Fluorescent Proteins and <i>in Vitro</i> Genetic Organization for Cell-Free Synthetic Biology

To facilitate the construction of cell-free genetic devices, we evaluated the ability of 17 different fluorescent proteins to give easily detectable fluorescence signals in real-time from <i>in vitro</i> transcription-translation reactions with a minimal system consisting of T7 RNA polymerase and <i>E. coli</i> translation machinery, i.e., the PUREsystem. The data were used to construct a ratiometric fluorescence assay to quantify the effect of genetic organization on <i>in vitro</i> expression levels. Synthetic operons with varied spacing and sequence composition between two genes that coded for fluorescent proteins were then assembled. The resulting data indicated which restriction sites and where the restriction sites should be placed in order to build genetic devices in a manner that does not interfere with protein expression. Other simple design rules were identified, such as the spacing and sequence composition influences of regions upstream and downstream of ribosome binding sites and the ability of non-AUG start codons to function <i>in vitro</i>

Two-Way Chemical Communication between Artificial and Natural Cells

Artificial cells capable of both sensing and sending chemical messages to bacteria have yet to be built. Here we show that artificial cells that are able to sense and synthesize quorum signaling molecules can chemically communicate with <i>V. fischeri</i>, <i>V. harveyi</i>, <i>E. coli</i>, and <i>P. aeruginosa</i>. Activity was assessed by fluorescence, luminescence, RT-qPCR, and RNA-seq. Two potential applications for this technology were demonstrated. First, the extent to which artificial cells could imitate natural cells was quantified by a type of cellular Turing test. Artificial cells capable of sensing and in response synthesizing and releasing <i>N</i>-3-(oxohexanoyl)­homoserine lactone showed a high degree of likeness to natural <i>V. fischeri</i> under specific test conditions. Second, artificial cells that sensed <i>V. fischeri</i> and in response degraded a quorum signaling molecule of <i>P. aeruginosa</i> (<i>N</i>-(3-oxododecanoyl)­homoserine lactone) were constructed, laying the foundation for future technologies that control complex networks of natural cells