Difference between revisions of "Part:BBa K1951008"

Revision as of 22:52, 15 October 2016 (view source) LISM JS (Talk | contribs) ← Older edit		Revision as of 19:50, 16 October 2016 (view source) Clacla02 (Talk | contribs) (→‎Experience summary) Newer edit →
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	* The promotor and RBS we have used [https://parts.igem.org/Part:BBa_K880005 K880005] were designed and tested for high level expression in <i>E.coli</i>		* The promotor and RBS we have used [https://parts.igem.org/Part:BBa_K880005 K880005] were designed and tested for high level expression in <i>E.coli</i>
−	== [https://parts.igem.org/Part:BBa_K1951005:Experience Experience summary] ==	+	== [https://parts.igem.org/Part:BBa_K1951008:Experience Experience summary] ==
	To test the correct functioning of this biobrick we have performed three different types of experiment.		To test the correct functioning of this biobrick we have performed three different types of experiment.

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Revision as of 19:50, 16 October 2016

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:

• K1951005
• K880005

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

FliC, the main flagella protein of E.coli

General

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 [http://2016.igem.org/Team:Aix-Marseille/Design#Biosorption_and_reduction_using_flagellin_and_peptides 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

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 promotor or RBS of our part so the flagellin is well expressed and functional. Unfortunately when the Glasgow team trie to make this part they picked up a mutation in the promotor.
• Second the sequence that we have used it a synthetic gene with codon optimisation, 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

.

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)