Mechanistic Engineering Strategies to Design and Create Synthetic Cellular Systems & Synthetic Biology Tools

A mechanistic understanding of living cells and the complex interplay between their biochemical and molecular components is a crucial cornerstone in the path towards rational and reproducible bioengineering of cellular and sub cellular systems in synthetic biology. Bioengineering approaches that couple such understanding with traditional synthetic biology tools can be exploited to create reconstituted complex molecular networks, artificial cells, and semi-living cellular systems with the desired traits of engineered biomaterials, but with the adaptability of living cells.The design and creation of such bioengineered systems will need the implementation of a holistic perspective that accounts for and exploits the crosstalk between metabolic networks occurring inside living cells, synthetic circuits, and synthetic materials. In the research presented in this dissertation, I use an integrative approach involving knowledge in a variety of disciplines such as cell, molecular, systems, and synthetic biology to rationally design synthetic systems using a holistic approach. Using this holistic approach, during my doctorate research I was able to create novel strategies to reconstitute metabolic networks in vitro, and biomimetic systems with different synthetic materials and engineered capabilities. My thesis research is comprised of four different projects, all within the central topic of developing engineering strategies that exploit the mechanistic understanding of bacteria and their modular components to develop new technologies that solve challenges in synthetic biology. The first part of my dissertation focuses on the use of a synthetic microbial consortia approach and a single co-purification step as a new strategy to reconstitute complex metabolic pathways ex vivo. Using this approach, we pioneered the rapid reconstitution of the 34-protein core translation machinery from Escherichia coli. The next project tackled a persistent problem in cellular engineering, the limited performance of synthetic gene circuits in different chemical contexts outside living cells. My strategy aimed to minimize the context dependency of gene circuits by using artificial cells, which are biological mimics engineered from the bottom-up by encapsulating specific cellular components and gene networks inside a synthetic liposome. The resulting artificial cellular systems are capable of robust gene expression, chemical communication, and detecting and killing bacteria in different chemical contexts. In the following project, I developed a new approach to tackle the decreased functionality of orthogonal synthetic circuits caused by crosstalk and noise within a cell. Using a holistic approach that integrates systems and synthetic biology concepts, I demonstrated how orthogonal synthetic circuits can be used to reprogram the host proteome and in turn enhance the function of the synthetic orthogonal circuit. I exploited this previously unknown approach to produce a superior cell-free system as a proof of concept aimed to highlight the potential of this new positive feedback loop approach. The last part of my dissertation research builds on my previous projects and incorporates methodologies from materials science to develop a new class of cell-derived semi-living organism. I developed an approach that uses a physically restrictive method to restrict bacterial growth without compromising other cellular processes. This results in the creation of metabolically active, non-growing, and stress resistant cell chassis that we termed Cyborg Cells with potential applications as living therapeutics, gene delivery vectors, live attenuated vaccines, and biosensors. Each one of the projects comprising my dissertation research has opened new research avenues in areas such as cancer research, biosensing within bacterial biofilms, and living therapeutics. Also, they have the potential to inspire and become the foundation of new technologies that can help us tackle challenges as complex as the precise engineering of mammalian cells, and the real-time detection & control of disease development