Cybergenetic circuits to test composability of gene networks

A key property of engineered systems is their composability: the behavior of a complex network is computable from the wiring diagram and the known characteristics of its constituent parts. Despite of all the advances in systems and synthetic biology, it is unknown if genetic regulatory networks are at all composable yet the answer is fundamental for our ability to understand the behavior of evolved biological networks, as well as to engineer complex new ones. We have recently developed a unique experimental platform that permits image-based tracking of hundreds of cells in a precisely manipulated microfluidic environment, on-line analysis of the observed data, as well as optogenetic signalling to individual cells. This gives us unprecedented opportunities to perturb, observe and control gene networks in single cells, to study stochastic gene regulation dynamics, and most importantly, to implement real-time communication between a computer and single cells. We can therefore construct hybrid bio-digital circuits in which a part of the genetic network is effectively implemented as a biological network whereas another part exists only virtually in the form of a model in the computer. In this project, we will make use of these novel technological capacities to resolve or to circumvent what is arguably the most profound problem in quantitative biology: our failure to understand, or even just to predict, the dynamics of non-trivial biochemical processes in living cells and our resulting inability to rationally design complex synthetic circuits that reliably fulfill quantitative specifications. There are thus two major goals that we want to reach: to construct models that explain and predict better how composed circuits function in vivo and to construct synthetic circuits that function in vivo as previously specified.

We established that relative gene order on the chromosome plays an essential role in determining the phenotype that a gene regulatory network can implement. We also identified the main molecular mechanism responsible for this behavior to be transcriptional read-through. As each gene has to start to be read in the process of transcription, it also needs to stop and this is being achieved through transcriptional terminators. This is a pervasive mechanism in bacteria as most transcriptional terminators are not very strong and thus offers a potential function for weak transcriptional terminators. What does this mean in a larger biological context? It means that the relative physical order of genes on the chromosome represents an extra degree of freedom that biological systems can use to encode more phenotypes, i.e. implementing more phenotypes with fewer genes. These findings ought to be placed in a wider context of what motivated us to do this work in the first place. The overarching idea of the project was to study the general concept of composability in biological systems. As such, composability represents a very general property of human engineered systems, without which human progress in the industrial age would not have been possible. In a very general sense, a complex engineered system, such as an electronic circuit diagram, is 'computable' or understandable from the wiring diagram on which it is built and based on the known properties of its constituent components. At an even more basic level, if we take something like a brick for example, we know its properties very well (size, weight, sturdiness), and can intuitively (or computationally if we talk about static properties in architecture) predict what we can build with it and how, e.g. a house. More generally, any engineered system is endowed with such property, by the very fact that engineered systems by definition are bottom-up, namely they are built one component at a time. This is very different to the way we go about understanding the natural world around us, where we are forced to start from the top to the bottom, by poking natural systems for the existence of any building blocks, be they theoretical or experimental. Thus, looking at living systems through the lens of evolution, a certain degree of composability of gene regulatory networks is necessary in order for biological systems to be able to function despite the accumulation of mutations between organisms of a species and between species. It is unclear though how far the concept of composability as we know if from human engineering endeavors works across biological scales. Our project had as a general goal to explore these questions and limits.