Fine tuning of synthetic gene expression systems is expected to improve their applications. In this perspective, BioBricks from the iGEM registry were used to implement a switch with a fast OFF to ON transition -when the control signal crosses the activation threshold- because of a positive feedback mechanism. In E. coli a molecular circuit controls with a positive feedback (LacY gene expression) the Lac operon activation for a fast switch from glucose to lactose metabolism. Thus, a recombinant circuit with similar properties was reproduced by inserting LacY (J22101) and GFP (J04631) downstream of the Lac promoter (R0010) in a high copy number plasmid (pSB1AK3), together with LacI repressor (S0100) under the control of Tet promoter (R0051). DH5alpha E. Coli strain - which lacks the genomic Lac operon- was engineered with the circuit. Bacteria were first characterized in an open-loop condition by using the simple switch pTet- LacI- pLac-GFP. As expected, fluorescence intensity increases exponentially after IPTG induction, and decreases after IPTG removal. A mathematical model of the circuit was implemented in Simulink and bistability, as well as hysteresis properties, were observed. The validation of the complete close-loop circuit is in progress.
A Synthetic Switch for Gene Expression Control in E. Coli
CERONI, FRANCESCA;PASINI, ALICE;GIORDANO, EMANUELE DOMENICO;CAVALCANTI, SILVIO
2008
Abstract
Fine tuning of synthetic gene expression systems is expected to improve their applications. In this perspective, BioBricks from the iGEM registry were used to implement a switch with a fast OFF to ON transition -when the control signal crosses the activation threshold- because of a positive feedback mechanism. In E. coli a molecular circuit controls with a positive feedback (LacY gene expression) the Lac operon activation for a fast switch from glucose to lactose metabolism. Thus, a recombinant circuit with similar properties was reproduced by inserting LacY (J22101) and GFP (J04631) downstream of the Lac promoter (R0010) in a high copy number plasmid (pSB1AK3), together with LacI repressor (S0100) under the control of Tet promoter (R0051). DH5alpha E. Coli strain - which lacks the genomic Lac operon- was engineered with the circuit. Bacteria were first characterized in an open-loop condition by using the simple switch pTet- LacI- pLac-GFP. As expected, fluorescence intensity increases exponentially after IPTG induction, and decreases after IPTG removal. A mathematical model of the circuit was implemented in Simulink and bistability, as well as hysteresis properties, were observed. The validation of the complete close-loop circuit is in progress.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
