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EnglishFine 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.
| Titolo: | A Synthetic Switch for Gene Expression Control in E. Coli | |
| Autore/i: | CERONI, FRANCESCA; PASINI, ALICE; GIORDANO, EMANUELE DOMENICO; CAVALCANTI, SILVIO | |
| Autore/i Unibo: | ||
| Anno: | 2008 | |
| Titolo del libro: | Advances in Synthetic Biology | |
| Pagina iniziale: | 7 | |
| Pagina finale: | 7 | |
| 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. | |
| Data prodotto definitivo in UGOV: | 23-nov-2008 | |
| Appare nelle tipologie: | 4.02 Riassunto (Abstract) |
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