Protein_Domain

Part:BBa_K1951009

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


FliC Desulfovibrio 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:

FliC, the main swimming protein of Desulfovibrio

General

Flagellin C (FliC) protein from Desulfovibrio vulgaris strain is the main protein constitutive of the flagellum filament and is involved to promote bacterial swimming. This sequence is conserved in many bacterial strains as the capacity of swimming given by the flagellum confers a great selective advantage.

Representative electron micrographs of nanoparticles produced by D. alaskensis G20. Pd is found on the surface of the cell [1]

Metal Biosorption Capacity

It has been demonstrated that Flagellin has the ability to adsorb precious metal on its surface such as platinum, palladium gold [2][3] and this was important for our project [http://2016.igem.org/Team:Aix-Marseille/Design#Biosorption_and_reduction_using_flagellin_and_peptides Highway to platinum]

Immune response capacity

The propensity of the immune response to flagellin may be explained by two facts:

  • Flagellin is an extremely abundant protein in flagellated bacteria.
  • There exists a specific innate immune receptor that recognizes flagellin, Toll-like receptor 5 (TLR5). [4]

Design summary

We found the sequence in online databases, and after processed it ordered it to IDT. The cloning was made fisrly in the pSB1C3 vector thanks to SLIC oligo designed by our team.

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

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.

Experience summary

Once sequencing data indicated that the cloning and transformation was successful, we assessed the production of the protein by a SDS page/comassie test. COOMASSIE However, no functionality test as swimming test has been carried out with this biobrick contrary to the FliC protein from E .coli

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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 362
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 461


  1. ↑ https://www.ncbi.nlm.nih.gov/pubmed/25686718
  2. ↑ Deplanche & al., 2007 http://onlinelibrary.wiley.com/doi/10.1002/bit.21688/abstract.
  3. ↑ Capeness & al. 2015, http://eprints.nottingham.ac.uk/27979/1/Michael%20Capeness%20-%20Thesis%20-%20PDF.pdf
  4. ↑ Kathrani A. & al, 2012 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030117)
[edit]
Categories
Parameters
None