Cell-Free Prototyping of AND-Logic Gates Based onHeterogeneous RNA Activators

RNA-based devices controlling gene expression bear great promise for synthetic biology, as they offer many advantages like short response times and light metabolic burden compared to protein-circuits. However, little work has been done regarding their integration to multi-level regulated circuits. In this work, we combined a variety of small transcriptional activator RNAs (STARs) and toehold switches to build highly effective AND-gates. To characterise the components and their dynamic range, we used an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets. We analysed a prototype gate in vitro as well as in silico, employing parameterised ordinary differential equations (ODEs), where parameters were inferred via parallel tempering, a Markov chain Monte Carlo (MCMC) method. Based on this analysis, we created nine additional AND-gates and tested them in vitro. The functionality of the gates was found to be highly dependent on the concentration of the ...