Browsing by Subject "Synthetic biology"

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Biology in the information age : computational methods to understand and engineer the central dogma
(2020-02-03) Shroff, Raghav; Ellington, Andrew D.; Davies, Bryan; Elber, Ron; Iyer, Vishwanath; Marcotte, Edward
The rise of NGS, big data, and ‘-omics’ has ushered biology into a new age, with the power to fundamentally change how research is approached. Rather than using a singular hypothesis, we can now incorporate more data-driven methods that drive new biological insights, explain emergent biological phenomena, and/or derive novel functionality. This thesis highlights the changing role of computation to both learn more about biological systems as well as leveraging data-intensive computational techniques to create new proteins and enzymes. The ability for computational approaches to drive biological understanding is presented in three studies. First, the laboratory evolution of DNA polymerases, the workhorses of replication, towards novel functionality is explored. In the three polymerases created, modeling and large scale approaches are used to demonstrate the additional capability of each new enzyme. Next, two independent studies in the genomic adaptations needed for E. coli cells to adapt a 21st amino acid (selenocysteine and nitrotyrosine) are presented. Next generation sequencing is used to better understand the mechanisms of how cells accommodate the increased fitness burden placed by an orthogonal translation system. Lastly, community-wide changes in the oral microbiome are studied in the progression towards periodontitis, with implications towards potential therapeutic targets. The capstone of this thesis leverages big data techniques to engineer novel proteins, the chief functional units within cells. Protein structural data is implemented into a convolutional neural network to associate amino acids with neighboring chemical microenvironments at state-of-the-art accuracy. This algorithm enables identification of gain-of-function mutations, and subsequent experiments confirm substantive improvements in stability-associated phenotypes in vivo across three diverse proteins. This work is the first demonstration of using deep learning to empirically improve protein function and opens a new avenue for protein engineering.
CRN++ : molecular programming language
(2018-05-03) Vasic, Marko; Khurshid, Sarfraz; Soloveichik, David
Synthetic biology is a rapidly emerging research area, with expected wide-ranging impact in biology, nanofabrication, and medicine. A key technical challenge lies in embedding computation in molecular contexts where electronic micro-controllers cannot be inserted. This necessitates effective representation of computation using molecular components. While previous work established the Turing-completeness of chemical reactions, defining representations that are faithful, efficient, and practical remains challenging. This work introduces CRN++, a new language for programming deterministic (mass-action) chemical kinetics to perform computation. We present its syntax and semantics, and build a compiler translating CRN++ programs into chemical reactions, thereby laying the foundation of a comprehensive framework for molecular programming. Our language addresses the key challenge of embedding familiar imperative constructs into a set of chemical reactions happening simultaneously and manipulating real-valued concentrations. Although some deviation from ideal output value cannot be avoided, we develop methods to minimize the error, and implement error analysis tools. We demonstrate the feasibility of using CRN++ on a suite of well-known algorithms for discrete and real-valued computation. CRN++ can be easily extended to support new commands or chemical reaction implementations, and thus provides a foundation for developing more robust and practical molecular programs.
Design and synthesis of synthetic UP elements for modulation of gene expression in Escherichia coli
(2017-01-05) Flexer Harrison, Madeleine Hannah; Alper, Hal S.
To limit both cost and environmental impact, microorganisms are exploited by synthetic biologists for ecologically friendly ways to produce biochemicals essential to both industry and health. Synthetic biologists and metabolic engineers work to hijack the metabolism of accommodating hosts in order to produce useful chemicals. The alteration and optimization of endogenous and heterologous metabolic pathways require careful balancing and tuning of gene expression. While there are numerous ways to control gene expression in the cell, by far the most common way to regulate expression is transcriptionally through the promoter. In this work we explore a particular region of the bacterial promoter, the UP element, as an effective way modulate gene expression in Escherichia coli. We use both rational and library design of UP elements to search the upstream sequence space of the core promoter, rrnD. Using FACS and flow cytometry, we screen multiple libraries to identify a group of UP elements capable of modulating expression in E. coli. Additionally, we explore the effects of modifying sequences adjacent and upstream to the UP element site. We report that expression strength can be tuned both positively and negatively through the modifications in the UP element sequence and through modifications of sequences upstream and adjacent of the UP element. The strongest sequence identified, TP-24, amplifies our core promoter strength up to 30-fold based on GFP fluorescence. Our final selection of UP elements activate rrnD from 8 –– 26 fold, each stronger than the commonly used IPTG inducible promoter, Ptac1. This work provides a novel and minimal way to control promoter strength in E. coli expression without major alteration to the core promoter.
