DNA assembly and rational design are cornerstones of synthetic biology. While many DNA assembly standards have been published in recent years, only the Modular Cloning standard, or MoClo, has the advantage of publicly available part libraries for use in plant, yeast, and mammalian systems. No multipart modular library has previously been developed for use in prokaryotes. Building upon the existing MoClo assembly framework, we developed a collection of DNA parts and optimized MoClo protocols for use in E. coli. We present this assembly standard and library along with part characterization, design strategies, potential applications, and troubleshooting. Developed as part of the Cross-disciplinary Integration of Design Automation Research (CIDAR) lab collection of tools, the CIDAR MoClo Library is publicly available and contains promoters, ribosomal binding sites, coding sequences, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from previously published protocols. The CIDAR MoClo Library is the first bacterial DNA part library compatible with a multipart assembly standard. To demonstrate the utility of the CIDAR MoClo system in a traditional biology context, we used the library and previous expression data to create a series of dual expression plasmids. In this manner, we produced a dual expression plasmid capable of expressing equimolar amounts of two variants of rabbit aldolase, a His-tagged wildtype protein and a single-amino-acid substitution mutant deficient in binding actin. This expression plasmid will enable the production of dimer-of-dimer heterotetramers needed for structural determination of the actin-aldolase interaction by electron microscopy. To employ CIDAR MoClo in a synthetic biology context, we produced a bioelectronic pH-mediated genetic logic gate with DNA circuits built using MoClo and integrated with Raspberry Pi computers, Twitter, and 3D printed components. Logic gates are an increasingly common biological tool with applications in cellular memory and biological computation. MoClo facilitates rapid iteration of genetic designs, better enabling the development of cellular logic. The CIDAR MoClo Library and assembly standard enable rapid design-build-test cycles in E. coli making this system advantageous for use in many areas of synthetic biology as well as traditional biological research.

Genetic regulatory circuits control metabolism, development, and environmental response across all kingdoms of life. Genetic circuit engineering facilitates sustainable and efficient production of biopharmaceutical, chemical, fiber, and food products that keep humans healthy, nourished, and clothed. However, the complexity of most genetic regulatory circuits, particularly in the context of multicellular eukaryotes, often prevents them from being leveraged as tools or applied technologies with bioeconomic relevance. However, synthetic biology enables the transfer of genes, circuits, networks, and even whole chromosomes between organisms. This approach can be leveraged to port genetic circuits into simple model organisms to control existing and engineer new cellular functions. Still, porting genes to non-native contexts can affect circuit function due to unknown factors. For this reason, iterative design-build-test-learn (DBTL) cycles are necessary for optimizing circuits in new contexts. To facilitate the DBTL cycle, automation approaches can be deployed for streamlining synthetic genetic circuits optimization. Here, I provide a case study for how using synthetic biology and automation – a biofoundry approach – has facilitated engineering of the auxin signaling pathway in a synthetic yeast system. Auxin is a phytohormone involved in nearly every aspect of plant growth and development, and this striking versatility designates it as a target for biotechnology development and a candidate for engineering. First, I provide a literature review of the history of synthetic auxin engineering in yeast, a survey of tools available for expanding yeast synthetic biology, and a summary of applicable automation tools and platforms. Next, I describe and validate a platform called AssemblyTron, which deploys liquid handling robotics for DNA assembly and can serve as the foundation of a biofoundry platform. I then introduce TidyTron, which is a protocol library for automated wash and reuse of single use lab plastics to promote biofoundry sustainability. Next, I expand the AssemblyTron package by providing protocols for mutant and modular indexed plasmid library assembly. Finally, I describe a modular indexed plasmid library (toolkit) for rapid assembly of auxin circuit variants and validate it by building and optimizing an auxin circuit. / Doctor of Philosophy / Genetic mechanisms allow humans, plants, and microbes to grow, breathe, speak, and survive. The DNA that encodes these genetic mechanisms produces protein machines that make chemicals, transfer them, and respond to them in other cells. This process is called signaling, and the protein machines involved make a circuit. In biotechnology, we harness natural genetic circuits to create important products like biopharmaceuticals, food, and clothes. However, the genetic circuits that make valuable proteins/chemicals are usually located on chromosomes along with every other gene involved in building an advanced, multicellular organism (called the genome). Synthetic biology allows us to choose just the DNA that encodes a genetic circuit of interest and put it into the chromosome of a simpler organism with faster growth, smaller genome, etc., which allow us to engineer it more easily. However, transferring a gene circuit to a new organism can cause problems, and it is usually necessary to try many versions of gene circuits to find one that works. Using robots to do synthetic biology can make it faster and less error-prone, which enables more versions of the genetic circuit to be tested. Here, I describe a biofoundry approach where I combined synthetic biology and robotics to speed up the process of building and optimizing the auxin plant hormone signaling pathway. Auxin is a small molecule that plants produce and transfer throughout their leaves, stems, and roots to turn growth on or off (e.g., auxin causes plants to do things like bend towards the sun). I focus on auxin because my goal is to manipulate the auxin pathway to rationally control plant growth. First, I provide a recap of existing work in the field of auxin synthetic biology, tools for transferring auxin circuits into simpler organisms, and available robotics that can speed up auxin synthetic biology. Next, I introduce a software called AssemblyTron, which I developed for building and modifying genes (a process called DNA assembly) with a robot. Next, I discuss how I used the same robot to wash and reuse plastic pipette tips and plates to improve lab sustainability. I then discuss an extended version of AssemblyTron that can be used for more advanced DNA assembly applications like making 10s – 100000s of versions of gene circuits at the same time. Finally, I introduce a collection of auxin circuit DNA parts that can be assembled interchangeably for rapid synthetic auxin engineering.