BioSysBio:abstracts/2007/Guillermo Rodrigo: Difference between revisions
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two transcription factors: cI and CRP [3]. In addition, this promoter can be | two transcription factors: cI and CRP [3]. In addition, this promoter can be | ||
understood as an AND logic gate (see promoter behavior in Fig. 4, and see its | understood as an AND logic gate (see promoter behavior in Fig. 4, and see its | ||
construction in Fig. 5). | construction in Fig. 5). | ||
==Results== | ==Results== | ||
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==Images/Tables== | ==Images/Tables== | ||
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|align="left"| [[Image:iGEM_Valencia_image001.png |thumb| 300px|Synthetic two-component signal transduction pathway used by Hellinga ]] | |||
| [[Image:iGEM_Valencia_image003.png |thumb| 200px|3D model of redesigned RBP binding to the target compound (vanillin). H-bonds are in dashed lines. Design made by Pablo Tortosa and Alfonso Jaramillo for iGEM2006 Valencia.]] | |||
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| [[Image:iGEM_Valencia_image005.png |thumb| 400px|Circuit design. When our redesigned rbp binds vanillin, the complex will bind trg that will cause phosphorylation of OmpR at the EnvZ domain (we use a chimeric trg-EnvZ fusion), which will regulate several designed promoters. We use an AND promoter. We don't expect (we hope) that for high-copy number plasmids the use of the promiscuous CRP will cause much trouble.]]<br><br> | |||
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| [[Image:iGEM_Valencia_image007.png |thumb| 200px|AND promoter's activity (Bintu et al. 2005) versus [CRP] and [lam-cI]]] | |||
| [[Image:iGEM_Valencia_image009.png |thumb| 300px|Details of the AND promoter construction (Joung et al. 1994)]] | |||
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==Materials/Methods== | ==Materials/Methods== |
Revision as of 08:50, 29 September 2006
iGEM 2006 Valencia: EcoliTaster
Author(s): G. Rodrigo1, P. Tortosa3, A.
Aparici2, MC. Aroca2, J. Carrera1, C. Edo1,2, G. Fuertes2, D. Jiménez2, C. Mata2, JV. Medrano2, A.
Montagud2, C. Navarrete2, E. Navarro1, M. Báguena1, P. Fernández de Córdoba1, A.
Ferrando2, J. Salgado2, J. Urchueguía1,A. Jaramillo3
Affiliations: 1Universidad Politecnica Valencia, Spain
- 2Universidad de Valencia, Spain
- 3Ecole Polytechnique, France
- 2Universidad de Valencia, Spain
Contact: http://www.enseignement.polytechnique.fr/profs/biochimie/Alfonso.Jaramillo/
Keywords: 'iGEM' 'Synthetic Biology' 'Computational Protein Design'
Background/Introduction
Our project for this iGEM edition in 2006 [1] is making a cellular biosensor. We use E. coli as cellular chassis, using a deficient EnvZ strain. We construct two different modules in order to assemble it: on the one hand, sensor devices, and on the other, actuators. Firstly, we use membrane proteins to perform the sensing function, inspired on Hellinga’s work sensing TNT and other molecules using a mutated periplasmic binding protein (PBP) [2]. Thus, our team thought in building a PBP that docks a vanillin molecule. It performs an allosteric motion that makes it binding to the trg protein (Fig. 1). When the PBP-vanillin complex (Fig. 2) binds trg, then an allosteric motion is propagated to the EnvZ kinase domain resulting in autophosphorylation and phosphate transfer to OmpR transcription factor (OmpR-P). Secondly, we use a genetic synthetic network as actuator that at high input levels has a given fluorescent response and at low levels other (Fig. 3). Therefore, for intermediate levels there is a gradient if we superpose the colors. To get that behavior, we construct two branches with different strengths, and we use a synthetic promoter activated by two transcription factors: cI and CRP [3]. In addition, this promoter can be understood as an AND logic gate (see promoter behavior in Fig. 4, and see its construction in Fig. 5).
Results
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Images/Tables
Materials/Methods
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Conclusion
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References
[1] http://parts.mit.edu/wiki/index.php/UPV-UV_Valencia%2C_Spain_2006
[2] L.L. Looger, M.A. Dwyer, J. Smith, and H.W. Hellinga. Computational design of receptor and sensor proteins with novel functions. Nature, 423, 185-190 (2003).
[3] J.K. Joung, D.M. Koepp, and A. Hochschild. Synergistic activation of transcription by bacteriophage lambda cI protein and E. coli cAMP receptor protein. Science, 265, 1863-1866 (1994).