DNA

Part:BBa_K1951008

Designed by: claire raynaud   Group: iGEM16_Aix-Marseille   (2016-10-13)


FliC E.coli producer

The purpose of this biobrick is to produce as much flagellin as possible, for incorporation in to flagella.

This biobrick was made from 2 parts:

This biobrick is an improvement of the biobrick BBa_K1463604 designed by Glasgow 2014 team.

FliC, the main flagella protein of E.coli

General

Flagellum structure
FliC of Escherichia coli is the main protein that makes up the flagella filament and is thus necessary for bacterial swimming

[1]. Flagellin is a globular protein that arranges itself in a hollow cylinder to form the filament in a bacterial flagellum. It has a mass of 30,000 to 60,000 daltons depending on the bacterium. In E.coli it has a mass of 51.3 kDa.

Metal biosorption capacity

It has been demonstrated that flagellin has the ability to adsorb precious metals on its surface such as platinum, gold... [2] and this was important for our project Highway to platinum

Immune response capacity

The immune system has a very stong response to flagellin, this is the result of:

  • Flagellin being an abundant extracellular protein in many bacterial pathogens;
  • the presence of an innate immune response to flagellin,

mediated by the Toll-like receptor 5 (TLR5). [3]

Design summary

To ensure high level flagellin expression:

  • The coding sequence we have used BBa_K1951005 has been codon optimized for expression in E.coli
  • The promotor and RBS we have used K880005 were designed and tested for high level expression in E.coli

Experience summary

To test the correct functioning of this biobrick we have performed three different types of experiment.

  • Protein production checked by SDS PAGE
  • Swimming phenotype complementation of a fliC mutant
  • Microscopic verification of flagellar formation
T--Aix-Marseille--swim.jpeg

Protein production

Protein production was confirmed by SDS page and coomassie blue staining in cells carrying, or not, a plasmid for expression of this biobrick.

Swimming test

We constructed a fliC deletion mutant that is unable to swim, from the wild-type strain W3110. The ability to swim was restored to this mutant by our biobrick, as can be seen in the illustration here. This demonstrated that the protein can be correctly inserted into flagella and functions.

Electron Microscopy

We also observed, using electron microscopy, the presence of multiple flagella on bacteria containing our biobrick. The flgaella in these bacteria appeared more numerous than in wild-type bacteria.

Biobrick BBa_K1463604improvement

This biobrick is an improvement on the biobrick designed by the Glasgow 2014 team. K1463604

The improvement of this part is multiple.

  • First there is no mutation in the promoter or RBS of our part so the flagellin is well expressed and functional. Unfortunately when the Glasgow team tried to make this part they picked up a mutation in the promoter.
  • Second the sequence that we have used it a synthetic gene with codon optimization, designed specifically for high level expression in E.coli. Thus as an expression part is improved over the initial design.
  • Third in the functional swimming assay, we see evidence for improved swimming (denser halo), in cells expressing our biobrick, a phenotype not observed by the Glasgow team in 2014.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1285
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 362
    Illegal AgeI site found at 770
  • 1000
    COMPATIBLE WITH RFC[1000]


.
  1. Capeness & al. 2015, http://eprints.nottingham.ac.uk/27979/1/Michael%20Capeness%20-%20Thesis%20-%20PDF.pdf
  2. Deplanche & al., 2007 http://onlinelibrary.wiley.com/doi/10.1002/bit.21688/abstract.
  3. Kathrani A. & al, 2012 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030117)
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