Synthetic Biology Interdisciplinary Challenge 3
Reconstructing gene circuitry: How can synthetic biology lead us to an understanding of the principles underlying natural genetic circuits and to the discovery of new biology?

Challenge Summary
Genetic circuits have traditionally been studied using genetics and biochemistry.  These studies underpin our current understanding of the regulatory wiring diagrams of organisms. They have also revealed that biological components like regulatory elements in DNA, genes, and proteins are intrinsically modular in nature. However, even when we believe we know the list of circuit components and their interactions, this knowledge often fails to explain/recapitulate the mechanism of the circuit. What is missing from these circuit diagrams? How can we infer those missing components if they have not been revealed by traditional experimentation? How can we test what parts of a given circuit are sufficient for a particular behavior?  How different are potential circuit designs, that we imagine, from the actual circuit designs that have evolved to solve biological problems?

Due to an enormous expansion in our knowledge about genetic components and interactions in a number of model systems, we are now in a position to pursue a complementary approach to understanding natural gene circuits, based on reconstruction of genetic circuits. Specifically, we can engineer synthetic genetic circuits out of well-characterized genetic components and analyze their behavior in cells and organisms.  These circuits can be based on their natural counterparts or on theories of how natural processes might work. Equally important, they can be engineered to operate as independently as possible from the corresponding endogenous cellular circuits. Circuits can also be created by ‘rewiring’ existing circuits (adding, deleting, or changing regulatory connections).  The goals of studying such reconstructed genetic circuits are to understand how different aspects of circuit architecture contribute to function, to determine what functional tradeoffs are inherent in the design of the circuit, and to establish the sufficiency of particular circuit designs for given biological functions.  More generally, they provide a complementary path to identifying both particular circuit interactions and general principles of gene circuit operation.

A reconstructive approach to genetic circuits may allow us to design circuits with unique properties and may provide insight into their underlying mechanisms. With a synthetic approach, it may be possible to construct a replica of a particular natural genetic circuit out of well-understood components and monitor its exact function in living cells. Using a synthetic approach, we could test the sufficiency of an arbitrary circuit made up of well-characterized components for generating a particular function. A major advantage to this approach is that we may be able to study the circuit mechanism without impairing cellular functions or inducing downstream consequences which are often drawbacks of traditional perturbation approaches. Finally, different circuit designs with similar functions can be directly compared to determine the precise properties each design grants a network as well as their relative advantages and disadvantages in particular cellular contexts. Ultimately, these studies may provide us with a deep enough understanding that we can design circuits that perform novel biological functions and we can exploit synthetic circuitry to reveal basic principles about natural circuit design.

Nonetheless, the synthetic approach faces many obstacles.  For example, while we often know the components in a circuit, we frequently do not have vivo information regarding kinetic parameters (affinities, binding and degradation rates, etc.). How can we infer these values if we cannot or have not measured them directly? Additionally, the intracellular environment is intrinsically “noisy,” and small copy numbers of molecular species limit the predictability of biochemical reactions.  How can we interpret or predict circuit functions in the face of such noise?  Can we devise synthetic circuits that suppress such noise to operate reliably, or take advantage of such noise to enable probabilistic cellular behaviors?

Key Questions

• What are the major advantages and limitations of synthetic circuits as a means of understanding the principles of genetic circuit design?
  
• How do we determine the basic principles underlying which circuit architectures can generate particular functions in cells and organisms?
• How do we identify missing components from natural circuits if they have not been revealed by traditional experimentation? How can we infer in vivo kinetic values if we cannot or have not measured them directly?
• To what extent can we analyze genetic circuits without comprehensive knowledge of all components and interactions? 
• How can we evaluate how a circuit operates in the context of a complete organism?
• What new challenges and opportunities do particular classes of circuits present?  In particular, what can synthetic biology do to better understand probabilistic behaviors, developmental circuits, neural circuits, immune circuits, and plant circuits?
• Can we delete natural circuits and replace them with synthetic counterparts within organisms?
• How can we engineer circuits that perform robustly in a noisy environment?
• If synthetic circuits completely fail to work, or work exactly as expected, they may appear to have taught us nothing. How do we develop synthetic projects that are as informative as possible?