Team:ETH Zurich/Decoder/Design

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=== Circuit Design ===
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== Circuit Design ==
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The presence of blue or red light leads to the activation of LovTAP and to the deactivation of Cph8, respectively. As a result the expression of lacI and tetR being controlled by the tryptophan repressor promoter and the red light promoter is terminated.  
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[[File:DecoderCircuit.png|frameless|500px|right|thumb|''Fig. 1'': Circuit for Decoder.  ]]
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The presence of blue or red light leads to the activation of LovTAP and to the deactivation of Cph8, respectively. As a result the expression of lacI and tetR being controlled by the tryptophan repressor promoter and the red light promoter is terminated.
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=== Dual input promoters ===
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The intermediate NOR gate represented by P<sub>L TetO/LacO</sub> is only active in the absence of TetR and LacI and controls ''pabAB'', the formation of the violet pigment and cI. cI ensures that the other ouputs are repressed.
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In our case we want the decoder ([https://2012.igem.org/Team:ETH_Zurich/Modeling/Construct1 construct 1] [https://2012.igem.org/Team:ETH_Zurich/Modeling/Construct2 construct 2])  to have NOT logics. Therefore we designed 3 different dual input promoters, all of them derived from the λ-phage promoters P<sub>R</sub> and P<sub>L</sub>:
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For the two cases where only one light-input is present merely one hybrid promoter is not repressed leading to the formation of the red or green pigment.
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1. a) [http://partsregistry.org/Part:BBa_K909012  Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and LacO<sub>1</sub>]
 
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    b)  [http://partsregistry.org/Part:BBa_K909016  Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and LacO<sub>1</sub>/]O<sub>2</sub>
 
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2. [http://partsregistry.org/Part:BBa_K909013 Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and TetO<sub>1</sub>]
 
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3. [http://partsregistry.org/Part:BBa_K909011 Modified P<sub>L</sub> promoter with operator sites TetO<sub>1</sub> and LacO<sub>1</sub>]
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<br style="clear: both" />
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== Hybrid promoters ==
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[[File: Promotersethzdecoder.jpg|center| 500px|thumb|''Figure'': .]]
 
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[[File:Promoterbothnativeethz2012.png|right| 300px|thumb|''Fig. 2'': Conserved regions of P<sub>R</sub> and P<sub>L</sub>.]]
 +
In our case we want the decoder ([https://2012.igem.org/Team:ETH_Zurich/Modeling/Construct1 construct 1] [https://2012.igem.org/Team:ETH_Zurich/Modeling/Construct2 construct 2])  to have NOR logics. Therefore we designed 3 different hybrid promoters (all in all 4 variants), all of them derived from the λ-phage promoters P<sub>R</sub> and P<sub>L</sub> <span class='eth_reference'>[Cox2007]</span>: <partinfo>BBa_K909011</partinfo>, <partinfo>BBa_K909012</partinfo>, <partinfo>BBa_K909013</partinfo> and <partinfo>BBa_K909016</partinfo>.
 +
Each hybrid promoter is repressible by two of the following three transcriptional repressors LacI, cI and TetR. Only in case neither of the two repressor is present, transcription of genes underlying the control of the hybrid promoter can start.
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=== Output: p-ABA ===
 
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We constructed one  [http://partsregistry.org/Part:BBa_K909015 ''pab'' operon] consisting of the ''pabA'' and ''pabB/C'' gene from ''Lactococcus lactis'' to overexpress p-ABA.
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===P<sub>R</sub> derived promoter ===
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The region upstream the -10 region of the P<sub>R</sub> derived promoters was conserved while the operator site of TetR or LacI was incorporated upstream to the -10 region <span class='eth_reference'>[Kinkhabwala2008]</span>.
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 +
 
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<br style="clear: both" /> [[File:Prprprprprprp2012ethz.png|right| 350px|thumb|''Fig. 3'': Top:  P<sub>R</sub> repressible by LacI and cI. Bottom: P<sub>R</sub> repressible by TetR and cI]]
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=====P<sub>R</sub> with Lac operator sites=====
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We decided to test two variants containing Lac operator composition with
 +
*[http://partsregistry.org/Part:BBa_K909012  Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and LacO<sub>1</sub>].
 +
   
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*[http://partsregistry.org/Part:BBa_K909016  Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and LacO<sub>1</sub>/O<sub>2</sub>]
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===== P<sub>R</sub> with Tet operator sites =====
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[http://partsregistry.org/Part:BBa_K909013 Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and TetO<sub>2</sub>]
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 +
 
 +
 
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 +
 
 +
 
 +
 
 +
 
 +
 
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 +
 
 +
 
 +
 
 +
 
 +
 
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=== P<sub>L</sub> derived promoter ===
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[[File:Plambdalefttetlac2012.png|frameless|350px|right|thumb|''Fig.4.'': P<sub>L</sub> promoter repressible by TetR and LacI.  ]]
 +
 
 +
 
 +
 
 +
The -35 and -10 regions of the P<sub>L</sub> derived promoters were conserved while the operator sites of TetR was incorporated between the -35 and  -10 region and the LacI binding sites downstream the -10 region. We decided to test two variants of that specific promoter. They differ only on the Lac operator composition.
 +
 
 +
3. [http://partsregistry.org/Part:BBa_K909011 Modified P<sub>L</sub> promoter with operator sites TetO<sub>2</sub> and LacO<sub>1</sub>]
 +
 
 +
 
 +
 
 +
 
 +
== Output: PABA ==
 +
 
 +
We constructed one  [http://partsregistry.org/Part:BBa_K909015 ''pab'' operon] consisting of the ''pabA'' and ''pabB/C'' gene from ''Lactococcus lactis'' to overexpress PABA.
Therefore we used the biobrick parts [http://partsregistry.org/Part:BBa_K137055 BBa_K137055] and [http://partsregistry.org/Part:BBa_S04039 BBa_S04039] as a template to construct the wished composites.
Therefore we used the biobrick parts [http://partsregistry.org/Part:BBa_K137055 BBa_K137055] and [http://partsregistry.org/Part:BBa_S04039 BBa_S04039] as a template to construct the wished composites.
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Biobrick [http://partsregistry.org/Part:BBa_K909014  K909014] will be used to show that p-ABA is produced, while the [http://partsregistry.org/Part:BBa_K909015 operon] can be cloned downstream to any promoter.
 
