Abstract

Genetic engineering has been widely used to reprogram cells for a variety of purposes, suggesting a wide range of possible applications in industrial and academic research. Although the techniques available are very well established, the mechanisms of cellular life are not completely understood. Therefore, despite offering a versatile tool, engineered cells are prone to possible unexpected behaviors. Investigations of more controllable systems are partially focused on the creation of cellular mimics assembled from discrete components with defined properties. The controlled assembly of molecules allows the creation of entities able to form compartments in water solutions and to carry out enzymatic reactions or gene expression. These artificial cells are able to establish communication pathways with natural cells and may be further developed to fight pathogens or cancer cells, for example. Despite these promising results, technological applications based on cellular mimics necessitate further technical improvements. A considerable defect of artificial cells is the lack of some mechanisms for selfsustainment that are instead present in engineered living cells. Besides few strategies aimed at energy restoration, artificial cells are not yet able to efficiently use the available resources in their environment. Considering these technical limitations, this thesis proposes to improve communication pathways between artificial and natural cells by exploiting multiple kinds of cellular mimics. Artificial cells can vary in composition and if engineered to coordinate activity, could be capable of overcoming individual weaknesses. To investigate the possibility of creating communities of artificial cells that collaborate with each other, the work described here was focused on establishing molecular communication pathways between two kinds of artificial cells. The designed communication was based on the exchange of chemical messages between two cellular mimics resulting either in genetic regulation or enzymatic reactions. On one side, lipid vesicles carrying gene expression through in vitro transcription and translation reactions and on the other side a novel structure composed of modified proteins, named proteinosome, to carry out enzymatic reactions. Each part of the communication pathway was separately investigated. Some efforts were put into the characterization of genetic switches so as to be able to better tune gene expression. All the other components were then singularly tested before combining together. One way that artificial cells, either alone or in a community, can function as a useful technology is if the artificial cells are able to sense and respond to environmental changes. The sensing functionality can be conferred by natural or synthetic transcriptional regulators. It is possible to modify biological macromolecules to interact with chemical messages released by natural cells. The second part of the thesis summarizes two distinct works aimed at developing two kinds of biosensors with potential applications within artificial cells. Several technical problems arose while testing the communication pathway, and it was necessary to change the initial strategy to include engineered cells. Nonetheless, the work presented here offers a method for the establishment of molecular communication pathways within communities of artificial cells that could serve as the basis for future implementation in more efficient communication systems.