Universal switch

Introduction of complex regulatory circuits in mammalian cells requires the availability of large number of designable orthogonal switches. We provide this tool to the synthetic biology community by creating a new type of bistable toggle switch based on designable TAL regulators.

We created several TAL repressors, activators and their reporter plasmids with their corresponding DNA-binding sites and promoters. The TAL repressor constructs exhibited over 90% repression and TAL activator constructs enhanced expression over 1500 fold.

We tested five induction systems for mammalian cells and adapted three of them for driving TAL regulators in the switch.

We designed and modelled a mutual repressor toggle switch based on two TAL repressors repressing each other.

Experimental results demonstrated low stability of the basic mutual repressor switch, based on the classical toggle switch topology - this was confirmed by modeling analysis, which indicated high sensitivity to leaky gene expression and a requirement for high cooperativity of TAL repressors in order to exhibit bistable behavior.

We designed an improved toggle switch that included additional positive feedback loops based on orthogonal TAL repressors and activators.

Models demonstrated higher robustness in comparison to the mutual repressor switch and exhibited bistability even without cooperative binding to DNA, since the positive feedback loop introduces nonlinearity.

Experimental results demonstrated stable states at induction with corresponding inducers and a clear bimodal distribution of fluorescence. The switch remained in a stable state after removal of inducers, which confirmed the epigenetic bistability of our system.

Our project is based on production of biopharmaceuticals by engineered cells and with tight but versatile regulation and safety in mind, we set out to contribute some fundamental advances to synthetic biology.

We considered two options of regulating production of therapeutic proteins in mammalian cells:

• The first would be a prosthetic network where the cells are engineered to sense endogenous signals such as for example blood glucose levels. This signal would control the production of a therapeutic protein (such as insulin), which in turn affects the level of the endogenous signal (i.e. glucose) and is thus regulated by a kind of a feedback loop. This type of system can replace the function of a defective tissue or organ, hence the name prosthetic. Although a very attractive option, such a system would need to be tailor-made for a specific disease.
• For the second option we regarded a cellular genetic network that produces the therapeutic protein of choice, but can be controlled by an external signal such as small molecular drugs or metabolites which can be administered orally or topically. Instead of requiring a continuous presence of an activating or repressing signal, the system should function as a bistable or multistable switch, requiring only a short signal pulse to change into any selected state. This type of a toggle switch has already been implemented in mammalian cells based on prokaryotic transcription factors fused to the eukaryotic transactivator (eg. VP16, VP64) or transrepressor (eg. KRAB) domains (Carlsson et al., 2012).

We consulted several physicians concerning different potential therapeutic applications of such a delivery system. As examples of medical problems we considered hepatitis C infection, wound healing and cardiovascular ischemia.

Physicians preferred the ability of the device to be controlled by the therapist rather than autonomous switching by sensing internal signals. An additional advantage of a controlled switch is that a very similar device core could be used for different therapeutic applications.

Synthetic biology has already demonstrated the ability of a genetic device to assume many different states. However, while prokaryotic DNA-binding domains appear to be the simplest tool for device construction, their number is limited and currently does not allow scalability to construct very complex logical functions. On the other hand, modular protein DNA-binding domains are known that can be designed to bind to almost any DNA sequence, providing high orthogonality.