Difference between revisions of "Part:BBa K1951008:Design"

 
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__NOTOC__
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<partinfo>BBa_K1951008 design</partinfo>
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<partinfo>BBa_K1951008 short</partinfo>
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High flagellin expression coding sequence has been designed using two parts :
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*
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== Optimization
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===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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===Codon optimization for <i>Escherichia coli</i>. ===
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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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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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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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Revision as of 12:49, 15 October 2016

BBa_K1951008 design Not understood High flagellin expression coding sequence has been designed using two parts :

== Optimization

Sequence optimization

We took fliC sequence from Glasgow 2014 team 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[1]. We changed a Bbs1 site 130 position by substitution of amino acid coding.

Codon optimization for Escherichia coli.

We obtimised codon for Escherichia coli. Codon optimization is a technique used to improve the protein expression in living organism by increasing the translational efficiency of gene of interest [1-4, 6-13, 15-18, 20-28]. This biobrick codon optimised increase the functionality of gene. To process it, we use codon optimization IDT software[2]. If you use this biobrick in Escherichia coli, you can be sure that the protein produced will be highly expressed and well solubilised.

Prefix and suffix addition

Prefix and suffix subsequences (containing restriction site EcoRI, XbaI and SpeI PstI respectively) have been added by a SLIC method with the following oligos :

FliC E. coli slic forward cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGCACAAGTCATTAAT
FliC E.coli slic reverse ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACCCTGGAGCAG
.


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]


Design Notes

On the BbaK1951004, the restriction site Bbs1 is mutated (substitution) to allow the control for a future Bbs1 site insertion.


Source

We made this part from 2 others :

- BbaK1951004

- Bba880005

References

  1. ↑ http://eu.idtdna.com/site
  2. ↑ https://eu.idtdna.com/CodonOpt