On the Deviant Behavior of Synthetic Gene Networks in Genomic and Transient Context or: How I Stopped Worrying about Stable Cell Lines and Started Loving Transient Transfections

Abstract

The mapping of inputs to their molecular outputs is of pivotal interest in biolog- ical research. In the field of synthetic biology, this became a standard analysis technique to characterize (novel) gene networks. Experimentally, these mappings can be obtained through transfections of gene circuits into cells – either stably inte- grated or transiently transfected. While stable integration harbors the advantage of maintaining gene circuits within a cell’s genome, they are oftentimes very labor intense and difficult to achieve. Transient transfections, on the other hand, are easy to implement, but have the downside to be only suitable for short term exper- iments as the gene circuits will eventually vanish due to dilution caused by cell division. Furthermore, the process of transient transfections generates additional noise that can make interpreting raw data very difficult. This limitations were overcome by developing an analysis tool (PFAFF) that is not only capable of extracting input/output mapping from stably integrated synthetic gene circuits but also from noisy transient transfection data. The aim was to inves- tigate, whether it is possible to infer input/output behavior of stably integrated gene circuits from transient transfection data. To achieve this, four three-node cir- cuits were modelled and in-silico simulations for both scenarios were performed. Analyses of the resulting data sets clearly showed that the behavior of simulated stably transfected gene circuits can be inferred from simulated transiently trans- fected gene circuit data. To this end the thesis shows the application of our analysis tool for four synthetic gene circuits of different complexities, studied in an experimental setup using a mammalian cell line – HEK tetON – for both (stable and transient) transfection scenarios. The inferred input/output behaviors match in both transfection scenar- ios. This work further highlights the differences between synthetic gene circuits condensed on a single-plasmid or distributed on a multiple plasmid setup and puts it in perspective to the corresponding stably integrated case. I close this work discussing the aims that were achieved, the role of our findings and the limitations to the presented approaches.