Difference between revisions of "Part:BBa K1951008"
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=== Metal biosorption capacity===
 
=== Metal biosorption capacity===
  
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It has been demonstrated that flagellin has the ability to adsorb precious metals on its surface such as platinum, gold... <ref>Deplanche & al., 2007 http://onlinelibrary.wiley.com/doi/10.1002/bit.21688/abstract. </ref> and this was important for our project [http://2016.igem.org/Team:Aix-Marseille Highway_to_platinum]
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It has been demonstrated that flagellin has the ability to adsorb precious metals on its surface such as platinum, gold... <ref>Deplanche & al., 2007 http://onlinelibrary.wiley.com/doi/10.1002/bit.21688/abstract. </ref> 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 ===  
 
=== Immune response capacity ===  
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== [https://parts.igem.org/Part:BBa_K1951005:Experience Experience summary] ==   
 
== [https://parts.igem.org/Part:BBa_K1951005:Experience Experience summary] ==   
 +
 +
To test the correct functioning of this biobrick we have performed three different types of experiment.
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* Protein production checked by SDS PAGE
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* Swimming phenotype complementation of a fliC mutant
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* Microscopic verification of flagellar formation
 +
 
[[File:T--Aix-Marseille--swim.jpeg|300px|right]]
 
[[File:T--Aix-Marseille--swim.jpeg|300px|right]]
 
===Protein production===
 
===Protein production===
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Protein production was confirmed by SDS page/ comassie blue
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 +
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===
 
===Swimming test===
  
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Complementation of FliC mutant <i>E. coli</i> 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
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We constructed a fliC deletion mutant that is unable to swim, from the wild-type strain W3110.
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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.
  
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===Motility and biosorption using flagellin ===
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===Electron Microscopy===
  
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To know more about the integration of this biobrick in our project, you can visit our website :
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We also observed, using electron microscopy, the presence of multiple flagella on bacteria containing our biobrick.  
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[http://2016.igem.org/Team:Aix-Marseille/Design#Biosorption_and_reduction_using_flagellin_and_peptides Biosorption and reduction using flagellin and biopeptides]
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The flgaella in these bacteria appeared more numerous than in wild-type bacteria.
  
 
==Biobrick [https://parts.igem.org/Part:BBa_K1463604 BBa_K1463604]improvement==
 
==Biobrick [https://parts.igem.org/Part:BBa_K1463604 BBa_K1463604]improvement==
  
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This biobrick is an improvement on the biobrick designed by the Glasgow 2014 team.
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[https://parts.igem.org/Part:BBa_K1463604 K1463604]
  
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This biobrick has been improved from a previous onimprovement==
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The improvement of this part is multiple.
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* 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.
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* Second the sequence that we have used it a synthetic gene with codon optimisation, designed specifically for high level expression in <i>E.coli</i>. Thus as an expression part is improved over the initial design.
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This biobrick has been improved from a previous one e designed by Glasgow 2014 team.
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* 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.
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[[File:T--Aix-Marseille--graph.jpeg|700px|left|thumb|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]]
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* 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 :
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[https://parts.igem.org/Part:BBa_K1463604 BBa_K1463604]Promotor they have used had a mutation which makes it unfunctionnal.
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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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<i>.</i>
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==Sequence and Features==
 
==Sequence and Features==
 
<partinfo>BBa_K1951008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1951008 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 22:52, 15 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:

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

[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
T--Aix-Marseille--swim.jpeg

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


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]


.
  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)