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Method for genetically modifying intestinal primitive bacteria

A technology of genetic modification and protozoa, applied in biochemical equipment and methods, microorganisms, pharmaceutical formulations, etc., can solve the problems of not fully meeting research needs, high price of germ-free mice, long transplantation period, etc., and achieve efficient and convenient colonization. , the effect of improving biological compliance and low cost

Inactive Publication Date: 2019-06-21
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the price of germ-free mice is high, and due to certain immunodeficiency after gene knockout, it cannot fully meet the research needs
Bacteria transplantation has problems such as long transplantation period and low colonization efficiency

Method used

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  • Method for genetically modifying intestinal primitive bacteria
  • Method for genetically modifying intestinal primitive bacteria
  • Method for genetically modifying intestinal primitive bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Using the method of synthetic biology, the screened intestinal native Escherichia coli is genetically modified and then microencapsulated:

[0039] 1) Screening of native Escherichia coli in the intestine of C57BL / 6 mice

[0040] ①Take fresh intestinal tissue from C57BL / 6 mice in a sterile EP tube;

[0041] ② Wash the intestinal contents with pre-cooled sterile PBS;

[0042] ③After cutting the intestinal wall, cut the intestinal wall with a sterile glass slide, mix the intestinal wall with sterile pre-cooled PBS at a mass volume ratio of m / v=1:10, and vortex for 15 minutes;

[0043] ④ Centrifuge the mixture at 4°C, 2000rpm for 5min, discard the precipitate, centrifuge the supernatant at 4°C, 12000rpm for 20min, discard the supernatant, and keep the precipitate;

[0044] ⑤ After resuspending the pellet with 100 μL PBS, take 10 μL and spread it on a solid LB plate for blue-white plate screening, and culture overnight at 37°C;

[0045] ⑥The next day, according to the gr...

Embodiment 2

[0064] A method for treating intestinal diseases with microencapsulated intestinal native bacteria, the specific steps are as follows:

[0065] 1) Using the method of synthetic biology to genetically modify the screened intestinal native Escherichia coli and then carry out microencapsulation treatment:

[0066] (1) Screening of Escherichia coli native to the intestinal tract of C57BL / 6 mice

[0067] ①Take fresh intestinal tissue from C57BL / 6 mice in a sterile EP tube;

[0068] ② Wash the intestinal contents with pre-cooled sterile PBS;

[0069] ③After cutting the intestinal wall, cut the intestinal wall with a sterile glass slide, mix the intestinal wall with sterile pre-cooled PBS at a mass volume ratio of m / v=1:10, and vortex for 15 minutes;

[0070] ④ Centrifuge the mixture at 4°C, 2000rpm for 5min, discard the precipitate, centrifuge the supernatant at 4°C, 12000rpm for 20min, discard the supernatant, and keep the precipitate;

[0071] ⑤ After resuspending the pellet wi...

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Abstract

The invention provides a method for genetically modifying intestinal primitive bacteria. Screened intestical primitive escherichia coli are genetically modified by a synthetic biology method and thenare microencapsulated. Mucin (Ag43) which is single functional bioactive molecule secreted by the primitive escherichia coli is taken as an example, and the application of mucin (Ag43) in the treatment of an inflammatory bowel disease (IBD) is studied. The invention provides a new idea for researching the interaction between intestinal microorganisms and organisms. A bio-safe synthetic biologicaltechnology and a non-invasive photogenetic technology are combined to be applied to the treatment of intestinal diseases, and compared with a traditional intestinal disease treatment method, the method has the advantages that the screened intestinal primative escherichia coli are genetically modified, so that the biological compliance of intestinal colonization of microencapsulated primitive bacteria in the later period is improved. A synthetic biological technology is combined with a photogenetic technology, so that bioactive molecules for treating the intestinal diseases can be accurately regulated and controlled in time and space.

Description

technical field [0001] The invention relates to the field of microorganisms and clinical treatment, in particular to a method for genetic modification of intestinal protobacteria. Background technique [0002] As the largest organ in an organism, the intestine plays an important role in digestion and absorption, immune regulation and other aspects. In addition to epithelial cells and immune cells that perform digestion, absorption, and immune regulation functions, the intestinal tract also contains many microorganisms. More than 99.9% of the microorganisms are bacteria, which are collectively known as the gut flora. [0003] Gut flora, as a "symbiont" in the gut of organisms, participates in the regulation of many important life processes and diseases. Most of the existing methods for studying the relationship between intestinal microbes and the body are to use germ-free mice or extensive flora transplantation, combined with microbiome and metabolomics analysis, to find ou...

Claims

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Application Information

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IPC IPC(8): C12N13/00C12N15/70A61K35/74A61K9/50A61K47/36A61K47/34A61K47/02A61P1/00
Inventor 王汉杰崔梅慧常津李树斌孙韬潘惠卓
Owner TIANJIN UNIV
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