BioSysBio:abstracts/2007/Guillermo Rodrigo

iGEM 2006 Valencia: EcoliTaster

Author(s): G. Rodrigo¹, P. Tortosa³, A. Aparici², MC. Aroca², J. Carrera¹, C. Edo^1,2, 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

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

Synthetic two-component signal transduction pathway used by Hellinga	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.
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.
AND promoter's activity (Bintu et al. 2005) versus [CRP] and [lam-cI]	Details of the AND promoter construction (Joung et al. 1994)

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).