Developing synthetic, minimal promoters in Saccharomyces cerevisiae
(2017-05) Redden, Heidi Rosemary; Browning, Karen S.; Barrick, Jeffrey; Hoffman, David; Keatinge-clay, Adrian
Promoters enable synthetic biologists to manipulate protein expression at the DNA level. For this reason, promoters are essential for almost all applications aiming to engineer an organism. Unfortunately, promoters available for eukaryotic organisms are derived directly from the genome. Such promoters are large and result in substantial and therefore, difficult DNA insertions to express a heterologous multi-gene pathway. Furthermore, their high sequence homology provides the organism the opportunity to perform homologous recombination resulting in undesirable gene deletions. For these reasons, there is a critical demand for short promoters with low sequence homology to the organism’s genome to continue synthetic biology advancements in eukaryotic hosts. This work addresses the need for yeast promoters by engineering the promoter’s two distinct DNA regions– the upstream activating sequence (UAS) and the downstream 3ˋ area comprised of the promoter’s core. The modularity of these regions is demonstrated in a non-conventional yeast, Yarrowia lipolytica by assembling multiple native UAS in tandem with a core. In doing so, the strongest promoters ever reported for Y. lipolytica were created. Drawing from these lessons, the length of promoters in the popular host strain Saccharomyces cerevisiae was minimized. The core region is first addressed by establishing promoter libraries with minimized de novo cores. Synthetic cores are isolated from a short promoter library and are evaluated in six DNA contexts to establish nine minimal cores with modularity, robustness, and context independence. Second, the UAS region was minimized. To do so, a randomized region of DNA was hybridized upstream of a synthetic minimal core to construct 18 de novo libraries of promoters. From these libraries, 26 short constitutive and inducible UAS elements were isolated. Collectively, this work highlights the utility of hybrid promoter engineering to increase the number of promoters available for host organisms, Y. lipolytica and S. cerevisiae. More importantly, it establishes a highly desirable set of 81 synthetic, minimal promoters of inducible and constitutive function that provides a 70-fold range of expression in S. cerevisiae. Furthermore, the workflow presented herein is generic enough for application in other eukaryotic host organisms to build their synthetic biology toolboxes.
Engineered plasmids : uncovering lost histories and improving annotation
(2023-04-13) McGuffie, Matthew James; Barrick, Jeffrey E.; Wilke, Claus O; Marcotte, Edward M; Davies, Bryan W
Engineered plasmids are a fundamental tool in many biological disciplines. They are the primary workhorses for molecular cloning, protein expression, genetic engineering, and more. Their utility stems from the fact that they are relatively easy to build using a standard set of well-understood DNA sequences. However, since engineered plasmids are created by scientists, they may contain artifacts from their construction histories that affect their functions in unexpected ways. Furthermore, engineered plasmids tend to receive less attention regarding the evolutionary pressures they face, compared to natural DNA sequences. In particular, the backbones of these plasmids, containing origins of replication and selection markers, are typically assumed to be immutable or at least tolerant of mutations. Nonetheless, engineered plasmids are subject to the same evolutionary forces that shape all DNA replicating within a cell, meaning that they will be subject to selection and may accrue mutations that affect their function, especially if doing so improves the fitness of the host cell. In Chapter 1 I reflect on the history of engineered plasmids and the inherent conflicts between engineered plasmids and their host bacteria. In Chapter 2 I describe pLannotate, a webserver and command line tool that is designed specifically for annotating engineered DNA and plasmids. I use this tool to demonstrate that most engineered plasmids contain fragments of functional genetic elements that are hidden relics from their construction histories. In Chapter 3 I develop a method for identifying variants of common functional elements used in engineered plasmids that likely arose multiple times independently by convergent engineering or evolution. I compile a list of hundreds of these undocumented variants and others that are found in plasmids from many labs. In Chapter 4 I analyze the ColE1 origin of replication, which is the most common functional element in engineered plasmids for evidence of unintended evolution. I show that a majority of convergent ColE1 variants have significantly altered copy numbers, which could be due to selection to reduce burden in some cases. In Chapter 5 I summarize lessons from each of the previous chapters, discuss caveats, and offer suggestions for future research in these areas.
