Generator

Part:BBa_K792012

Designed by: Manuel Giménez   Group: iGEM12_Buenos_Aires   (2012-09-25)

Yeast exportable Trp-rich peptide w/enhanced import (2)

This composite device produces and exports a Tryptophan rich peptide, that is import enhanced (thanks to a trojan peptide). This device is intended to be used with Yeast chassis, so it is composed by subparts designed also to be use in yeast.


The high level structure of the device is:

  • BamHI restriction site (introduced to facilitate cloning into yeast expression vectors (pEG202, pCM180 series) - this is not essential for the device function)
  • Kozak consensus sequence for initiation of translation
  • Signal peptide that targets the product of the gene for secretion
  • Trojan peptide, to increase internalization in target cell (Polyarginine)
  • Payload: this is the exported Tryptophan rich domain of the protein (a custom designed peptide that we called PolyWb)


The device's sequence has inherited HindIII restriction site from the parts it's made of.


Important note: this part has been submitted to the registry using a direct synthesis DNA sample, so it does not contain scars from BB assembly standard.
See also: Another Trp-rich export and enhanced import composite device Part:BBa_K792010.
Similar design His-rich export and enhanced import composite device Part:BBa_K792011.
BsAs2012-Biohazard.png This device contains BBa_K792004, a trojan peptide. Extreme your safety measures when dealing with strains containing this part: avoid direct contact with them or conditioned medium and discard them as pathogenic residues.
The device was originally designed and built to be used in iGEM BsAs 2012 project to implement a cross-feeding regulatory system between two different yeast strains with specific auxotrophy. Visit our wiki to read details about the designing process and implementation details. Also feel free to contact us for any question regarding this part. Igem.bsas.png

Data

To determined the devices Tryptophan's secretion rate we used a simple model that describes the behaviour of a strain transformed with the device that produces and secretes this amino acid and how it accumulates in the medium. The secretion rate is the ratio between the concentration of tryptophan at a given time and the time integral of the culture density.


  • We measured the amount of tryptophan in the medium acquiring the florescence spectra of the medium and compering with a calibration curve.
  • We used W303a yeast strains, and pCM182 plasmid.
  • To take into account aminoacid natural diffusion through cell's membrane, we used a strain transformed with the pCM182 plasmid but without BBa K792010 device, as a control.


As shown in the figure, the tryptophan secretion rate in the strain with the device is almost 10x higher than the control. More information about experiments we performed with this device can be found in BsAs 2012 wiki.


BsAs2012-result-K792012.jpg


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/eukaryote/yeast
Parameters
None