Difference between revisions of "Part:BBa K1951008:Design"
 
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===Design Notes===
  
 
High level flagellin expression:
 
High level flagellin expression:
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This biobrick is composed of two parts
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This biobrick is composed of two parts:
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* [https://parts.igem.org/Part:BBa_K880005 BBa_K880005 design] 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_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 BBa_K1951005 Design] 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 of Bba_K1951008==
 
== Optimization of Bba_K1951008==
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===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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===Optimized codons 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:
 +
* it has already been successfully used in the past,
 +
* 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 of promotor BBa_J23100==
 
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Parts J23100 are a family of constitutive promoter parts isolated from a small combinatorial library.
 
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This promoter part can be used to tune the expression level of constitutively expressed parts. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification.
 
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== Design of RBS BBa_B0034==
 
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Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. 
 
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No secondary structures are formed in the given RBS region. Users should check      for secondary structures induced in the RBS by upstream and downstream elements      in the +50 to -50 region, as such structures will greatly affect the strength of the RBS.
 
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Contact info for this part:
 
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Primers: Note: EcoRI sites removed. Must use XP. Base pairs in CAPS are the truncated prefix and the suffix. Sequences provided by Gingko BioWorks.
 
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{| class="wikitable"
 
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| Fwd : 
 
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| GCTTCTAGAGATACATGAACATGCAATACttgacggctagctcagtcctaggtacagtgctagc
 
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|-
 
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| Rv :
 
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| CTGCAGCGGCCGCTACTAGTAGAGAGCGTTCtataaacgcagaaaggcccacccg
 
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|}.
 
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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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- [https://parts.igem.org/Part:BBa_K1951005 BBa_K1951005]
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- [https://parts.igem.org/Part:BBa_K880005 BBa_K880005]
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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