Characterization of Synthetic Gene Circuits with Absolute Quantification in Continuous Culture.

Despite rapid improvements in our ability to engineer novel biological systems, robust biodesign of synthetic gene circuits-networks of synthetic genes regulating each other-has been limited by a lack of standardized methods for understanding and reproducibly characterizing their behavior in complex cellular contexts and over long timescales. The challenges underlying this include the complexity of biological interactions and the cellular contexts, changes in the cell culture environment over time, and the use of inconsistent measurement techniques in synthetic biology. Here, we describe a methodology for characterizing engineered biological systems while addressing these issues with the help of mathematical modeling, continuous cell culture, and absolute quantification of protein and cell numbers. As a case study, the characterization of a simple small RNA circuit in the Chi.Bio bioreactor platform is considered. We describe the biological system design choices, preparation of calibrants, running an experiment in Chi.Bio, the use of resulting data to obtain calibrated measurements in absolute units, and parameterization of a mathematical model of the engineered system. By coupling computational methods with precise control of cellular environments and robust experimental measurements, this interdisciplinary approach can produce more informative data and new insights into the design of engineered biological systems.