Team:ETH Zurich/Decoder/Design

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[[File:DecoderCircuit.png|frameless|500px|right|thumb|''Fig. 1'': Circuit for Decoder.  ]]
[[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. Each output is controlled by a two-input-promoter that can be described by a NOR gate.
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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.
 +
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.
 +
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.
<br style="clear: both" />
<br style="clear: both" />
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== Dual input promoters ==
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== Hybrid promoters ==
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[[File:Promoterbothnativeethz2012.png|right| 300px|thumb|''Fig. 2'': Conserved regions of P<sub>R</sub> and P<sub>L</sub>.]]  
[[File:Promoterbothnativeethz2012.png|right| 300px|thumb|''Fig. 2'': Conserved regions of P<sub>R</sub> and P<sub>L</sub>.]]  
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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 NOR logics. Therefore we designed 3 different dual input promoters (all in all 4 variants), all of them derived from the λ-phage promoters P<sub>R</sub> and P<sub>L</sub>.
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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 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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=== P<sub>R</sub> derived promoter ===
 
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----
 
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The region upstream the -10 region of the P<sub>L</sub> derived promoters was conserved while the operator site of TetR or LacI was incorporated upstream to the -10 region.
 
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<br style="clear: both" /> [[File:Lacpror2012ethz.jpg|right| 350px|thumb|''Fig. 3'': P<sub>R</sub> repressible by LacI and cI]]  
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===P<sub>R</sub> derived promoter ===
 +
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>.
 +
 
 +
 
 +
<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]]
 +
 
=====P<sub>R</sub> with Lac operator sites=====
=====P<sub>R</sub> with Lac operator sites=====
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We decided to test two variants containing Lac operator composition with  
We decided to test two variants containing Lac operator composition with  
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*[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_K909012  Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and LacO<sub>1</sub>].
      
      
*[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>]  
*[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 =====
===== 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>1</sub>]
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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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[[File:Prtetoorethz2012.jpg|right| 350px|thumb|''Fig. 4'': P<sub>R</sub> repressible by TetR and cI]]
 
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=== P<sub>L</sub> derived promoter ===
=== P<sub>L</sub> derived promoter ===
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----
+
 
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[[File:Lactetplethz2012basel.png|frameless|350px|right|thumb|''Fig.5.'': P<sub>L</sub> promoter repressible by TetR and LacI.  ]]
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[[File:Plambdalefttetlac2012.png|frameless|350px|right|thumb|''Fig.4.'': P<sub>L</sub> promoter repressible by TetR and LacI.  ]]
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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.
+
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>1</sub> and LacO<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>2</sub> and LacO<sub>1</sub>]
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== Output: PABA ==
== Output: PABA ==
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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.
+
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.
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.
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.
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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>1</sub> and LacO<sub>1</sub>] to incorporate the ''pab'' genes into our system.
+
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:2012ethzpllacootetowithopaboperon.png|400 px|center| thumb |''Fig. 6'': Dual input promoter controling the ''pab'' genes and by that PABA expression ]]
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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




