Team:ETH Zurich/UVR8/Design

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== UVR8 - TetR<sub>DBD</sub> fusion: a UV sensing protein ==
== UVR8 - TetR<sub>DBD</sub> fusion: a UV sensing protein ==
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We wanted to keep the TetR<sub>DBD</sub> as intact as possible, thus we fused  TetR<sub>DBD</sub> its C term to the N term of UVR8. Three different fusion strategies were carried out to test for the best UVR8-TetR<sub>DBD</sub> fusion protein [[http://partsregistry.org/Part:BBa_K909008 link to the partsregistry]]:
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We wanted to keep the TetR<sub>DBD</sub> as intact as possible, thus we fused  TetR<sub>DBD</sub> its C term to the N term of UVR8. Three different fusion strategies were carried out to test for the best UVR8-TetR<sub>DBD</sub> fusion [[http://partsregistry.org/Part:BBa_K909008 link to the partsregistry]]:

Revision as of 23:33, 26 September 2012

Eth ecolipseeth logo.png
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Navigate through Overview, Design and Results


UVR8 - TetRDBD fusion: a UV sensing protein

We wanted to keep the TetRDBD as intact as possible, thus we fused TetRDBD its C term to the N term of UVR8. Three different fusion strategies were carried out to test for the best UVR8-TetRDBD fusion http://partsregistry.org/Part:BBa_K909008 link to the partsregistry:


  • 1. Full length UVR8 without a linker, a.k.a. TetRDBD-full UVR8
Uvr82ndeth.jpg
  • 2. Full length UVR8 were extended with [GGS]x2 linker (+6 aa) between TetRDBD
Uvr83rdeth.jpg
  • 3. Truncated UVR8 by 13 aa
Ethuvr81.jpg
Uvr8uv.jpg


Constructs were tested in two plasmid system: repressor and reporter plasmids. As repressor plasmid, a medium copy number pSEVA183 derived plasmid (provided by [http://www.bsse.ethz.ch/bpl/people/bosandre Andreas Bosshart]), containing constitutively expressed LacI, were used. UVR8-TetRDBD fusions and TetRDBD and full length TetR for control measurements were cloned after LacI a regulating Ptac promoter.

For the reporter plasmid, the GFP producing biobrick from Ptet promoter (<partinfo>BBa_I13522</partinfo>) was cloned into low copy number pSB4K5 plasmid.

PSB4K5.jpg
Pseva 183.jpg



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