Part:BBa_K1951008

FliC E.coli producer

FliC, the main flagella protein of E.coli

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.

General

Flagellum structure

FliC of Escherichia coli is the main protein constitutive of the flagellum filament and is involved to promote 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 about 30,000 to 60,000 daltons. In the possession of flagellum, bacteria can move what confers them a selective advantage.

Metal biosorption capacity

It has been demonstrated that Flagellin has the ability to adsorb precious metal on its surface such as platinum, gold...

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).

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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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Design summary

Bba_K1951008 is an improvement of the [BBa_K1463604. Contrary to the biobrick from Glasgow 2014, our biobrick contains a strong promotor allowing a high level expression.

Codons have been optimized for Escherichia coli allowing a high transcription level.

Every forbidden restriction sites have been removed and sequence is optimital.

Prefix and suffix have been added using SLIC oligos designed by our team Clic here to see the SLIC oligos table

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]

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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