Difference between revisions of "Part:BBa K1951008:Design"
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<partinfo>BBa_K1951008 design</partinfo>
 
 
High flagellin expression coding sequence has been designed using two parts :  
 
High flagellin expression coding sequence has been designed using two parts :  
 
* [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]
 
* [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]

Revision as of 13:02, 15 October 2016

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