Engineering and evolution of Saccharomyces cerevisiae for muconic acid production
(2016-09-14) Leavitt, John Michael; Alper, Hal S.; Barrick, Jeffrey E.; Bull, James J; Whiteley, Marvin; Wilke, Claus O
The advent of metabolic engineering and synthetic biology has resulted in a proliferation of microbial cell factories capable of producing valuable chemical products in diverse microbial hosts. This promises to provide a means to produce many of the chemical products which are currently derived from petroleum in an alternative, environmentally friendly, renewable process. Muconic acid is a chemical of particular interest for bioproduction as it can serve as a precursor for many compounds including the polymers nylon and polyethylene terephthalate. My initial research resulted in importing the biosynthetic capacity for muconic acid into the yeast host Saccharomyces cerevisiae. Through this work, we demonstrated the novel production of muconic acid for the first time in yeast and performed subsequent strain engineering to increase titers to 140mg/L, then the highest titer of any product from the shikimate pathway in yeast [1]. To further improve muconic acid titers, we chose to use adaptive laboratory evolution to complement initial, rational metabolic engineering efforts. To facilitate the screening of mutant strains with increased muconic acid production, a transcription-factor based biosensor was created. This biosensor was created to detect aromatic amino acids as a surrogate for flux through the shikimate pathway, the precursor pathway also used for muconic acid biosynthesis. This biosensor was based on the Aro80p transcription factor and demonstrated both tunable induction upon aromatic amino acids as well as a constitutive mode that created ultra-strong promoters capable of two-fold stronger expression that TDH3 (GPD), one of the strongest promoters available in yeast [2]. Finally, the utility of this biosensor coupled with adaptive laboratory evolution was demonstrated in a further approach to increase muconic acid production. Namely, this sensor was used in a biosensor-enabled adaptive laboratory evolution scheme to increase titers in our original strain to over 550 mg/L muconic acid in shake flask and 1.94g/L in a fed-batch bioreactor. This work represents a 14-fold improvement in titer over our previously engineered strain and nearly a 400-fold increase over simple heterologous expression of the pathway. These results demonstrate the power of coupling rationale engineering with adaptive engineering to increase product titers.
Engineering the gut microbiome of honey bees
(2020-06-22) Leonard, Sean Patrick; Moran, Nancy A., Ph. D.; Barrick, Jeffrey E.; Ochman, Howard; Marcotte, Edward; Davies, Bryan
Honey bees are critically important commercial pollinators and model systems for insect physiology and behavior. Honey bees are also suffering dramatic declines worldwide due to many factors, including agricultural practices, parasites, and pesticide use. These bees house a simple, conserved gut microbiome that is important for their health. Can we use this gut microbiome to protect bees in new ways? Synthetic biology combines recombinant DNA technology and rational design principles to redesign biological processes. Microbiome engineering applies synthetic biology and engineering principles to microbial communities to improve or expand their functions. Because of their agricultural importance, history as a model organism, and simple gut microbiome, honey bees are a promising testbed for the nascent field of microbiome engineering. In Chapter 1 I provide a brief introduction to the host-associated microbiomes, honey bees, and synthetic biology. In Chapter 2, I develop broad-host-range tools for genetic manipulation of bacteria from honey bees and show that genetically engineered bacteria can recolonize and function in bees. This lays the groundwork for follow-on efforts to both study and further engineer the bee gut microbiome. In Chapter 3, I describe the application of these genetic tools to engineer core microbiome member Snodgrassella alvi to produce double-stranded RNA (dsRNA) and thereby induce RNA-interference (RNAi) in bees. Activating RNAi enables bee researchers to study specific bee genes. In the future this technique may be used to protect honey bee hives from viruses and parasitic mites. In Chapter 4, I describe a computational approach for designing and evaluating defined bacterial communities and discuss using these defined communities in honey bees. These chapters together demonstrate how the bacterial community native to an organism can be modified and address several technical limitations of microbiome engineering in honey bees. Finally, I discuss the next steps for continuing this work.
Exploring the design principles of orthgonal transcription control systems
(2021-08-11) Kar, Shaunak; Ellington, Andrew D.; Davies, Bryan; Alper, Hal; Soloveichik, David; Barrick, Jeffrey
The last two decades has witnessed an unprecedented growth in our ability to engineer biological systems for a wide range of applications ranging from the development of smart therapeutics, production of valued products and chemicals and engineering crops with programmable traits and much more. At the core of these capabilities has been the design and characterization of synthetic genetic programs that has enabled the predictable programming of cellular behavior and phenotypes. A fundamental challenge in the construction of such circuits and programs is being able to design and model them against a variety of organismal backgrounds, which can be often difficult to predict and can lead to circuit failure when systems are ported across organisms. Such failure modes can potentially be mitigated by embedding orthogonal modes of transcriptional control and regulation in genetic programs to drive the expression of the circuit components in both prokaryotes as well as eukaryotes. Specifically, in prokaryotes, we demonstrate how an autoregulated network controlling the expression of an orthogonal RNA polymerase – T7 RNA polymerase, can be utilized to precisely express target genes in a highly predictable manner dictated by mutant T7 RNAP promoters. Furthermore, with the use of a modular architecture we show how such expression systems can be readily ported across diverse prokaryotes. In each species, the relative strength of expression obtained from the T7 RNAP homeostasis circuit is nearly identical, suggesting T7 RNAP driven expression systems can be utilized as predictable cross-species gene expression platform. In another example, orthogonal transcriptional regulation was engineered in a complex eukaryote (plants) using a programmable transcription factor - dCas9:VP64 and a set of designed synthetic promoters whose activity can precisely regulated with the expression of specific guide RNAs (gRNAs). This strategy was used to construct three mutually orthogonal promoters, allowing multiplexed control of gene expression in plants. Overall, the design strategies and architectures described in this work can be used to explore the design of more complex circuits where the activity of T7 RNAP can be coupled to regulate the activity of dCas9 based transcription to generate circuits operating across kingdoms of life.
