Precise determination of input-output mapping for multimodal gene circuits using data from transient transfection

The mapping of molecular inputs to their molecular outputs (input/output, I/O mapping) is an important characteristic of gene circuits, both natural and synthetic. Experimental determination of such mappings for synthetic circuits is best performed using stably integrated genetic constructs. In mammalian cells, stable integration of complex circuits is a time-consuming process that hampers rapid characterization of multiple circuit variants. On the other hand, transient transfection is quick. However, it is an extremely noisy process and it is unclear whether the obtained data have any relevance to the input/output mapping of a circuit obtained in the case of a stable integration. Here we describe a data processing workflow, Peakfinder algorithm for flow cytometry data (PFAFF), that allows extracting precise input/output mapping from single-cell protein expression data gathered by flow cytometry after a transient transfection. The workflow builds on the numerically-proven observation that the multivariate modes of input and output expression of multi-channel flow cytometry datasets, pre-binned by the expression level of an independent transfection reporter gene, harbor cells with circuit gene copy numbers distributions that depend deterministically on the properties of a bin. We validate our method by simulating flow cytometry data for seven multi-node circuit architectures, including a complex bi-modal circuit, under stable integration and transient transfection scenarios. The workflow applied to the simulated transient transfection data results in similar conclusions to those reached with simulated stable integration data. This indicates that the input/output mapping derived from transient transfection data using our method is an excellent approximation of the ground truth. Thus, the method allows to determine input/output mapping of complex gene network using noisy transient transfection data.Author summary: One of the key features of a gene circuit is its input/output behavior. A few earlier publications attempted to develop methods to extract this behavior using transient transfection of circuit components in mammalian cells. However, the hitherto developed methods are only suitable for circuit with monomodal output distribution. Moreover, the relationship between the extracted I/O mapping and the "ground truth" that would have obtained with stably-integrated circuits, has not been addressed. Here we explore cell populations easily identifiable in flow cytometry data, namely, the peaks of fluorescent readout distribution in cells binned by the common expression value of the transfection reporter, or marker, gene. Using numerical simulations, we find that the distribution of circuit copy number in these cells deterministically depends on marker fluorescence in the noise-dependent manner. Moreover, we find that this is true also in the case of bi-modal output distribution. Using the peaks of input and output distributions, we are able to reconstruct the I/O mapping of the circuit and relate it to the I/O mapping of the stably-integrated circuit. The reconstruction is enabled by a new computational method we call PFAFF. The method is extensively validated with forward-simulated flow cytometry data from stable and transient transfections, with up to seven different circuits. The results show excellent correlation between the I/O behavior extracted by PFAFF from simulated transient transfection data, and the data simulated for stably integrated circuit.