Abstract

Previous attempts to control cellular behavior were mainly based on genetic engineering. While useful, such an approach suffers from several complications. Living cells grow and evolve which could lead to modifications of the engineered circuits, causing not only the loss of their functions but also an altering of the environment. However, other methods are possible. All living cells can naturally sense and respond to their environment and to each other. Thus, artificial, non- living cells can be engineered to activate already existing natural cellular pathways. In this way, the genetic engineering component moves from the natural to completely artificial, laboratory-made cells. Moreover, synthetic systems operating in living organisms also depend on elements with unknown function, leaving many gaps in the understanding of how living cells work. Building life- like systems with non-living components could help reveal unrecognized but necessary cellular mechanisms. However, the design of functional, genetically encoded cell-free systems is difficult, because biological parts have been evolved to function optimally inside of living cells. In vitro conditions are different. First, some practical rules for the construction of functional synthetic circuits in vitro were defined. The Influences of the organization of genetic elements within a synthetic operon on protein expression levels were studied and optimal sequence compositions and lengths between genes to assemble genetic circuits were found. Then, artificial cells that can control the behavior of living systems were built. The artificial cells were able to sense a molecule that Escherichia coli cannot sense on its own and translate that molecule into a chemical message that E. coli can sense and respond to. The natural sensing of E. coli was expanded without genetically modifying the bacteria. Finally, to better integrate artificial with natural cells, a complete communication pathway was constructed. Bacteria speak to each other by quorum sensing. Such mechanisms mediate cell-cell communication among bacteria and regulate several cell density related processes, such as virulence. Various synthetic quorum sensing mechanisms were constructed in vitro within artificial cells. Artificial cells able to sense the presence of living cells were built. In addition, the artificial cells were capable to synthesize quorum sensing molecules for E. coli, Vibrio harveyi, Vibrio fischeri and Pseudomonas aeruginosa. When integrated together, artificial cells successfully mediated interspecies communication with natural cells. Such artificial systems could be useful as therapeutic tools to defeat pathogenic infections. Moreover, the achievement of such functions represents a new way to better understand the potential of the artificial cells to mimic cellular life.