Generalizable approaches for gene expression regulation in Saccharomyces cerevisiae
(2018-08-03) Morse, Nicholas J.; Alper, Hal S.; Contreras, Lydia; Georgiou, George; Ellington, Andrew; Iyer, Vishwanath
There is a current surge of interest in using synthetic biology for biotechnology applications. Metabolic engineers, for example, are interested in synthetic biology for its modular and well characterized transcriptional “parts”, such as synthetic gene promoters and terminators, which enable fine tuning in metabolic pathway optimization. Likewise, emerging gene editing methods, such as CRISPR-Cas9, are enabling quicker and more precise genomic integrations. Using both of these advances, there is an increase in the throughput for which altered pathway conditions can be screened. While some advances are being made, there are still several technological gaps, especially for eukaryotic yeast hosts. Therefore, this dissertation work focused on developing engineering methodologies for the yeast Saccharomyces cerevisiae to expand capacity in each of these areas. There were three main areas explored in this work. First, we developed a method for synthetic promoter design which establishes de novo upstream activating sequences (UAS) capable of regulating gene expression by growth phase. These UAS elements, discovered through a transcriptome mining approach, show an over 30-fold activation of a core promoter with completely synthetic designs. Secondly, we improved synthetic terminator design, whereby both minimal synthetic terminators and larger native terminators were improved by adjusting nucleosome occupancy in adjacent sequence space. Using this methodology, de novo synthetic terminators were designed for increased termination efficiency. Lastly, we developed a method for guide RNA expression in yeast organisms using T7 RNA polymerase in vivo. This method allowed guide RNA expression to be exportable across yeast hosts and enabled more complex regulation designs, such as dCas9 logic gates. Together, these approaches improved synthetic promoter design, synthetic terminator design, and guide RNA expression regulation in ways that both complement current ongoing research in S. cerevisiae and enable a generalized approach to be established for other yeast organisms.
Genetic and biochemical dissection of complex evolved traits in bacteria
(2014-08) Quandt, Erik Michael; Georgiou, George; Ellington, Andrew D.; Barrick, Jeffrey E.; Alper, Hal S; Molineux, Ian J
Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability (Cit⁺ ) to utilize abundant citrate in the growth medium after ~31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit⁺ variants achieve a much higher population density in this environment. All Cit⁺ individuals share a mutation that activates aerobic expression of the citT citrate:C₄-dicarboxylate antiporter, but this mutation confers only an extremely weak Cit⁺ phenotype on its own. To determine which of the other >70 mutations in early Cit⁺ clones were needed to take full advantage of citrate, we developed a Recursive Genome-Wide Recombination and Sequencing (REGRES) method and performed genetic backcrosses to purge mutations not required for Cit⁺ from an evolved strain. We discovered a mutation that increased expression of the dctA C₄-dicarboxylate transporter greatly enhanced the Cit⁺ phenotype after it evolved, implicating the intracellular supply of succinate or other C₄-dicarboxylates to be a critical factor for the expression of the phenotype. The activity level of citrate synthase (CS), encoded by the gltA gene, was also found to be important for Cit⁺. A mutation to gltA (gltA1) occurred before the evolution of Cit⁺ and led to an increase in CS activity by diminishing allosteric inhibition by NADH. This mutation was found to be deleterious for high-level citrate utilization, a situation that was remedied shortly after the evolution of Cit⁺ by the evolution of compensatory mutations to gltA which decreased CS activity. We speculate that the gltA1 mutation may have been important to 'potentiate' the evolution of a weak Cit⁺ phenotype by increasing succinate production via an upregulated glyoxylate pathway but that as cells became able to import succinate by virtue of the dctA mutation that this pathway became maladaptive, prompting this evolutionary reversal. Overall, our characterization of this metabolic innovation highlights the degree to which interactions between alleles shape the evolution of complex traits and emphasizes the need for novel whole-genome methods to explore such relationships.
