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Automatic Control of Gene Expression in Mammalian Cells

Research output: Contribution to journalArticle

  • Chiara Fracassi
  • Lorena Postiglione
  • Gianfranco Fiore
  • Diego di Bernardo
Original languageEnglish
Pages (from-to)296-302
Number of pages7
JournalACS Synthetic Biology
Volume5
Issue number4
DOIs
DatePublished - 28 Sep 2015

Abstract

Automatic control of gene expression in living cells is paramount importance to characterize both endogenous gene regulatory networks and synthetic circuits. In addition, such a technology can be used to maintain the expression of synthetic circuit components in an optimal range in order to ensure reliable performance. Here we present a microfluidics-based method to automatically control gene expression from the tetracycline-inducible promoter in mammalian cells in real time. Our approach is based on the negative-feedback control engineering paradigm. We validated our method in a monoclonal population of cells constitutively expressing a fluorescent reporter protein (d2EYFP) downstream of a minimal CMV promoter with seven tet-responsive operator motifs (CMV-TET). These cells also constitutively express the tetracycline transactivator protein (tTA). In cells grown in standard growth medium, tTA is able to bind the CMV-TET promoter, causing d2EYFP to be maximally expressed. Upon addition of tetracycline to the culture medium, tTA detaches from the CMV-TET promoter, thus preventing d2EYFP expression. We tested two different model-independent control algorithms (relay and proportional-integral (PI)) to force a monoclonal population of cells to express an intermediate level of d2EYFP equal to 50% of its maximum expression level for up to 3500 min. The control input is either tetracycline-rich or standard growth medium. We demonstrated that both the relay and PI controllers can regulate gene expression at the desired level, despite oscillations (dampened in the case of the PI controller) around the chosen set point.

    Research areas

  • Microfluidics, SYNTHETIC BIOLOGY, Control Theory, Fluorescence Microscopy, control engineering, gene expression, mammalian cell

    Structured keywords

  • Bristol BioDesign Institute

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    Rights statement: This is the accepted author manuscript (AAM). The final published version (version of record) is available online via American Chemical Society at http://dx.doi.org/10.1021/acssynbio.5b00141. Please refer to any applicable terms of use of the publisher.

    Accepted author manuscript, 252 KB, PDF document

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