Device

Part:BBa_K2040121

Designed by: Chilam Poon   Group: iGEM16_NYMU-Taipei   (2016-10-14)


PgpdA + KillerRed + TtrpC

A KillerRed expression cassette was created with a fungal promoter PgpdA and a fungal terminator TtrpC.


Usage and Biology

KillerRed,always used for precise inactivation of selected proteins in chromophore-assisted light inactivation (CALI) technique and for the light-induced cell killing, is rarely used to express in fungal chassis.

NYMU 2016 construct this new device, for fungal expression, to test wheather KillerRed protein can be used in entomopathogenic fungi Metarhizium anisopliae, our chassis organism.

We demonstrated the functionality of this part via transforming this expression cassette into M.anisopliae. The transformants were under a fluorescence microscope(Magnification: 400X):


  • Wildtype:
WT FL.png
  • KillerRed transformants:
KR FL.png


This new device was successfully expressed in M.anisopliae.

As we observedFig1., the growth situations of M.anisopliae KR transformants on media will not be affected greatly since irradiation of KillerRed localized in cell cytosol has a weak effect on cell survival in eukaryotic cells. In advance, we decided to fuse a SV40 NLS to the KillerRed protein (BBa_K2040122)so that KillerRed can function in the ROS-sensitive intracellular localizations, the chromatin in nucleus, due to the NLS.

Figure1: Wildtype(left) and KR transformant(right) growing on PDA



Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 880
    Illegal BsaI.rc site found at 1171


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Parameters
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