Harnessing bacterial electroactivity with materials science and synthetic biology
(2020-08-10) Dundas, Christopher Michael; Keitz, Benjamin K.; Chemical Engineering; Alper, Hal; Georgiou, George; Werth, Charles J
Biotechnology has been transformed by increased cellular control over molecular products and processes. The fields of metabolic engineering and synthetic biology have standardized methods for biosynthesizing chemicals and progressed development of novel vaccines, genetic therapies, and transgenic organisms. Despite these significant advances, cellular programmability has largely stopped short of controlling the non-biological world – forward bioengineering of abiotic and inorganic processes, such as bio-electronic interfaces and material syntheses, remains a substantial challenge. To realize the benefits of engineered biology with such technologies, chassis organisms and extracellular pathways that connect genetics with inorganic transformations are required. In this work, we develop the electroactive bacterium Shewanella oneidensis and its extracellular electron transfer pathways as a genetically tunable platform to control redox-driven reactions. To engineer S. oneidensis electroactivity for redox chemistry, we accomplish two overarching objectives. For the first objective, we establish genotype-phenotype relationships between S. oneidensis and material syntheses/transformations that tune material properties and functioning. In Chapter 2, we determine that S. oneidensis activates metal catalysts involved in atom-transfer radical polymerization and that polymerization rate depends on carbon metabolism and expression of key electron transfer proteins. In Chapter 3, we show that S. oneidensis reductively precipitates palladium nanoparticles onto the cell surface and that expression of outer membrane cytochromes and availability of redox-active metabolites alters particle size and cellular localization. In Chapter 4, we demonstrate that S. oneidensis respires onto synthetic mineral analogs (metal-organic frameworks) via outer membrane cytochromes and that these bio-functionalized materials enable chromium remediation. For the second objective, we develop genetic tools to program extracellular electron flux and material formation by S. oneidensis. In Chapter 5, we use transcription/translation regulators and high throughput redox analysis to build plasmid-based logic gates in S. oneidensis that express single extracellular electron transfer proteins and exhibit inducible electroactivity responses, similar to fluorescent protein circuits. In Chapter 6, we leverage one of these S. oneidensis circuits to control radical crosslinking of a semi-synthetic hydrogel and predictably modulate gel stiffness via inducer molecule concentration. Taken together, this work provides new knowledge of S. oneidensis-controlled material transformations and presents a workflow to program extracellular electron transfer using synthetic biology
Insights into the functions of RNA post-transcriptional modifications gained through studies in cellular stress
(2017-08-29) Baldridge, Kevin Charles; Contreras, Lydia M.; Sanchez, Isaac C., 1941-; Alper, Hal; Georgiou, George; Ren, Pengyu
In recent years, the explosion of knowledge regarding functional roles for RNA in biology have uncovered the need for detailed molecular understanding of the relationship between the chemistry of RNA and its function. Post-transcriptional modifications in RNA are changes to the chemistry of the four basic RNA nucleotides, which can be thought of as expansions to the genetic alphabet of RNA. By subtly changing the chemistry of the standard RNA nucleotides, post-transcriptional modifications (PTMs) can act to modulate RNA function through a variety of mechanisms ranging from altering the basic physicochemical properties of an RNA molecule to fine-tuning complex regulation processes in higher organisms such as humans. While a number of functions have been ascribed to RNA post-transcriptional modifications, we still lack a comprehensive picture of how subtle changes in RNA chemistry can alter cellular function. Therefore, my dissertation focuses on applying cellular stress models as a way to interrogate how RNA post-transcriptional modifications can modulate RNA function. First, I provide a thorough discussion of how ribosomal RNA methylations help Escherichia coli adapt to stressful environmental conditions by protecting the vital protein translation process. Furthermore, by performing a targeted meta-analysis of publicly available stress-induced gene expression data, I identify specific ribosomal RNA methylations which may be important for E. coli adaptation to particular stresses including oxidative, heat, and cold stresses. Next, I examined the question of how RNA chemistry is affected directly by an external stress using a model system for simulated air pollution exposure with human lung cell cultures to demonstrate that cellular RNA oxidation is a reliable consistent indicator of oxidative stress from air pollution exposure. Building upon this air pollution exposure model system, I further explored how chemical oxidation of RNAs can be specifically enriched in certain RNA transcripts under oxidative stress induced by air pollution exposure. This work highlights specific oxidation of mRNAs involved in regulatory pathways as a possible mechanism for air-pollution related disease, suggesting that accumulation of oxidized mRNAs might interfere with processing or gene regulation. Additionally, examination of specific RNA oxidation associated with pre- and post-stress differential gene expression after exposure to air pollution highlights the potential that the oxidized RNA base 8-oxoguanosine might function as an epitranscriptomic mark affecting transcription. Lastly, I characterized how RNA modification machinery can contribute to cellular stress associated with expression of engineered transfer RNAs for applications in expanding the genetic code in E. coli. Using a suite of suppressor tRNAs designed by directed evolution, I demonstrated that the evolved lower expression level minimizes the impact of stress caused by interactions with E. coli post-transcriptional modification machinery, resulting in high performing tRNAs that remain functional regardless of their interactions with host PTM machinery. Collectively, the studies described in this dissertation demonstrate the power of employing cellular stress models to interrogate functional roles of RNA post-transcriptional modifications and highlight the myriad roles that PTMs can play in responding to and contributing to cellular stress.
