Difference between revisions of "Part:BBa K2040121"
 
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<partinfo>BBa_K2040121 short</partinfo>
 
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KillerRed([https://parts.igem.org/Part:BBa_K1184000 BBa_K1184000]) is a red fluorescent protein that produces reactive oxygen species (ROS) in the presence of yellow-orange light (540-585 nm). KillerRed is engineered from anm2CP to be phototoxic.  Expression of KillerRed and irradiation with light may act a kill-switch for biosafety applications. More details about KillerRed seen [http://2013.igem.org/Team:Carnegie_Mellon/KillerRed 2013 Carnegie_Mellon].
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A KillerRed expression cassette was created with a fungal promoter PgpdA and a fungal terminator TtrpC.
  
 
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===Usage and Biology===
 
===Usage and Biology===
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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.
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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.
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We demonstrated the functionality of this part via transforming this expression cassette into ''M.anisopliae''. The transformants were under a fluorescence microscope(Magnification: 400X):
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*<b>Wildtype:</b>
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[[Image:WT FL.png|center|810px]]
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*<b>KillerRed transformants:</b>
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[[Image:KR FL.png|center|850px]]
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This new device was successfully expressed in ''M.anisopliae''.
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As we observed<sup>Fig1.</sup>, 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 ([https://parts.igem.org/Part:BBa_K2040122 BBa_K2040122])so that KillerRed can function in the ROS-sensitive intracellular localizations, the chromatin in nucleus, due to the NLS.
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[[Image:KR-WT.jpeg|center|500px|thumb|Figure1: Wildtype(left) and KR transformant(right) growing on PDA]]
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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BBa_K2040121 SequenceAndFeatures
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<partinfo>BBa_K2040121 SequenceAndFeatures</partinfo>
  
  

Latest revision as of 14:57, 19 October 2016


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