Part:BBa_K1951008

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

Flagellum structure

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

. 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... and this was important for our project [http://2016.igem.org/Team:Aix-Marseille 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).

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

Protein production

Protein production was confirmed by SDS page/ comassie blue

Swimming test

Complementation of FliC mutant E. coli W3110 with Bba_K151008 recovers the swimming ability and makes the motility higher than in the wild type strain. It has been tested by a swimming test

Motility and biosorption using flagellin

To know more about the integration of this biobrick in our project, you can visit our website : [http://2016.igem.org/Team:Aix-Marseille/Design#Biosorption_and_reduction_using_flagellin_and_peptides Biosorption and reduction using flagellin and biopeptides]

Biobrick BBa_K1463604improvement

This biobrick has been improved from a previous onimprovement==

This biobrick has been improved from a previous one e designed by Glasgow 2014 team.

Mesure of the bacterial motility (y axe left) in function of the incubation time (x axe) and density of swimmer cells (y axe right) in different backgrounds. Density is based on a scale from 0 to 3 : 0=transparent, 1=slighty turbidity, 2=medium turbidity, 3=high turbidity

• For this biobrick design, Glasgow team results has shown that their FliC wasn't able to recover the swimming capacity showing a bad quality of the synthetised flagellin protein. Please find the link of this biobrick below :

BBa_K1463604Promotor they have used had a mutation which makes it unfunctionnal.

Instead of Bba_J23106 and Bba_J23116, we used strong promoter, strong RBS combination for high expression levels of the flagellin. By the combination of Bba_K880005 and Bba_K1951005, we made a high flagellin expression vector able to highly recover swimming and even surexpress this pattern.

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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]

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