Measuring chemicals with biology : engineering genetic biosensors for chemical analysis
(2021-08-10) d'Oelsnitz, Simon; Ellington, Andrew D.; Alper, Hal S.; Davies, Bryan W; Barrick, Jeffrey E; Liu, Hung-wen
Nature has evolved incredibly efficient and sustainable technology over millions of years, and we are just now learning how to reprogram it for our needs. In the past century this has been manifested in our ability to create proteins for breaking down and building up chemicals. Common laundry detergents contain a cocktail of enzymes for removing lipids, starches, and sugars from textiles. The pharmaceutical industry is embracing enzymes for more efficient drug synthesis. We are already adopting nature’s solution for chemical manipulation. The next frontier will involve adopting nature’s solution to chemical measurement. All living organisms use proteins, or genetic biosensors, to sense and respond to chemical cues. In the past two decades these natural biosensors have been repurposed for monitoring biomarkers, detecting chemical threats, and screening for improved catalysts. However, just as with enzymes, biosensors from nature oftentimes must be tailored for a desired application. This thesis describes novel methods for engineering genetic biosensors, as well as demonstrations of applications enabled by them, over three independent studies. In the first study, a new evolutionary approach to genetic biosensor design is described and subsequently used to create a series of highly specific sensors for five therapeutic alkaloids. One of these sensors is then used to rapidly evolve a plant methyltransferase enzyme capable of producing tetrahydropapaverine, an immediate precursor to four FDA-approved drugs. Next, the same evolutionary approach is used to create a generalist biosensor responsive to a wide range of otherwise intractable monoterpenes that are commonly used in the fragrance, flavor, cosmetic, and pharmaceutical industries. Finally, the last study describes two separate genetic biosensors engineered to monitor the incorporation of the noncanonical amino acids selenocysteine and L-DOPA. The utility of these sensors is then demonstrated by measuring the incorporation efficiency of thousands of seleno-competent bacterial strains and hundreds of L-DOPA aminoacyl-tRNA synthetases in hours, which would otherwise require months using traditional equipment. Scaling the approaches described herein will facilitate the industrialization of genetic biosensors for next- generation chemical analysis.
New tools and applications for genetically engineered insect symbionts
(2022-08-04) Elston, Katherine Marie; Barrick, Jeffrey E.; Moran, Nancy A; Davies, Bryan W; Havird, Justin C
Insects play a broad range of roles in natural ecosystems. They pollinate plants, recycle resources, and spread diseases. One integral component of the biology of many of these insects is the symbiotic bacteria that live inside of them. The relationships between symbionts and their insect hosts are well-studied in model systems like aphids, but despite this work, the ability to study genetic components of these relationships has been lacking. A major factor in this limitation is the deficit of tools that are available for genetically manipulating both non-model bacteria and insect species. In this work, I examine these limitations, along with the possibilities for the study of insect biology and control of insect pests that may arise if we can overcome them (Chapter 1). I develop methods to engineer recently isolated symbionts of aphids (Chapter 2), and fruit flies (Chapter 3). I then adapt these engineered aphid symbionts to try to alter aphid gene expression through symbiont-mediated RNAi (Chapter 4). I conclude with a summary of our results and the implications this work may have for the future of insect symbiont engineering (Chapter 5)
Of yeast and men : insights into evolution and human health from 1 billion years of divergence
(2021-05-08) Garge, Riddhiman Kannan; Marcotte, Edward M.; Wallingford, John B.; Wilke, Claus O.; Johnson, Arlen W.; Barrick, Jeffrey E.
