Positive feedback loop switch in mammalian cells based on noncooperative designed DNA binding domains

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Tina Lebar, Urban Bezeljak, Anja Golob, Miha Jerala, Lucija Kadunc, Botjan Pir, Maja Somrak, Martin Straar, Uro Zupani, Mojca Benina, Vida Forstneri, Rok Gaber, Jan Lonzari, Andreja Majerle, Alja Oblak, Ane Smole, Roman Jerala

National Institute of Chemistry, Slovenia

Introduction of complex regulatory circuits into mammalian cells requires the availability of several designable orthogonal switches. While prokaryotic regulators appear to be an efficient tool for construction of genetic devices in mammalian cells, their number is limited and their properties differ, which does not allow direct scalability required for the construction of complex logical functions. Modular DNA-binding protein domains such as zinc finger proteins or TAL effectors can be designed to bind almost any DNA sequence, providing high orthogonality. An orthogonal switch based on such designable elements would allow simultaneous introduction of several multistable switches into mammalian cells for an advanced level of regulation in different applications. Epigenetic toggle switches have been constructed based on bacterial transcription factors. Here we present a bistable toggle switch based on designable and freely scalable elements. The functional core of our device is composed of TAL effector-based transcriptional regulators. Simple wiring of mutual repressors as used in previous genetic toggle switches did not result in a functional switch due to the lack of cooperativity as demonstrated also by modeling. Functional switch was prepared by the addition of two positive feedback loops in a switch combining a pair of mutual repressors and a pair of activators that compete for the same operator sequence. This switch is robust and many different orthogonal switches can function in the complex system of mammalian cells. Switches can be used as the basic building blocks of memory cells, analogous to electronic components. A set of orthogonal bistable switches could be used to build biological memory of significantly higher complexity than previous attempts.