(32g) Synthetic Transcriptional Motifs in Distributed Systems

A grand challenge within the field of developmental biology is to determine the rules by which cells in tissues adopt the proper fates. From an engineer’s perspective, these rules, which often involve gene regulation, could be exploited to aid in the construction of synthetic tissue. And while gene regulation -- especially regulatory motifs composed of multiple genetic interactions -- has been extensively studied in both natural and synthetic systems, the vast majority of these studies have focused on single cell systems. In contrast, tissue patterning is intrinsically a spatially-distributed, multicellular phenomenon. In naturally developing tissues, spatial concentration gradients of key signaling factors, called morphogens, initialize the patterns within the tissue. Further regulation downstream of the morphogen can confer robustness to the gene expression patterns, which in turn become the patterns of cell fates in the tissue. However, naturally-occurring morphogen/gene regulation systems can be difficult to decipher due to complexity and unknown interactions. Therefore, we used the Drosophila melanogaster embryo as a spatially-distributed model system, and analyzed a synthetic negative feedback motif that operated in the 2-4 hr old embryo. Our rationale is that a synthetic system would have less complexity and fewer unknown interactions.We hypothesized that the negative feedback motif would add robustness to the system with respect to morphogen fluctuations. However, we found that the qualitative behavior of the system depended on the balance among the synthetic components. When the activator (synthetic morphogen) was in excess over the negative feedback inhibitor, a small increase in robustness was observed. When the two were more balanced, the negative feedback motif actually potentiated morphogen signaling in a spatially non-uniform fashion. These sometimes counterintuitive results highlight the need to study synthetic biology not just in single-cell systems, but also in spatially distributed systems. As the qualitative behavior depended on the balance of protein abundances, we tested the ability of self-cleaving ribozymes to regulate mRNA levels. We found these ribozymes can span a wide dynamic range of activity and can be used in diverse applications. Our ultimate goal with these synthetic network motifs in the fly embryo is to optimize their behavior, and thus and advance our understanding of multicellular gene regulation for tissue engineering applications.