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Systems and methods for genetic manipulation of akkermansia species

A kind of Akkermansia gene technology, applied in Akkermansia bacteria, genetic modification Akkermansia bacteria, treatment of diseases, can solve the problem of Akkermansia difficult molecular manipulation and so on

Pending Publication Date: 2021-02-19
DUKE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Akkermansia has been found to be difficult to molecularly manipulate

Method used

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  • Systems and methods for genetic manipulation of akkermansia species
  • Systems and methods for genetic manipulation of akkermansia species
  • Systems and methods for genetic manipulation of akkermansia species

Examples

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example 1

[0078] Example 1: Modified Tn mutagenesis vector for use in Akkermansia: pSAM_Akk

[0079] To enable genetic screening for genes with altered phenotypes and altered bacteria, tools for mutating bacteria are needed. A modified version of the previously described vector was generated for use in Akkermansia. Original vector pSAM_Bt 1 Designed for use with Bacteroides thetaiotaomicron. The vector encodes both the modified mariner transposon himar1C9 with the erythromycin resistance gene and the transposase required to catalyze transposition. The plasmid uses a lamba pir-dependent origin of replication and cannot replicate in strains lacking the pir gene such as Akkermansia.

[0080] For compatible use of pSAM_Bt in Akkermansia, the original erythromycin resistance marker (ermG) on the transposon was replaced with a chloramphenicol resistance cassette (cat). Initial attempts to use erythromycin as a selectable marker in Akkermansia were unsuccessful, and growth with erythromyci...

example 2

[0081] Example 2: Methods for Mutagenesis and Transposon (Tn) Library Construction

[0082] The transposon vector (SEQ ID NO: 1 ) was introduced into Akkermansia by conjugation to an E. coli donor strain. Akkermansia starter cultures were subcultured at a ratio of 1:5 in 30ml of synthetic medium 2 Medium and grow to OD600=0.6-1.0. Cells were then harvested by centrifugation at 10 000 xg in 1.5 ml tubes for 5 minutes at 4°C. At the same time, the Escherichia coli S17 pSAM_Akk culture was aerobically cultured in LB+100ug / ml ampicillin, 35ug / ml chloramphenicol at 37°C and 200rpm until the optical density (OD) OD600=0.4-0.7. To avoid shearing of conjugated pili, E. coli were centrifuged at 2000 xg for 3 minutes and washed once with sterile PBS. The E. coli and Akkermansia pellets were combined in synthetic medium to a total volume of 0.5 ml, and the suspension was used to make 100 μl puddles on pre-reduced synthetic medium plates. Plates were incubated aerobically at 37°C for ...

example 3

[0085] Example 3: Transposon mutant screening - transposon mutants were screened for genes required for mucin utilization

[0086] To screen for genes required for mucin utilization, array Tn mutants were used to inoculate mucin-containing media 3 Or replicate 96-well plates with synthetic media. Plates were incubated anaerobically at 37°C for 3 days. After growth, measure OD600 using a microplate reader. Mutants that grew in synthetic media but not in mucin were selected for additional characterization. To confirm the initial screen, mutants of interest were tested for mucin growth defects by performing a growth curve assay in a microplate reader, with measurements taken every 60 min for 72 h ( image 3 ). Random PCR was used to map transposon insertion sites and identify genes required for growth on mucins. The screen led to the identification of genes specifically required for growth on mucins but not on monosaccharides (Table 1).

[0087] Table 1. Genes identified as...

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Abstract

The present disclosure provides methods and systems for genetically altering and screening Akkermansia bacteria, including Akkermansia muciniphila. The methods also provide genetically altered bacteria, libraries of genetically altered bacteria and use of such bacteria for treatment of diseases.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 671,614, filed May 15, 2018, the contents of which are incorporated by reference in their entirety. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with Government support under Federal Grant No. 5R21DK110496-02 awarded by the NIH. The Federal Government has certain rights in this invention. Background technique [0005] The field of the invention relates to the genetic modification and selection of gut microbes with altered phenotypes and the use of these microbes in the treatment of disease. [0006] Akkermansia muciniphilia is a bacterium found on the mucosal surface of the human intestinal tract. This bacterium uses mucin as its sole source of nutrition. The bacterium accounts for 1-4% of the intestinal bacteria in adults and is a bacterium that inhabits the large intestine. The bacterium is a Gram-...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/90C12N15/65C12N15/10C12N1/21C40B50/06C40B40/02C12Q1/689C12Q1/04C12R1/01
CPCC12Q1/04C12N15/1082C12N2800/101C12N2800/90G01N2333/195C12N1/20C12N15/1079C12N15/74
Inventor R·瓦尔迪维亚P·马尔库斯L·戴维
Owner DUKE UNIV
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