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In a second step the pab operon will be cloned downstream of the [http://partsregistry.org/Part:BBa_K909011  P<sub>L</sub> promoter with operator sites TetO<sub>1</sub> and LacO<sub>1</sub>]. In case blue and red light is present, p-ABA will be produced as a blue and red light response.
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Biobrick [http://partsregistry.org/Part:BBa_K909014  BBa_K909014] will be used to show that PABA is produced. In that case  the ''pab'' operon is controled by the constitutive promoter [http://partsregistry.org/Part:BBa_J23100  J23100] while the [http://partsregistry.org/Part:BBa_K909015 BBa_K909015 operon] can be cloned downstream to any promoter.
 +
We will clone the ladder operon downstream of the [http://partsregistry.org/Part:BBa_K909011  P<sub>L</sub> promoter with operator sites TetO<sub>2</sub> and LacO<sub>1</sub>] to incorporate the ''pab'' genes into our system.
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[[File:Eth2012pabagoal.jpg|500 px|center]]
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[[File:Pabagenes2012ethzbasellast.png|400 px|center| thumb |''Fig. 5'': Hybrid promoter controling the ''pab'' genes and by that PABA expression ]]

Latest revision as of 01:10, 27 September 2012

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Navigate through Overview, Design and Results

Contents

Circuit Design

Fig. 1: Circuit for Decoder.

The presence of blue or red light leads to the activation of LovTAP and to the deactivation of Cph8, respectively. As a result the expression of lacI and tetR being controlled by the tryptophan repressor promoter and the red light promoter is terminated.

The intermediate NOR gate represented by PL TetO/LacO is only active in the absence of TetR and LacI and controls pabAB, the formation of the violet pigment and cI. cI ensures that the other ouputs are repressed.

For the two cases where only one light-input is present merely one hybrid promoter is not repressed leading to the formation of the red or green pigment.



Hybrid promoters

Fig. 2: Conserved regions of PR and PL.

In our case we want the decoder (construct 1 construct 2) to have NOR logics. Therefore we designed 3 different hybrid promoters (all in all 4 variants), all of them derived from the λ-phage promoters PR and PL [Cox2007]: <partinfo>BBa_K909011</partinfo>, <partinfo>BBa_K909012</partinfo>, <partinfo>BBa_K909013</partinfo> and <partinfo>BBa_K909016</partinfo>. Each hybrid promoter is repressible by two of the following three transcriptional repressors LacI, cI and TetR. Only in case neither of the two repressor is present, transcription of genes underlying the control of the hybrid promoter can start.


PR derived promoter

The region upstream the -10 region of the PR derived promoters was conserved while the operator site of TetR or LacI was incorporated upstream to the -10 region [Kinkhabwala2008].



Fig. 3: Top: PR repressible by LacI and cI. Bottom: PR repressible by TetR and cI
PR with Lac operator sites

We decided to test two variants containing Lac operator composition with

  • [http://partsregistry.org/Part:BBa_K909012 Modified PR promoter with operator sites OR1,OR2 and LacO1].
  • [http://partsregistry.org/Part:BBa_K909016 Modified PR promoter with operator sites OR1,OR2 and LacO1/O2]
PR with Tet operator sites

[http://partsregistry.org/Part:BBa_K909013 Modified PR promoter with operator sites OR1,OR2 and TetO2]








PL derived promoter

Fig.4.: PL promoter repressible by TetR and LacI.


The -35 and -10 regions of the PL derived promoters were conserved while the operator sites of TetR was incorporated between the -35 and -10 region and the LacI binding sites downstream the -10 region. We decided to test two variants of that specific promoter. They differ only on the Lac operator composition.

3. [http://partsregistry.org/Part:BBa_K909011 Modified PL promoter with operator sites TetO2 and LacO1]



Output: PABA

We constructed one [http://partsregistry.org/Part:BBa_K909015 pab operon] consisting of the pabA and pabB/C gene from Lactococcus lactis to overexpress PABA. Therefore we used the biobrick parts [http://partsregistry.org/Part:BBa_K137055 BBa_K137055] and [http://partsregistry.org/Part:BBa_S04039 BBa_S04039] as a template to construct the wished composites.


Biobrick [http://partsregistry.org/Part:BBa_K909014 BBa_K909014] will be used to show that PABA is produced. In that case the pab operon is controled by the constitutive promoter [http://partsregistry.org/Part:BBa_J23100 J23100] while the [http://partsregistry.org/Part:BBa_K909015 BBa_K909015 operon] can be cloned downstream to any promoter. We will clone the ladder operon downstream of the [http://partsregistry.org/Part:BBa_K909011 PL promoter with operator sites TetO2 and LacO1] to incorporate the pab genes into our system.

Fig. 5: Hybrid promoter controling the pab genes and by that PABA expression




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