References

  • Brown, B. a, Headland, L. R., & Jenkins, G. I. (2009). UV-B action spectrum for UVR8-mediated HY5 transcript accumulation in Arabidopsis. Photochemistry and photobiology, 85(5), 1147–55.
  • Christie, J. M., Salomon, M., Nozue, K., Wada, M., & Briggs, W. R. (1999): LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proceedings of the National Academy of Sciences of the United States of America, 96(15), 8779–83.
  • Christie, J. M., Arvai, A. S., Baxter, K. J., Heilmann, M., Pratt, A. J., O’Hara, A., Kelly, S. M., et al. (2012). Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science (New York, N.Y.), 335(6075), 1492–6.
  • Cloix, C., & Jenkins, G. I. (2008). Interaction of the Arabidopsis UV-B-specific signaling component UVR8 with chromatin. Molecular plant, 1(1), 118–28.
  • Cox, R. S., Surette, M. G., & Elowitz, M. B. (2007). Programming gene expression with combinatorial promoters. Molecular systems biology, 3(145), 145. doi:10.1038/msb4100187
  • Drepper, T., Eggert, T., Circolone, F., Heck, A., Krauss, U., Guterl, J.-K., Wendorff, M., et al. (2007). Reporter proteins for in vivo fluorescence without oxygen. Nature biotechnology, 25(4), 443–5
  • Drepper, T., Krauss, U., & Berstenhorst, S. M. zu. (2011). Lights on and action! Controlling microbial gene expression by light. Applied microbiology, 23–40.
  • EuropeanCommission (2006). SCIENTIFIC COMMITTEE ON CONSUMER PRODUCTS SCCP Opinion on Biological effects of ultraviolet radiation relevant to health with particular reference to sunbeds for cosmetic purposes.
  • Elvidge, C. D., Keith, D. M., Tuttle, B. T., & Baugh, K. E. (2010). Spectral identification of lighting type and character. Sensors (Basel, Switzerland), 10(4), 3961–88.
  • GarciaOjalvo, J., Elowitz, M. B., & Strogatz, S. H. (2004). Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proceedings of the National Academy of Sciences of the United States of America, 101(30), 10955–60.
  • Gao Q, Garcia-Pichel F. (2011). Microbial ultraviolet sunscreens. Nat Rev Microbiol. 9(11):791-802.
  • Goosen N, Moolenaar GF. (2008) Repair of UV damage in bacteria. DNA Repair (Amst).7(3):353-79.
  • Heijde, M., & Ulm, R. (2012). UV-B photoreceptor-mediated signalling in plants. Trends in plant science, 17(4), 230–7.
  • Hirose, Y., Narikawa, R., Katayama, M., & Ikeuchi, M. (2010). Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter. Proceedings of the National Academy of Sciences of the United States of America, 107(19), 8854–9.
  • Hirose, Y., Shimada, T., Narikawa, R., Katayama, M., & Ikeuchi, M. (2008). Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proceedings of the National Academy of Sciences of the United States of America, 105(28), 9528–33.
  • Kast, Asif-Ullah & Hilvert (1996) Tetrahedron Lett. 37, 2691 - 2694., Kast, Asif-Ullah, Jiang & Hilvert (1996) Proc. Natl. Acad. Sci. USA 93, 5043 - 5048
  • Kiefer, J., Ebel, N., Schlücker, E., & Leipertz, A. (2010). Characterization of Escherichia coli suspensions using UV/Vis/NIR absorption spectroscopy. Analytical Methods, 9660. doi:10.1039/b9ay00185a
  • Kinkhabwala, A., & Guet, C. C. (2008). Uncovering cis regulatory codes using synthetic promoter shuffling. PloS one, 3(4), e2030.
  • Krebs in Deutschland 2005/2006. Häufigkeiten und Trends. 7. Auflage, 2010, Robert Koch-Institut (Hrsg) und die Gesellschaft der epidemiologischen Krebsregister in Deutschland e. V. (Hrsg). Berlin.
  • Lamparter, T., Michael, N., Mittmann, F., & Esteban, B. (2002). Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site. Proceedings of the National Academy of Sciences of the United States of America, 99(18), 11628–33.
  • Levskaya, A. et al (2005). Engineering Escherichia coli to see light. Nature, 438(7067), 442.
  • Mancinelli, A. (1986). Comparison of spectral properties of phytochromes from different preparations. Plant physiology, 82(4), 956–61.
  • Nakasone, Y., Ono, T., Ishii, A., Masuda, S., & Terazima, M. (2007). Transient dimerization and conformational change of a BLUF protein: YcgF. Journal of the American Chemical Society, 129(22), 7028–35.
  • Orth, P., & Schnappinger, D. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature structural biology, 215–219.
  • Parkin, D.M., et al., Global cancer statistics, 2002. CA: a cancer journal for clinicians, 2005. 55(2): p. 74-108.
  • Rajagopal, S., Key, J. M., Purcell, E. B., Boerema, D. J., & Moffat, K. (2004). Purification and initial characterization of a putative blue light-regulated phosphodiesterase from Escherichia coli. Photochemistry and photobiology, 80(3), 542–7.
  • Rizzini, L., Favory, J.-J., Cloix, C., Faggionato, D., O’Hara, A., Kaiserli, E., Baumeister, R., et al. (2011). Perception of UV-B by the Arabidopsis UVR8 protein. Science (New York, N.Y.), 332(6025), 103–6.
  • Roux, B., & Walsh, C. T. (1992). p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA. Biochemistry, 31(30), 6904–10.
  • Strickland, D. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences of the United States of America, 105(31), 10709–10714.
  • Sinha RP, Häder DP. UV-induced DNA damage and repair: a review. Photochem Photobiol Sci. (2002). 1(4):225-36
  • Sambandan DR, Ratner D. (2011). Sunscreens: an overview and update. J Am Acad Dermatol. 2011 Apr;64(4):748-58.
  • Tabor, J. J., Levskaya, A., & Voigt, C. A. (2011). Multichromatic Control of Gene Expression in Escherichia coli. Journal of Molecular Biology, 405(2), 315–324.
  • Thibodeaux, G., & Cowmeadow, R. (2009). A tetracycline repressor-based mammalian two-hybrid system to detect protein–protein interactions in vivo. Analytical biochemistry, 386(1), 129–131.
  • Tschowri, N., & Busse, S. (2009). The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli. Genes & development, 522–534.
  • Tschowri, N., Lindenberg, S., & Hengge, R. (2012). Molecular function and potential evolution of the biofilm-modulating blue light-signalling pathway of Escherichia coli. Molecular microbiology.
  • Tyagi, A. (2009). Photodynamics of a flavin based blue-light regulated phosphodiesterase protein and its photoreceptor BLUF domain.
  • Vainio, H. & Bianchini, F. (2001). IARC Handbooks of Cancer Prevention: Volume 5: Sunscreens. Oxford University Press, USA
  • Quinlivan, Eoin P & Roje, Sanja & Basset, Gilles & Shachar-Hill, Yair & Gregory, Jesse F & Hanson, Andrew D. (2003). The folate precursor p-aminobenzoate is reversibly converted to its glucose ester in the plant cytosol. The Journal of biological chemistry, 278.
  • van Thor, J. J., Borucki, B., Crielaard, W., Otto, H., Lamparter, T., Hughes, J., Hellingwerf, K. J., et al. (2001). Light-induced proton release and proton uptake reactions in the cyanobacterial phytochrome Cph1. Biochemistry, 40(38), 11460–71.
  • Wegkamp A, van Oorschot W, de Vos WM, Smid EJ. (2007 )Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis. Appl Environ Microbiol. Apr;73(8):2673-81.
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