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
. 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
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). 
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
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 was confirmed by SDS page and coomassie blue staining in cells carrying, or not, a plasmid for expression of this biobrick.
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.
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.
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
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21Illegal BamHI site found at 1285
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 362
Illegal AgeI site found at 770
- 1000COMPATIBLE WITH RFC
- Capeness & al. 2015, http://eprints.nottingham.ac.uk/27979/1/Michael%20Capeness%20-%20Thesis%20-%20PDF.pdf
- Deplanche & al., 2007 http://onlinelibrary.wiley.com/doi/10.1002/bit.21688/abstract.
- Kathrani A. & al, 2012 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030117)