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Bacteria-based protein delivery

A technology of bacteria and proteins, applied in the direction of bacteria, bacterial peptides, fusion with protease sites, etc., can solve the problems that hinder the wide application of the system, and no cloning code is designed

Active Publication Date: 2018-11-23
UNIVERSITY OF BASEL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

None of the above systems allow for single protein delivery, as in each case, one or more endogenous effector proteins are encoded
In addition, the vectors used were not designed to allow easy cloning of additional DNA fragments encoding the selected proteins, preventing widespread use of this system

Method used

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  • Bacteria-based protein delivery
  • Bacteria-based protein delivery
  • Bacteria-based protein delivery

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] A) Materials and methods

[0200] Bacterial strains and growth conditions. The strains used in this study are listed in Figure 15A to N. E. coli Top10 for plasmid purification and cloning, and E. coli Sm10λ pir for conjugation, and E. coli BW19610 for propagation of pKNG101 [36] were routinely grown on LB agar plates and in LB medium at 37 °C . Expression vectors were selected using ampicillin at a concentration of 200 μg / ml (Yersinia) or 100 μg / ml (E. coli). Streptomycin was used at a concentration of 100 μg / ml to select for suicide vectors. Yersinia enterocolitica MRS40 [22], non-ampicillin-resistant E40-derivatives [21] and their derivative strains were routinely grown on brain heart infusion (BHI; Difco) at room temperature. Nalidixic acid (35 μg / ml) was added to all Y. enterocolitica strains and all Y. enterocolitica strains were additionally supplemented with 100 μg / ml meso-2,6-diaminoheptane acid (mDAP, Sigma Aldrich). Salmonella enterica SL1344 is routin...

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Abstract

The present invention relates to recombinant Gram-negative bacterial strains and the use thereof for delivery of repeated domains of a heterologous protein or two or more domains of different heterologous proteins into eukaryotic cells.

Description

technical field [0001] The present invention relates to recombinant Gram-negative bacterial strains and their use for the delivery of repeat domains of heterologous proteins or two or more domains of different heterologous proteins into eukaryotic cells. Background technique [0002] Transient transfection techniques have been used in cell biology research for many years to address protein function. These methods often result in massive overexpression of the protein being studied, which can lead to oversimplified signaling models. For proteins that control short-lived signaling processes, the protein of interest exists far longer than the signaling event it controls. Furthermore, transient overexpression based on DNA transfection results in heterogeneous and asynchronous cell populations, which complicates functional studies and hampers omics approaches. Beyond that, scaling up these assays to larger scales is prohibitively expensive. These points mentioned above are cove...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/74C12N1/21C07K14/195C12N15/62
CPCC07K14/195C12N15/74C07K2319/036C07K2319/33C12N15/70Y02A50/30C07K2319/50
Inventor 西蒙·伊泰格马利斯·阿姆斯特茨克里斯托夫·卡斯帕
Owner UNIVERSITY OF BASEL