Difference between revisions of "Part:BBa K1951008:Design"
 
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===Design Notes===
  
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<partinfo>BBa_K1951008 design</partinfo>
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High level flagellin expression:
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High flagellin expression coding sequence has been designed using two parts :  
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This biobrick is composed of two parts:
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* [https://parts.igem.org/Part:BBa_K880005 design BBa_K880005 ] Strong promoter, strong RBS combination for high expression levels of proteins (composed of sub-parts [https://parts.igem.org/Part:BBa_J23100 design BBa_J23100 ] and [https://parts.igem.org/Part:BBa_B0034 design BBa_B0034]
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* [https://parts.igem.org/Part:BBa_K880005 BBa_K880005] A strong promoter, strong RBS combination specifically conceived and tested for high expression levels of proteins in <i>E.coli</i> (this part is itself composed of sub-parts [https://parts.igem.org/Part:BBa_J23100 BBa_J23100 ] and [https://parts.igem.org/Part:BBa_B0034 BBa_B0034]
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* [https://parts.igem.org/Part:BBa_K1951005:Design BBa_K1951005 ] the FliC coding sequence designed by our team
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* [https://parts.igem.org/Part:BBa_K1951005 BBa_K1951005] the FliC coding sequence designed by our team for high level expression in <i>E.coli</i> with sequence optimization.
  
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== Optimization ==
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== Optimization of Bba_K1951008==
  
 
===Sequence optimization===
 
===Sequence optimization===
  
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We took fliC sequence from Glasgow 2014 team [https://parts.igem.org/Part:BBa_K1463601 BBa_K1463601]. We used Snapgene to remove the forbidden site by changing in the substitution which didn't change the amino acid. The sequence has been sythetised by IDT<ref>http://eu.idtdna.com/site</ref>. We changed a Bbs1 site 130 position by substitution of amino acid coding.
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This part is optimized for expression in <i>E.coli</i> by codon optimization and synthesis of a synthetic gene.
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Codon optimization was done using the tool provided by IDT <ref> https://eu.idtdna.com/CodonOpt </ref>.
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Codon optimization has been shown to <u>improve protein expression</u> by increasing the translational efficiency of gene of interest, and consists of choosing for each amino acid the codon that works best in the target species <b>Claire a ref or 2</b>.
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We optimized the sequence for expression in <i>E.coli</i>.
  
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===Codon optimization for <i>Escherichia coli</i>. ===
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In addition to changing the codons from the <i>E.coli</i> sequence we also ensured that all forbidden restriction sites were absent to ensure compatibility with the biobrick standard.
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We also designed the insertion of a Bbs1 site at amino acid 130 to allow insertion of additional sequences in the loop at this position in the sequence. Unfortunately we did not get round to using this feature.  
  
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We obtimised codon for <i>Escherichia coli</i>. Codon optimization is a technique used to <u>improve the protein expression</u> in living organism by increasing the translational efficiency of gene of interest [1-4, 6-13, 15-18, 20-28]. This biobrick codon optimised <u>increase the functionality of gene</u>. To process it, we use codon optimization IDT software<ref> https://eu.idtdna.com/CodonOpt </ref>.
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===Promoter and RBS selection===
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If you use this biobrick in <i>Escherichia coli</i>, you can be sure that the protein produced will be highly expressed and well solubilised.
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=== Prefix and suffix addition===
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As the purpose of this part is high level protein expression we selected the part [https://parts.igem.org/Part:BBa_K880005 BBa_K880005] to drive expression of our coding sequence. We chose this part as:
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* it has already been successfully used in the past,
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* it was available in the distribution,
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* it combines a strong promotor and a RBS (so avoiding the separate incorporation of 2 parts).
  
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Prefix and suffix subsequences (containing restriction site EcoRI, XbaI and SpeI PstI respectively) have been added by a SLIC method with the following oligos :
 
  
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{| class="wikitable"
 
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| FliC E. coli slic forward 
 
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| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGCACAAGTCATTAAT
 
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|-
 
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| FliC E.coli slic reverse
 
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| ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACCCTGGAGCAG
 
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|}.
 
 
<partinfo>BBa_K1951008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1951008 SequenceAndFeatures</partinfo>
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===Design Notes===
 
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On the BbaK1951004, the restriction site Bbs1 is mutated (substitution) to allow the control for a future Bbs1 site insertion.
 
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===Source===
 
===Source===
  
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We made this part from 2 others :  
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We constructed this part from 2 others :  
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- BbaK1951004
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- Bba880005
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* [https://parts.igem.org/Part:BBa_K1951005 BBa_K1951005]
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* [https://parts.igem.org/Part:BBa_K880005 BBa_K880005]
  
 
===References===
 
===References===

Latest revision as of 14:30, 17 October 2016

Design Notes

High level flagellin expression: This biobrick is composed of two parts:

  • BBa_K880005 A strong promoter, strong RBS combination specifically conceived and tested for high expression levels of proteins in E.coli (this part is itself composed of sub-parts BBa_J23100 and BBa_B0034
  • BBa_K1951005 the FliC coding sequence designed by our team for high level expression in E.coli with sequence optimization.


Optimization of Bba_K1951008

Sequence optimization

This part is optimized for expression in E.coli by codon optimization and synthesis of a synthetic gene. Codon optimization was done using the tool provided by IDT [1]. Codon optimization has been shown to improve protein expression by increasing the translational efficiency of gene of interest, and consists of choosing for each amino acid the codon that works best in the target species Claire a ref or 2. We optimized the sequence for expression in E.coli.

In addition to changing the codons from the E.coli sequence we also ensured that all forbidden restriction sites were absent to ensure compatibility with the biobrick standard. We also designed the insertion of a Bbs1 site at amino acid 130 to allow insertion of additional sequences in the loop at this position in the sequence. Unfortunately we did not get round to using this feature.

Promoter and RBS selection

As the purpose of this part is high level protein expression we selected the part BBa_K880005 to drive expression of our coding sequence. We chose this part as:

  • it has already been successfully used in the past,
  • it was available in the distribution,
  • it combines a strong promotor and a RBS (so avoiding the separate incorporation of 2 parts).



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]

Source

We constructed this part from 2 others :

References

  1. ↑ https://eu.idtdna.com/CodonOpt