Project

Part:BBa_K5248021

Designed by: Songlin Ye   Group: iGEM24_SZPU-China   (2024-10-02)
Revision as of 11:06, 2 October 2024 by Registry (Talk | contribs)

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FimE Knockout

Overview

In order to develop a collective protective mechanism centered on the production of type I fimbriae in Escherichia coli Nissle 1917. Firstly we try to knock out FimE by using the lambda red recombinase system protocol. After two failed attempts at this method, we analyzed that there may be an A/T-rich region with probability a serious secondary structure near the FimE gene in Escherichia coli Nissle 1917. Then, the mutant Escherichia coli Nissle 1917, ΔfimE, was constructed through homologous recombination of a suicide plasmid (pCVD442), and validated the knockout method in Escherichia coli DH5α.

FimE knockout

FimE forms a transient covalent Tyr linkage to DNA. FimE, along with FimB, is a recombinase in Escherichia coli which catalyzes the site-specific recombination required for inversion of a 314-bp invertible DNA element, the fim switch (fimS), to control transcription of the type I fimbrial structural genes--a process known as phase-variation switching. fimS contains the promoter for expression of the fimbrial structural genes.

Strain construction Gene deletion in Cronobacter malonaticus 3267 was done using the lambda red recombinase system protocol with minor modifications. The ΔfimE mutant overexpressed type 1 fimbriae leading to the formation of microcolonies. Target cells within these microcolonies were protected from contact-dependent killing mediated by the type 6 secretion system (T6SS), leading to their survival. This defense mechanism could represent a nonspecific resistance mechanism, unlike immunity proteins, which could potentially lead to resistance against a broad range of assailants. Reference: Margot Marie Dessartine et al. Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks. mBio. 2024 Apr 10;15(4):e0255323.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 22
  • 1000
    COMPATIBLE WITH RFC[1000]


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