Autonomous translation modulation in a homeostatic artificial cell

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Cell-free gene expression have been used to answer fundamental biological question by deconstructing the cell. More recently we can employ cell-free based platforms towards the reconstruction of a cell. The reaction network responsible for gene expression in bacteria is tightly connected and consumes more than half of the cells energy. A great amount of components are invested in proof-reading mechanisms for protein synthesis and gene regulation. In this project, we will design and study genetic circuits that create homeostasis within a synthetic cell through the modulation of gene expression rates. The circuit will be using components from the gene expression machinery and therefore be coupled to core processes such as aminoacylation. We will thus set a steady-state for gene expression and make the synthetic cell more autonomous. The synthetic cells are based on surface-bound genes inside microfluidic compartments that are connected to a flow channel with reconstituted gene expression solution. The set-up allows for observing gene expression for prolonged time periods (>12h) with single molecule resolution using TIRF microscopy. The integration of components from core processes of gene expression into genetic circuit will show us the limitations and the amount of our understanding of these processes. We will gain detailed insights into the dynamics of the regulation of components that are directly involved in gene expression such as amino acid charged tRNAs molecules. The hypothesis in this project is that we are able to construct genetic circuits to create homeostasis in a synthetic cell, which is a fundamental working principle of living organisms.