Difference between revisions of "Part:BBa K1951009:Design"

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__NOTOC__
 
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<partinfo>BBa_K1951009 short</partinfo>
 
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<partinfo>BBa_K1951009 SequenceAndFeatures</partinfo>
 
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===Design Notes===
 
===Design Notes===
  
 
High level flagellin expression:
 
High level flagellin expression:
 
This biobrick is composed of two parts:
 
This biobrick is composed of two parts:
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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_K1951006 BBa_K880006] 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] the FliC coding sequence designed by our team for high level expression in <i>E.coli</i> with sequence optimization.
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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>Desulfovibrio vulgaris</i> with sequence optimization.
  
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We found the raw sequence from online data base '''QUEL DATA BASE YOANN????''
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===Sequence optimization===
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We found the raw sequence from online data base [http://www.ncbi.nlm.nih.gov/nucleotide/46451220?report=genbank&log$=nucltop&blast_rank=3&RID=WDB55ADB014 see here]. We removed all illegal sites (EcoRI, XbaI, PstI SpeI) and BbsI. Then we optimized the codon use for expression in''E. coli'',with an online [https://eu.idtdna.com/CodonOpt  codon optimizer]. As the optimization is random, some illegal or BbsI sites has appeared and we did the optimization until obtain a sequence with no such sites.
  
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===Source===
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The sequence amplified from the synthetized one by IDT was amplified and cloned into the pSB1C3 vector thanks to SLIC primers designed by our team.
  
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We used previous biobrick :
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===Promoter and RBS selection===
  
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- BBa_K880005
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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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===Source===
  
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- BBa_K1951006
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We constructed this part from 2 others :
  
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===References===
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* [https://parts.igem.org/Part:BBa_K1951006 BBa_K1951006]
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* [https://parts.igem.org/Part:BBa_K880005 BBa_K880005]

Latest revision as of 14:31, 17 October 2016

Design Notes

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

  • BBa_K880006 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 Desulfovibrio vulgaris with sequence optimization.

Sequence optimization

We found the raw sequence from online data base [http://www.ncbi.nlm.nih.gov/nucleotide/46451220?report=genbank&log$=nucltop&blast_rank=3&RID=WDB55ADB014 see here]. We removed all illegal sites (EcoRI, XbaI, PstI SpeI) and BbsI. Then we optimized the codon use for expression inE. coli,with an online codon optimizer. As the optimization is random, some illegal or BbsI sites has appeared and we did the optimization until obtain a sequence with no such sites.

The sequence amplified from the synthetized one by IDT was amplified and cloned into the pSB1C3 vector thanks to SLIC primers designed by our team.

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).

Source

We constructed this part from 2 others :