Functional Robustness of Synthetic Gene Circuits Under Mutation Pressure

Understanding the effect of genetic robustness, a fundamental property of biological systems allowing to maintain the systems functions against internal and external perturbations, is one of the key issues of synthetic biology. In this study, we hypothesized that metabolic load change, rather than the absolute load level, is one of the important factors determining the genetic robustness. We designed two different classes of gene circuits – activation and inhibition. We verified that the robustness is significantly dependent on the classes and furthermore that the inhibition circuits are more robust than the activation circuits. Specifically, we investigated the functional and genetic changes in gene circuits in E. coli by performing dilution experiments for evolution. The regulation (activation and inhibition) strength was continuously monitored by measuring the expression level of fluorescent proteins that are genetically fused with the transcription factors. We observed different patterns of functional and genetic changes for the two classes. For the activation circuits, the activation by luxR::Venus was turned off by mutation in the luxR coding region. When the activation was strong (+AHL), the observed mutation was built up more quickly than the case that the activation was weak (-AHL). On the other hand, for the inhibition circuits, the inhibition by lacI::Venus was turned off by mutation in the ribosome binding site and the start codon of lacI. When the inhibition was weak (+IPTG), the observed mutation was built up more quickly than the case that the inhibition was strong (-IPTG). This result shows that inhibition is more stable than activation. We explain this circuit topology effect on robustness by performing metabolic load analysis, which support that a metabolic load ‘change’ is more important factor than the absolute load levels. Findings in this study imply the possibility of understanding of gene circuit robustness at the network level and the rational design of gene circuits with enhanced genetic robustness.