A tunable dual-input system for on-demand dynamic gene expression regulation
Dynamic gene expression patterns orchestrate cellular functions. To understand the link between dynamic behaviour and cellular phenotype, and to direct cell fate in a user-defined way, tools enabling precise tuning of gene activation levels and temporal dynamics were developed.
Cells continuously process information to perform their functions and adapt to changes; triggered by extracellular molecules, such information is transduced to the intracellular compartments by activating a sequence of chemical reactions. Information processing is indeed performed at multiple levels through control of both the mRNA and protein abundance of a gene, but also of the intracellular protein localisation, and mRNA and protein stability.
Proteins are key elements of cellular functions; they are produced from a copy of specific sequences of the genome called messenger RNA (mRNA). The process of mRNA synthesis is known as transcription, whereas the process of protein production is known as translation.
The half-life of a protein can vary depending on its function: some proteins are rapidly degraded, and others are highly stable. The transcriptional rate and the stability of the mRNA molecule sometimes compensate for protein stability; for example, the mRNA encoding for ‘unstable’ proteins can be constantly transcribed or slowly degraded, and vice versa.
The interplay between different mRNA/protein production and turnover dynamics, together with feedback regulations (i.e., a gene can regulate another gene that, in turns, regulates it), fine-tune protein abundance and spatiotemporal patterning, which guarantee prompt and adaptive cellular responses.
Synthetic Biology aims at the forward engineering of these processes: programmable control of gene expression can unambiguously explain gene function both in vivoand in vitroand, ultimately, allow the direct engineering of specific cellular behaviours.
For example, inducible systems such as TET-On/Off permit precise and reversible regulation of transcription (i.e., turning the mRNA of a gene on or off) with drugs. However, these systems do not allow precise temporal control (e.g., fast switch on/off) in case of stable proteins, reducing the possibility to generate more complex dynamics similar to those observed in vivo.
To overcome these limitations, we designed a dual-input inducible system that allows fine-tuning of the amount and dynamics of a gene of interest by simultaneous regulation of transcriptional rate and protein stability.
Our dual-input system is based on two modules. The first module is an inducible promoter (i.e., TET-on) that drives the transcription of a gene of interest in response to an antibiotic (i.e., doxycycline); the second module consists of a destabilising domain (DD) which increases the degradation of the transcribed and translated protein unless the cells are exposed to another drug (the protein stabiliser Trimethoprim, TMP). We show that the adequate combination of inducer molecules doxycycline and TMP permits improved control of expression dynamic range andprotein dynamics across different culture platforms, from petri dishes to microfluidic devices. Moreover, we demonstrated, both with experiments and mathematical modelling, that our system is suitable for regulating a variety of protein regardless of their molecular size and stability.
We provided a novel combined system for controllable and predictable perturbation of a gene of interest, which could be used later used also in vivo. Indeed, both doxycycline and TMP can cross the placental barrier and therefore our approach could be applied for targeted and modulable gene therapy in vivo.
This interdisciplinary research, carried out at the University of Bristol in the Engineering Mathematics Department, the School of Cellular and Molecular Medicine and the Bristol Centre for Synthetic Biology (BrisSynSBio), was funded by BBSRC, EPSRC and MRC, and involved an international collaboration with the Telethon Institute for Genetics and Medicine (Italy) and the Centre for Genomic Regulation (Spain).