Construction and Modelling of an Inducible Positive Feedback Loop Stably Integrated in a Mammalian Cell-Line

Velia Siciliano, Filippo Menolascina, Lucia Marucci, Chiara Fracassi, Immacolata Garzilli, Maria Nicoletta Moretti and Diego di Bernardo

PLOS Computational Biology, 2011, vol. 7, issue 6, 1-11

Abstract: Understanding the relationship between topology and dynamics of transcriptional regulatory networks in mammalian cells is essential to elucidate the biology of complex regulatory and signaling pathways. Here, we characterised, via a synthetic biology approach, a transcriptional positive feedback loop (PFL) by generating a clonal population of mammalian cells (CHO) carrying a stable integration of the construct. The PFL network consists of the Tetracycline-controlled transactivator (tTA), whose expression is regulated by a tTA responsive promoter (CMV-TET), thus giving rise to a positive feedback. The same CMV-TET promoter drives also the expression of a destabilised yellow fluorescent protein (d2EYFP), thus the dynamic behaviour can be followed by time-lapse microscopy. The PFL network was compared to an engineered version of the network lacking the positive feedback loop (NOPFL), by expressing the tTA mRNA from a constitutive promoter. Doxycycline was used to repress tTA activation (switch off), and the resulting changes in fluorescence intensity for both the PFL and NOPFL networks were followed for up to 43 h. We observed a striking difference in the dynamics of the PFL and NOPFL networks. Using non-linear dynamical models, able to recapitulate experimental observations, we demonstrated a link between network topology and network dynamics. Namely, transcriptional positive autoregulation can significantly slow down the “switch off” times, as comparared to the nonautoregulatated system. Doxycycline concentration can modulate the response times of the PFL, whereas the NOPFL always switches off with the same dynamics. Moreover, the PFL can exhibit bistability for a range of Doxycycline concentrations. Since the PFL motif is often found in naturally occurring transcriptional and signaling pathways, we believe our work can be instrumental to characterise their behaviour. Author Summary: Synthetic Biology aims at designing and building new biological functions in living organisms. At the same time, Synthetic Biology approaches can be used to uncover the design principles of natural biological systems through the rational construction of simplified regulatory networks. Mathematical models of the networks are then derived from physical considerations and can be used to explain the observed dynamical behaviours. We have characterised a regulatory motif often found in transcriptional and signalling pathways. We constructed a positive feedback loop motif in mammalian cells, consisting of a protein controlling its own expression. We have shown that this motif exhibits a dynamic behaviour which is very different from that obtained when the autoregulation is removed. This difference is intrinsic to the specific wiring diagram chosen by the cell to control its behaviour (feedback versus non-feedback configurations), and can be instrumental in understanding the complex network of regulation occurring in a cell.

Date: 2011
References: View references in EconPapers View complete reference list from CitEc
Citations:

Downloads: (external link)
https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002074 (text/html)
https://journals.plos.org/ploscompbiol/article/fil ... 02074&type=printable (application/pdf)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:plo:pcbi00:1002074

DOI: 10.1371/journal.pcbi.1002074

Access Statistics for this article

More articles in PLOS Computational Biology from Public Library of Science
Bibliographic data for series maintained by ploscompbiol ().