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.

Plants often live in association with growth-promoting bacteria, which provide them with several benefits. One such benefit is the lowering of plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that cleaves the immediate biosynthetic precursor of ethylene, ACC. The plant hormone ethylene is responsible for many aspects of plant growth and development but under stressful conditions ethylene exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4, isolated from the rhizosphere of reeds, is a potent plant growth-promoting strain and as such was used, along with an ACC deaminase minus mutant of this strain, to study the role of ACC deaminase in plant growth-promotion. Also, transgenic plants expressing a bacterial ACC deaminase gene were used to study the role of this enzyme in plant growth and stress tolerance in the presence and absence of nickel. Transcriptional changes occurring within plant tissues were investigated with the use of an Arabidopsis oligonucleotide microarray. The results showed that transcription of genes involved in hormone regulation, secondary metabolism and the stress response changed in all treatments. In particular, the presence of ACC deaminase caused genes for auxin response factors to be up-regulated in plant tissues suggesting a de-repression of auxin signaling in the absence of high levels of ethylene. Also, transgenic plants had longer roots and grew faster than the non-transformed plants and genes involved in the stress response and secondary metabolism were up-regulated. Plants inoculated with bacteria had lower levels of secondary metabolism gene expression and slightly higher stress response gene expression than uninoculated plants. Yet, inoculation with the ACC deaminase-expressing bacterium caused less up-regulation of plant genes involved in stress and defense responses and the down-regulation of genes involved in nitrogen metabolism in comparison to plants inoculated with the ACC deaminase minus mutant. Nickel stress caused the down-regulation of genes involved in photosynthesis and carbon fixation and the up-regulation of genes involved in stress responses, and amino acid and lipid breakdown suggesting energy starvation. When transgenic plants expressing ACC deaminase in the roots were exposed to nickel stress, plant stress symptoms were significantly lower and biomass was significantly higher suggesting that lowering the level of ethylene relieved many of the stress symptoms. In fact, genes involved in photosynthesis, secondary metabolism and nitrate assimilation were up-regulated in transgenic plants compared with non-transformed plants in the presence of nickel, suggesting that ACC deaminase is effective at reducing the severe effects of this metal stress.

Plants often live in association with growth-promoting bacteria, which provide them with several benefits. One such benefit is the lowering of plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that cleaves the immediate biosynthetic precursor of ethylene, ACC. The plant hormone ethylene is responsible for many aspects of plant growth and development but under stressful conditions ethylene exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4, isolated from the rhizosphere of reeds, is a potent plant growth-promoting strain and as such was used, along with an ACC deaminase minus mutant of this strain, to study the role of ACC deaminase in plant growth-promotion. Also, transgenic plants expressing a bacterial ACC deaminase gene were used to study the role of this enzyme in plant growth and stress tolerance in the presence and absence of nickel. Transcriptional changes occurring within plant tissues were investigated with the use of an Arabidopsis oligonucleotide microarray. The results showed that transcription of genes involved in hormone regulation, secondary metabolism and the stress response changed in all treatments. In particular, the presence of ACC deaminase caused genes for auxin response factors to be up-regulated in plant tissues suggesting a de-repression of auxin signaling in the absence of high levels of ethylene. Also, transgenic plants had longer roots and grew faster than the non-transformed plants and genes involved in the stress response and secondary metabolism were up-regulated. Plants inoculated with bacteria had lower levels of secondary metabolism gene expression and slightly higher stress response gene expression than uninoculated plants. Yet, inoculation with the ACC deaminase-expressing bacterium caused less up-regulation of plant genes involved in stress and defense responses and the down-regulation of genes involved in nitrogen metabolism in comparison to plants inoculated with the ACC deaminase minus mutant. Nickel stress caused the down-regulation of genes involved in photosynthesis and carbon fixation and the up-regulation of genes involved in stress responses, and amino acid and lipid breakdown suggesting energy starvation. When transgenic plants expressing ACC deaminase in the roots were exposed to nickel stress, plant stress symptoms were significantly lower and biomass was significantly higher suggesting that lowering the level of ethylene relieved many of the stress symptoms. In fact, genes involved in photosynthesis, secondary metabolism and nitrate assimilation were up-regulated in transgenic plants compared with non-transformed plants in the presence of nickel, suggesting that ACC deaminase is effective at reducing the severe effects of this metal stress.

