Team:ETH Zurich/Decoder/Results

From 2012.igem.org

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In a first step two promoters were cloned together.
In a first step two promoters were cloned together.
The promoter controling mCherry expression is repressible by TetR and cI while the promoter controling eFCP expression is repressible by LacI and cI. To verify the expectations we used the following plasmid to introduce 4 distinct expression conditions:
The promoter controling mCherry expression is repressible by TetR and cI while the promoter controling eFCP expression is repressible by LacI and cI. To verify the expectations we used the following plasmid to introduce 4 distinct expression conditions:
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* IPTG & aTc: mCherry as well as eCFP are expressed.
* IPTG & aTc: mCherry as well as eCFP are expressed.
[[File: Pseva 183.jpg|frameless|210px|right]]
[[File: Pseva 183.jpg|frameless|210px|right]]
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[[File: ETHZ2012decoderpart1.png|frameless|200px|right]]
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[[File: 2012ethzDecoderodffacs.png|frameless|400px|center]]
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[[File: 2012ethzDecoderodffacs.png|frameless|200px|center]]
Single cell analysis revealed that the repression by LacI and TetR occurs and that the promoters work in absence of the repressors. The second step (full decoder assembly) was carried out after the FACS experiments.
Single cell analysis revealed that the repression by LacI and TetR occurs and that the promoters work in absence of the repressors. The second step (full decoder assembly) was carried out after the FACS experiments.

Revision as of 02:28, 26 October 2012

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Navigate through Overview, Design and Results


Hybrid promoters

Assembly part 1

In a first step two promoters were cloned together. The promoter controling mCherry expression is repressible by TetR and cI while the promoter controling eFCP expression is repressible by LacI and cI. To verify the expectations we used the following plasmid to introduce 4 distinct expression conditions:

Experimentsetuppart1ethz2012.png



  • No induction: LacI as well as TetR (due to the leakiness of the tac promoter) are present and bind to the LacO & TetO operator regions.
  • IPTG: LacI cannot repress the tac promoter anymore, TetR is expressed. TetR represses the expression of mCherry. On the other hand the promoter controling eCFP is active.
  • aTc: LacI is expressed, TetR is not there, no repression of mCherry occurs. eCFP cannot be produced as LacI binds to the LacO operator region.
  • IPTG & aTc: mCherry as well as eCFP are expressed.
Pseva 183.jpg
ETHZ2012decoderpart1.png
2012ethzDecoderodffacs.png

Single cell analysis revealed that the repression by LacI and TetR occurs and that the promoters work in absence of the repressors. The second step (full decoder assembly) was carried out after the FACS experiments.

Top: decoder part1; Left: P(TetO,LacO) controling cI and YFP; Right:Final decoder.

A first glimpse at the plates revealed already that the hybrid promoters are repressible by cI. While colonies containing only the hybrid promoters controling mCherry and eCFP were shining mainly red, the ones containing the whole decoder were hardly red.



Fig.3.: Digests left to right: Ladder, 3 screened colonies containing the decoder plasmid.


Hybrid promoters combined to the decoder.
Pseva 183.jpg


Fig.4: Boolean logic for testing of Decoder.


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