Protein_Domain

Part:BBa_K1951009:Design

Designed by: claire raynaud   Group: iGEM16_Aix-Marseille   (2016-10-13)
Revision as of 13:51, 16 October 2016 by Loulou (Talk | contribs) (→‎Sequence optimization)


FliC Desulfovibrio producer


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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 362
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 461


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

Source

We used previous biobrick :

- BBa_K880005

- BBa_K1951006

References