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RNA-based devices controlling gene expression bear great promise for synthetic biology, as they offer many advantages such as short response times and light metabolic burden compared to protein-circuits. However, little work has been done regarding their integration to multilevel 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 characterize 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 analyzed a prototype gate in vitro as well as in silico, employing parametrized ordinary differential equations (ODEs), for which parameters were inferred via parallel tempering, a Markov chain Monte Carlo (MCMC) method. On the basis of this analysis, we created nine additional AND-gates and …