Composite

Part:BBa_K1175100

Designed by: Nicholas K. Goldner   Group: iGEM13_WLC-Milwaukee   (2013-09-17)


BBa_K1175028 pBAD Promoter and SecI pump attached to Composite norvirus antibody BBa_K875004

These are the three essential parts prtD, prtE, prtF, that make up the Type I secretion system from Erwinia chrysanthemi.This part is made of 3 genes: prtD, prtE, and prtF, that constitute a type I Erwinia chrysanthemi secretion system. The Pump is expressed from a strong constitutive promoter, BBa_k206000, and has the translational terminator BBa_B0014. In pSB1C3.

Used to secrete proteins containing prtB C-terminal tag. The prtB C-terminal tag is built into the protein generator [BBa_K215002]. Any protein of interest can be inserted into the protein generator and then secreted when used in conjunction with this secretion system.

This construct is designed for the expression of an already descibed engeneered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. The antibody is expressed in a single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA-scFv 54.6 antinorovirus, Histidine tag (6HIS), Terminator (B0015).

The part combines a strongly expressed secretion pump with a antinoroviral antibody that is expressed in the outer-membrane can/will be combined with BBa_K1175012 A composite part formed between BBa_K206000 strong pBAD promoter, BBa_B0034 strong RBS, and BBa_K215001 a purification and secretion tag.

For full characterization data on this part, please see the University of Washington 2009 iGEM project page, Team Trieste iGEM 2012 and, Wisconsin Lutheran College 2013 iGEM project page.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1582
    Illegal BsaI.rc site found at 1228
    Illegal BsaI.rc site found at 3832


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Parameters
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