Coding
fecA-prhA

Part:BBa_K367006:Experience

Designed by: Fernando Govantes   Group: iGEM10_UPO-Sevilla   (2010-06-30)
Revision as of 17:22, 26 October 2010 by Dcabpra (Talk | contribs) (→‎Applications of BBa_K367006)

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Applications of BBa_K367006

FecA/PrhA artificial coding sequence spans the first 92 codons of fecA, encoding the signal peptide, NH2 terminal extension and the proposed Ton-box, fused to the distal end of the prhA coding sequence at the conserved GSGL motif (aa. 89-92). We synthesized this biobrick using MrGene services, so we also optimized the sequence to be expressed in Escherichia coli. To this way the hybrid protein include domains from both OM proteins:

  • Signal peptide of FecA which will help to the accurate emplacement of the hybrid protein in the outer membrane of E. coli.
  • NH2-terminal extension of FecA. Including this domain of FecA means including the periplasmic signaling domain of this protein. The signal transfer between the OM and the IM proteins is performed between the N-terminus of FecA and the C-terminus of FecR (both are shown in the periplasmic). The hybrid protein includes the N-terminus of FecA so our expectation is that FecA/PrhA protein was able to interact with FecR.
  • Most of the Secretin and TonB N-terminus Short Domain of FecA. This domain helps FecA to interact with TonB. If TonB interaction is required for the OM-IM signal transfer, our hybrid protein includes this domain. Also, doing this fusion the hybrid protein loses the unknown function domain set in the N-terminus of PrhA.
  • TonB-dependent Receptor Plug Domain of PrhA. In 89th codon there is a conserved motif which was used to fuse FecA with PrhA. The function of the Plug domain is to propagate allosteric transitions through the outer membrane signaling the occupancy of the receptor.
  • TonB Dependent Receptor Domain of PrhA. From the conserved motif GSGL to the end of the protein amino acids are the same that in PrhA protein. The hybrid protein includes most of the PrhA protein, from 92sd codon to the C-terminus. The aim of it is that FecA/PrhA was able to interact with the non-diffusible plant wall signal that PrhA detects. The mechanism of this interaction is unknown.

For more information see: "http://2010.igem.org/Team:UPO-Sevilla/Project/Sensing".

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