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 the C-terminus of TetR<sub>DBD</sub> to the N-terminus of UVR8. Three different fusion strategies were carried out to test for the best TetR<sub>DBD</sub>-UVR8 construct:
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*1. Full length UVR8 without a linker, a.k.a. TetR<sub>DBD</sub>-full UVR8
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*1. TetR<sub>DBD</sub>-full UVR8 - Full length UVR8 fusion with TetR<sub>DBD</sub> without a linker 
[[File:Uvr82ndeth.jpg|frameless|400px|center]]
[[File:Uvr82ndeth.jpg|frameless|400px|center]]
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*2. Full length UVR8 were extended with [GGS]x2 linker (+6 aa) between TetR<sub>DBD</sub>
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*2. TetR<sub>DBD</sub>-GGS-UVR8 - Full length UVR8 fusion with TetR<sub>DBD</sub> with extended [GGS]<sub>2</sub>-linker (6 additional amino acids)
[[File:Uvr83rdeth.jpg|frameless|400px|center]]
[[File:Uvr83rdeth.jpg|frameless|400px|center]]
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*3. Truncated UVR8 by 13 aa
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*3. [http://partsregistry.org/Part:BBa_K909008 TetR<sub>DBD</sub>-dUVR8] - Truncated version of UVR8 lacking 13 amino acids in N-terminus fusion with TetR<sub>DBD</sub>
[[File:Ethuvr81.jpg|frameless|400px|center]]
[[File:Ethuvr81.jpg|frameless|400px|center]]
[[File:Uvr8uv.jpg|frameless|400px|center]]
[[File:Uvr8uv.jpg|frameless|400px|center]]
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=== Plasmids ===
Constructs were tested in two plasmid system: repressor and reporter plasmids.  
Constructs were tested in two plasmid system: repressor and reporter plasmids.  
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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-TetR<sub>DBD</sub> fusions and TetR<sub>DBD</sub> and full length TetR for control measurements were cloned after LacI a regulating P<sub>tac</sub> promoter.
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We used a medium copy number pSEVA183 derived plasmid (provided by [http://www.bsse.ethz.ch/bpl/people/bosandre Andreas Bosshart]) constitutively expressing LacI as the repressor-plasmid. UVR8-TetR<sub>DBD</sub> fusions, TetR<sub>DBD</sub> and full length TetR (as a control) were cloned under the control of the P<sub>tac</sub> promoter.
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For the reporter plasmid, the GFP producing biobrick from P<sub>tet</sub> promoter (<partinfo>BBa_I13522</partinfo>) was cloned into low copy number pSB4K5 plasmid.   
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GFP controlled by P<sub>tet</sub> promoter (<partinfo>BBa_I13522</partinfo>) was cloned into pSB4K5 - a low copy number plasmid - and used as the reporter system.   
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Latest revision as of 01:49, 27 October 2012

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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 the C-terminus of TetRDBD to the N-terminus of UVR8. Three different fusion strategies were carried out to test for the best TetRDBD-UVR8 construct:


  • 1. TetRDBD-full UVR8 - Full length UVR8 fusion with TetRDBD without a linker
Uvr82ndeth.jpg
  • 2. TetRDBD-GGS-UVR8 - Full length UVR8 fusion with TetRDBD with extended [GGS]2-linker (6 additional amino acids)
Uvr83rdeth.jpg
  • 3. [http://partsregistry.org/Part:BBa_K909008 TetRDBD-dUVR8] - Truncated version of UVR8 lacking 13 amino acids in N-terminus fusion with TetRDBD
Ethuvr81.jpg
Uvr8uv.jpg

Plasmids

Constructs were tested in two plasmid system: repressor and reporter plasmids. We used a medium copy number pSEVA183 derived plasmid (provided by [http://www.bsse.ethz.ch/bpl/people/bosandre Andreas Bosshart]) constitutively expressing LacI as the repressor-plasmid. UVR8-TetRDBD fusions, TetRDBD and full length TetR (as a control) were cloned under the control of the Ptac promoter.

GFP controlled by Ptet promoter (<partinfo>BBa_I13522</partinfo>) was cloned into pSB4K5 - a low copy number plasmid - and used as the reporter system.

PSB4K5.jpg
Pseva 183.jpg



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