Life on the planet is incredibly diverse and it is often easy to compare and contrast the many features that distinguish any two pairs of species from each other. Despite this diversity, all organisms on Earth share a common origin. This shared ancestry establishes conservation at the core of biology. The concept of conservation (or what’s equivalent) across species organizes biology and stems from the natural selection of favorable traits in organisms. Evolutionary conservation extends even to the genetic and molecular level with genes, proteins, and the networks they constitute also sharing common ancestry. This property enables biologists to study conserved genes (orthologs) in simpler model organisms and relate them to their corresponding human equivalents. Despite this, it is largely unclear the extents to which orthologs between species are functionally compatible. The dissertation aims to directly address this question via cross-species gene swaps. By systematically humanizing yeast genes, this dissertation provides insights into how orthologous genes between species functionally diverge and evolve over vast timescales. In chapter one, I present conservation as a powerful organizing principle in biology and the roles orthologous biological systems play in connecting genotype and phenotype. In chapters two and three, I describe efforts to apply humanized yeast as a platform to study functional divergence in orthologs constituting expanded gene families and examine the trends that underlie them. In chapter four, I describe the synthesis of observations from multiple research threads including humanized yeast, model organisms, evolutionary conservation of biological systems, and global signatures of pesticide resistance to uncover a novel class of antifungals all capable of functioning as vascular disrupting agents. Finally, in chapter five, I discuss the future of cross-species gene swaps, humanized yeast, and their utility to human health and disease
Programming the form and function of living materials using extracellular electron transfer
(2021-08-10) Graham, Austin Joseph; Keitz, Benjamin K.; Rosales, Adrianne; Maynard, Jennifer; Cosgriff-Hernández, Elizabeth
Living systems exist in a state of dynamic reciprocity, wherein bidirectional flow of chemical, mechanical, and electrical information directs the functional relationship between cells and their extracellular matrix. These matrices, often networks of various polymers, have macroscopic properties that are directly controlled by cellular activity, including gene and protein expression. This capability guides the development of cellular “living materials,” including biofilm growth and tissue formation. However, synthetic materials offer alternative function and diversity that may be unavailable to natural materials. To enhance living material design, we employ biological electron transfer as a programmable interface between living bacteria and synthetic materials. Coupling a variety of metal-catalyzed redox reactions to extracellular electron transfer from Shewanella oneidensis brings the corresponding synthetic reactions under genetic and metabolic regulation. This enables direct cellular control over a variety of abiotic materials and their properties, ranging from metallic nanoparticles to polymer networks. We actualize the proposed concepts in two parts. First, we explore the diversity of materials that can be programmed using extracellular electron transfer and investigate which properties can be tuned via genetic engineering of specific electron transfer pathways. We then focus on synthetic polymer networks, drawing from synthetic biology to develop design rules for living materials controlled by extracellular electron transfer. Throughout, we establish genetic and transcriptional links to abiotic material properties, expanding the synthetic reaction scope available to living organisms. This bottom-up approach to establishing dynamic and functional relationships between cells and their extracellular environment will eventually enable forward engineering of living materials whose specific properties are autonomously controlled by their constituent cells.
Simplified engineering of Acinetobacter baylyi ADP1 and evolutionary strategies for genome minimization
(2019-09-24) Suárez, Gabriel Antonio; Barrick, Jeffrey E.; Alper, Hal S; Moran, Nancy A; Miller, Kyle M; Davies, Bryan W
Our ability to engineer and domesticate microbes to give them useful properties promises grand rewards in the energy, agriculture, chemical and health industries. Yet, synthetic biologists often struggle to engineer bacterial genomes despite ever-improving genome-scale models of how they function. Often, they are stymied by the sheer complexity of the cell’s underlying systems biology and by how these continue to evolve rapidly after they are engineered. Recent advances in genome stabilization and genome simplification promise to overcome these barriers and profoundly extend our understanding of basic molecular biology and cellular life. Both the natural instability of bacterial genomes and their unexplored complexity (e.g., the presence of many genes with unknown functions) underlie major challenges to be reckoned with that often lead synthetic biologists to rely on extensive experimental trial and error. The construction of cells with minimal genomes to make microbiology more predictable is riddled with difficulties. There are sometimes advantages and sometimes disadvantages for removing more and more genes to simplify a bacterial cell. Similarly, evolution is a process that may both frustrate or enable synthetic biology. It can be slowed down by removing selfish DNA elements from a genome or it can be applied to compensate for suboptimal designs. The work in this thesis explores these interactions between genome design and evolution. It asserts that rational engineering and simplification principles can lay stronger foundations for engineering microbial cells so that more complex and ambitious designs can be successfully built, but that evolution is also a necessary tool to achieve extreme simplification of a living cell to make it robust enough for research and industrial demands to achieve the potential of synthetic biology. Our model organism is Acinetobacter baylyi ADP1, a highly naturally transformable and metabolically versatile soil bacterium. Chapter 1 provides an introduction to A. baylyi genetic engineering and the current state-of-the-art in bacterial genome stabilizing and streamlining projects. Chapter 2 describes our rational engineering efforts to reduce A. baylyi ADP1 genome instability– mainly by deleting all transposable elements from its genome–and the beneficial phenotypes in the ADP1-ISx strain that resulted from this work. Chapter 3 describes improved A. baylyi genome engineering methods and how they were used in the first stage of a genome streamlining project. We also describe a “Golden Transformation” protocol that speeds up and simplifies the steps needed to make precise edits to the A. baylyi genome and also show that the native CRISPR-Cas system is functional and can be reprogrammed using this method. Chapter 4 describes how we begin to test how compensatory evolution of reduced genomes can open new pathways to more extreme genome minimization by restoring fitness that is lost after deleting many dispensable genes from a genome. Chapter 5 discusses future directions for making improvements that further stabilize and streamline the A. baylyi genome. Together, the work presented in this dissertation presents concepts, tools, and insights into strategies that were successful and unsuccessful for building a better and simpler Acinetobacter baylyi ADP1 genome. These approaches can also be applied to other bacterial species to propel the goals of synthetic biology forward
Synthetic transcription systems
(2010-05) Davidson, Eric Alan; Ellington, Andrew D.; Bull, James J.; Marcotte, Edward M.; Whitman, Christian P.; Yin, Yuhui W.
