a total volume of 15 μL, heat and denature it in a boiling water bath for 10 minutes, and immediately place it in an ice-salt bath to cool. Then add 2 μL of primer mixture, 2 μL of dNTP mixture, and 1 μL of enzyme, mix well, and place in a 37° C. water bath for about 16 hours. Add 0.8 μL 0.8M EDTA (pH 8.0) to terminate the reaction, add 2.5 μL 4M LiCl and mix well, then add 75 μL pre-cooled absolute ethanol to precipitate the labeled DNA, and place it at -80°C for 40 minutes. The DNA was collected by centrifugation at 12000 rpm for 20 minutes at 4°C, washed with pre-cooled 70% ethanol, dried in vacuo and redissolved in 50 μLTE (pH 8.0).

[0107] 2) Quality detection after DIG DNA probe labeling: Dilute the labeled DNA probe to the following six gradients, 1, 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 . Dilute the labeled control DNA to the following conce...

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 clone, sequencing, analysis and function study of biosynthesis gene cluster of antibiotics-Tectrocarcin A which has anti-tumor activity and is generated by Micromonospora chalcea NRRL11289 and the application thereof. The whole gene cluster contains 36 genes: 5 non-reused I type polyketone synthetase genes; 4 special three-carbon biosynthesis genes; 11 desoxy sugar biosynthesis genes; 9 post modifying genes; 2 regulator genes; 1 resistance gene; and 4 genes with uncertain function. Due to the genetic manipulation of biosynthesis genes, the biosynthesis of Tectrocarcin A can be prevented. The genes and the proteins thereof which are provided by the invention can also be used for searching and discovering the compounds, genes or proteins that can be applies in medicine, industry or agriculture.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic engineering, in particular to the cloning, analysis, function research and application of the biosynthetic gene cluster of the antibiotic Tetrocarcin A. technical background: [0002] Tetrocarcins are a new class of spirocyclic acetoacetolactone antibiotics, which were first isolated from the fermentation product of Micromonospora chalcea KY11091 (Micromonospora chalcea KY11091) [J. Antibiot. (1980) 33, 668-670]. Titrocacin A (such as figure 1 (shown) is one of the components, its molecule contains two parts of aglycone and sugar group: aglycon Tetronolide structure is very unique, which contains a trans-naphthalene ring and a spiro-4-hydroxyacetoacetolactone (Spirotetronate) unit; the C9 position of the aglycone is connected with a four-sugar side chain formed by two L-amishose and two L-digitonin sugars alternately connected, and the 4-position hydroxyl of one L-digitonin sugar is Acetylation...

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): C12N15/11C12N15/10C12P19/02C12P17/04
Inventor 刘文方洁
Owner SHANGHAI INST OF ORGANIC CHEM 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