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Regulation of Gene Expression and Signaling Pathway Activity in Mammalian Cells by Automated Microfluidics Feedback Control

Postiglione, Lorena; Napolitano, Sara; Pedone, Elisa; Rocca, Daniel L.; Aulicino, Francesco; Santorelli, Marco; Tumaini, Barbara; Marucci, Lucia; di Bernardo, Diego


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            <foaf:name>Department of Engineering Mathematics, University of Bristol, Bristol BS8 1UB, U.K. School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1UB, U.K.</foaf:name>
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            <foaf:name>Department of Engineering Mathematics, University of Bristol, Bristol BS8 1UB, U.K. School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1UB, U.K. BrisSynBio, Bristol BS8 1TQ, U.K.</foaf:name>
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            <foaf:name>Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy Department of Chemical, Materials and Industrial Engineering, University of Naples Federico II, Piazzale V. Tecchio 80, 80125 Naples, Italy</foaf:name>
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    <dct:description>&lt;p&gt;Abstract&lt;/p&gt; &lt;p&gt;Gene networks and signaling pathways display complex topologies and, as a result, complex nonlinear behaviors. Accumulating evidence shows that both static (concentration) and dynamical (rate-of-change) features of transcription factors, ligands and environmental stimuli control downstream processes and ultimately cellular functions. Currently, however, methods to generate stimuli with the desired features to probe cell response are still lacking. Here, combining tools from Control Engineering and Synthetic Biology (cybergenetics), we propose a simple and cost-effective microfluidics-based platform to precisely regulate gene expression and signaling pathway activity in mammalian cells by means of real-time feedback control. We show that this platform allows (i) to automatically regulate gene expression from inducible promoters in different cell types, including mouse embryonic stem cells; (ii) to precisely regulate the activity of the mTOR signaling pathway in single cells; (iii) to build a biohybrid oscillator in single embryonic stem cells by interfacing biological parts with virtual&amp;nbsp;&lt;em&gt;in silico&lt;/em&gt;&amp;nbsp;counterparts. Ultimately, this platform can be used to probe gene networks and signaling pathways to understand how they process static and dynamic features of specific stimuli, as well as for the rapid prototyping of synthetic circuits for biotechnology and biomedical purposes.&lt;/p&gt;</dct:description>
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