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Genetically engineered bacteriophage

A bacteriophage and gene technology, applied in the direction of bacteriophage, virus/bacteriophage, medical raw materials derived from virus/bacteriophage, etc., can solve the problem of bacteriophage DNA damage

Pending Publication Date: 2020-10-16
噬菌体技术公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Prophages may also cause damage to incoming phage DNA

Method used

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  • Genetically engineered bacteriophage
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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0164] Example 1-Phage isolation

[0165] Collect and purify samples from sewers and waste, environmental soil and animal feces for the isolation of bacteriophages. The purified sample is then screened for the presence of phages against specific bacteria. According to Bonilla et al. (2016) and Bourdin et al. (2014), the scheme and method of separating bacteriophages from water samples was adopted; according to Silankorva (2018), Pausz et al. Solutions and methods.

[0166] Rehydrate the solid sample with sterile water for at least 1 hour to allow the phage to spread. The sample is then centrifuged to remove solid matter and large particles, and the supernatant is collected. The centrifuged environmental samples and water samples are then further processed and purified using a filter (0.2 μM) to remove bacteria and smaller harmful particles. The filtered sample can be further concentrated using a filter tube, or stored at 4°C for future use.

[0167] The filtered samples were the...

example 2

[0169] Example 2-Platform Development

[0170] Using environmental samples EV31 / PP8, after phage isolation, the genomic material was purified using PureLink viral DNA / RNA extraction kit. Amplify the full-length genome (EV31 / Full / F / pYESIL and EV31 / Full / R / pYESIL, see sequence EV31) to have 30bp homology with the pYESIL sapphire vector. Phusion high-fidelity DNA polymerase was used for PCR amplification (modification using touchdown technology, the primers were annealed from 69°C, and each cycle decreased by 0.5°C). The PCR products were separated on an agarose gel, and the bands were cut, extracted and assembled. The resulting construct EV31pYES (unmodified) can be used for genetic modification of EV31 and the determination of mutation function in a phage rescue-based system.

example 3

[0171] Example 3-Full-length genome assembly

[0172] In short, the following provides a method for genetic manipulation of yeast (Kluyveromyces lactis and Pichia pastoris) cells to include T7 DNA (deoxyribonucleic acid) dependent from E.coli phage T7 The RNA (ribonucleic acid) polymerase is transcribed, and then the phage is expressed in yeast. A method for genetic manipulation of yeast (Kluyveromyces lactis and Pichia pastoris) cells is also provided, so that it includes bacteria (E. coli) and RNA (ribonucleic acid) polymerase (P ) And then express the phage in yeast.

[0173] See image 3 , Divide the genomic complement into fragments that overlap with adjacent fragments obtained by PCR amplification. Insert foreign genes into their respective fragments. The fragments were combined into a full-length genome by homologous recombination, and the yeast-based plasmid (as an additional PCR fragment) was combined with the T7 promoter in the yeast strain Pichia pastoris. The stable...

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PUM

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Abstract

There is disclosed a method of engineering bacteriophages comprising: identifying a bacteriophage with only one attachment gene; isolating said bacteriophage; removing said attachment gene from the genome of said bacteriophage; and inserting a non-natural attachment gene into the genome of said bacteriophage wherein said non-natural attachment gene is specific for attaching to a selected bacteria.There is also disclosed a mutant bacteriophage comprising a heterologous nucleic acid sequence encoding a first specific attachment gene, the first specific attachment gene being different than an inactivated attachment gene and being specific for a selected bacteria. In another embodiment, there is disclosed a method of eliminating a microbial contaminant, the method comprising: obtaining one ormore lytic enzymes produced by a mutant bacteriophage; applying the one or more lytic enzymes to a bacterial contaminant, without prior infection of the bacterial contaminant with a bacteriophage, toeliminate the bacterial contaminant.

Description

Technical field [0001] Prevention, diagnosis and treatment of bacterial infections of humans, animals and plants. Background technique [0002] Bacteria are single-celled biological entities, most of which are harmless to humans, and less than one percent of different types can make people sick. Many bacteria are beneficial to humans, such as those that help digest food, destroy disease-causing cells and provide necessary vitamins. [0003] Infectious bacteria (the harmful one percent) can cause diseases in humans and animals. They multiply rapidly in the body and produce toxic proteins that can cause tissue damage and disease. [0004] Bacteriophages (also called bacteriophages) were discovered by Ernest Hankin in 1896 and used as antibacterial agents against cholera. These bacteria-specific viruses can infect and destroy bacterial cells. [0005] Bacteriophages are composed of proteins that wrap the DNA or RNA genome. By injecting its viral genetic material (DNA or RNA) into the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N7/00C12N15/81A01N63/40A01P1/00A61K35/76A61L2/16A61P31/04
CPCA61K35/76A61P31/04C12N7/00C12N15/63C12N2795/00021C12N2795/00022C12N2795/00032A61L2/0005A61L2202/25A01N63/40A01P1/00C12N15/52C12N15/62C12N15/87C12N2795/00062C12N2800/10
Inventor 史蒂文·特里奥特
Owner 噬菌体技术公司
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