The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.

The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.

During embryogenesis and post-embryonic patterning, active transport of the phytohormone auxin, reflected in the expression of the Arabidopsis PIN family of auxin efflux mediators, generates local auxin distributions that are crucial for correct organ and tissue specification. Polar auxin transport routes have also long been postulated to regulate vein formation in the leaf. The molecular identification of PIN proteins has made it possible to investigate this hypothesis further by visualizing auxin transport routes in developing leaves. In Arabidopsis leaf primordia, PIN1 is expressed before the earliest known markers of vascular identity, in domains that are gradually restricted to sites of vein formation. PIN1 polarity indicates that auxin is directed towards distinct “convergence points” (CPs) in the marginal epidermis, from which it defines the sites of major vein formation. Within incipient veins, PIN1 polarity indicates drainage of auxin into preexisting veins, such that veins connected at both ends display two divergent polarities. Local auxin application triggers the formation of ectopic CPs and new veins, demonstrating the sufficiency of auxin as a vein-specifying signal. However, not all PIN1-labeled auxin transport routes differentiate as veins: Minor veins are initially unstable, suggesting local competition for auxin transport. Expression of ATHB8, a marker of vascular cell selection, correlates with enhanced PIN1 expression domain (PED) stability and vascular differentiation. Auxin application and auxin transport inhibition reveal that both CP formation in the epidermis and subepidermal PED dynamics are auxin-dependent and self-organizing. Furthermore, normal auxin perception through the ARF-Aux/IAA signaling pathway is required for the restriction of PIN1-mediated auxin transport to narrow subepidermal domains. ARF-Aux/IAA signaling is known to control auxin transport through the regulation of PIN1 dynamics, but the mechanism of this regulation is unclear. It is here shown that two redundantly acting AUXIN RESPONSE FACTOR (ARF) transcription factors, ARF5/MONOPTEROS (MP) and ARF7/NPH4, jointly regulate both PIN1 expression and localization during lateral root patterning in Arabidopsis, in part through the direct transcriptional activation of PIN1 by MP. Taken together, these results indicate that feedback between PIN-mediated auxin transport and ARF-Aux/IAA signaling regulates the patterning of root and shoot organs.

During embryogenesis and post-embryonic patterning, active transport of the phytohormone auxin, reflected in the expression of the Arabidopsis PIN family of auxin efflux mediators, generates local auxin distributions that are crucial for correct organ and tissue specification. Polar auxin transport routes have also long been postulated to regulate vein formation in the leaf. The molecular identification of PIN proteins has made it possible to investigate this hypothesis further by visualizing auxin transport routes in developing leaves. In Arabidopsis leaf primordia, PIN1 is expressed before the earliest known markers of vascular identity, in domains that are gradually restricted to sites of vein formation. PIN1 polarity indicates that auxin is directed towards distinct “convergence points” (CPs) in the marginal epidermis, from which it defines the sites of major vein formation. Within incipient veins, PIN1 polarity indicates drainage of auxin into preexisting veins, such that veins connected at both ends display two divergent polarities. Local auxin application triggers the formation of ectopic CPs and new veins, demonstrating the sufficiency of auxin as a vein-specifying signal. However, not all PIN1-labeled auxin transport routes differentiate as veins: Minor veins are initially unstable, suggesting local competition for auxin transport. Expression of ATHB8, a marker of vascular cell selection, correlates with enhanced PIN1 expression domain (PED) stability and vascular differentiation. Auxin application and auxin transport inhibition reveal that both CP formation in the epidermis and subepidermal PED dynamics are auxin-dependent and self-organizing. Furthermore, normal auxin perception through the ARF-Aux/IAA signaling pathway is required for the restriction of PIN1-mediated auxin transport to narrow subepidermal domains. ARF-Aux/IAA signaling is known to control auxin transport through the regulation of PIN1 dynamics, but the mechanism of this regulation is unclear. It is here shown that two redundantly acting AUXIN RESPONSE FACTOR (ARF) transcription factors, ARF5/MONOPTEROS (MP) and ARF7/NPH4, jointly regulate both PIN1 expression and localization during lateral root patterning in Arabidopsis, in part through the direct transcriptional activation of PIN1 by MP. Taken together, these results indicate that feedback between PIN-mediated auxin transport and ARF-Aux/IAA signaling regulates the patterning of root and shoot organs.