Exploring the potential of synthetic cell-free systems for low-cost bioproduction

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PhD project strategic theme: Bioscience for renewable resources and clean growthFor the purposes of bioengineering, the complexity of the cell poses a great number of challenges. The major issue encountered when attempting to introduce synthetic gene circuits into a host cell is unpredictability. There are numerous ways in which a synthetic circuit can fail inside a host cell, with some common examples being; issues with initiation and termination of transcription, cross-talk between circuit and host, and overburdening of the host cell through competition for a shared resource. An alternative route for the application of synthetic gene circuits is found in cell-free systems, which can be used to harness cellular biochemistry without the need or constraints of anintact cell.Cell-free systems involve the use of cell-lysates, prepared from whole-cells through a variety of means including sonication, purification, and bead lysis. These lysates contain the transcription and translation machinery which can be combined with a DNA template to perform protein synthesis in vitro.Cell-free systems offer an unparalleled tool for the rapid and facile prototyping of synthetic genetic circuits. This is a feature which is invaluable for the development of bioprocesses to be used in bioproduction. It has already seen applications in the production of therapeutic proteins, along with renewable fuels and commodity platform chemicals.There is the potential of cell-free systems to achieve higher levels of productivity than whole-cell based systems, at a lower cost. These systems can also facilitate the production of products which would either be toxic to cells or impossible for them to produce, while also alleviating some of the biosecurity concerns associated with engineered organisms. The ability to freeze dry cell-free systems for long term storage and transport opens the door for applications in point of usediagnostics and in education.A plant-based system would also be an ideal target for low-cost bioproduction, particularly if synthetic circuits could be targeted to the chloroplast. Plant systems offer access to agricultural-level scaling of production, with the infrastructure already in place for growth and harvest. However, until recently the transformation of chloroplast genomes has been a difficult task that represents a bottleneck in the application of synthetic biology to plant systems. Recent work has shown that pentatricopeptide repeat proteins (PPR proteins) can be engineered to promote transgene expression in chloroplasts.There clearly offers great potential for the application of synthetic gene circuits within the chloroplast. I propose to explore and expand upon the cell-free platform as a tool for gene circuit prototyping, and also to investigate its utility as a testbed for circuits that could be introduced into the chloroplast.