In this work, we seek to expand synthetic in vitro biological systems by using water-in-oil emulsions to provide an environment conducive to directed evolution. We approach this primarily by utilizing a model transcription system, the T7 RNA polymerase and promoter, which is orthogonal to both bacterial and eukaryotic transcription systems and is highly functional in vitro. First, we develop a method to identify functional promoter sequences completely in vitro. This method is tested using the T7 RNA polymerase-promoter model system. We then configure the T7 transcription system as an ‘autogene’ and investigate how this positive feedback circuit (whereby a T7 promoter expresses a T7 RNA polymerase gene) functions across various in vitro platforms, including while compartmentalized. The T7 autogene can be envisioned as a self-replicating system when compartmentalized, and its use for directed evolution is examined. Finally, we look towards future uses for these in vitro systems. One interesting application is to expand the utilization of unnatural base pairs within the context of a synthetic system. We investigate the ability of T7 RNA polymerase to recognize and utilize unnatural base pairs within the T7 promoter, complementing existing work on the utilization of unnatural base pairs for in vitro replication and transcription with an investigation of more complex protein-dependent regulatory function. We envision this work as a foundation for future in vitro synthetic biology efforts.
The impact of synthetically expanded genetic codes on evolvability
(2016-07-07) Hammerling, Michael J.; Barrick, Jeffrey E.; Bull, James; Ellington, Andrew; Marcotte, Edward; Molineux, Ian; Wilke, Claus
Synthetic biology allows researchers to address previously unanswerable biological questions through the wholesale reengineering of living systems. Even one of the most fundamental properties of biology – the canonical genetic code for the translation of genetic information into proteins – may now be altered to include nonstandard amino acids (nsAAs) because synthetic incorporation systems for nsAAs now match native host machinery in terms of efficiency and fidelity. However, the evolutionary stability of these systems and the potential for them to promote the evolvability of the organisms in which they are deployed remains mostly unexplored. In chapter one of this dissertation, I explore how Escherichia coli strains that incorporate a 21st nsAA at the recoded amber (TAG) stop codon evolve resistance to the antibiotic rifampicin. I found a variety of mutations that lead to substitutions of nsAAs in the essential RpoB protein confer robust rifampicin resistance, and I interpret these results in a framework in which an expanded code can increase evolvability in two distinct ways. I consider the implications of these results for the evolution of alternative genetic codes, in particular their support for the codon capture model of genetic code evolution. In chapters 2 and 3, I employ bacteriophage T7, which utilizes the translation apparatus of its E. coli host, as a model organism for whole-genome evolution with an expanded genetic code. I show that phages evolved on an E. coli host that incorporates 3-iodotyrosine became dependent on an alternative genetic code as they adapted to higher fitness. In particular, phages with a 3-iodotyrosine substitution in the type II holin protein outcompete phages with canonical amino acids at this position. Inspired by this surprising outcome, I performed massively parallel evolution experiments in T7 with six expanded genetic codes, finding over a thousand substitutions to nsAAs in these experiments, many of which displayed genomic signatures of positive selection. Two of the nsAAs accumulated in genomes at a rate comparable to a mock genetic code expansion with the canonical amino acid tyrosine. Together these results show that expanded genetic codes alter functional sequence space and create new possibilities for adaptive mutations, thereby improving evolvability in these organisms.
Towards a predictive functional synthesis : routing top-down and bottom-up approaches in biology
(2020-08-13) Cole, Austin Woodrow; Ellington, Andrew D.; Bull, James; Barrick, Jeffrey; Pogue, Gregory; Davies, Bryan
Nothing in evolution makes sense except in the light of biology. The collection of experiments described here attempts to engineer biology to influence evolution — this builds on and inverts an approach to parse historical evolutionary realities through molecular experimentation. Several different avenues are described here to prospectively anticipate the patterns of evolution, and a small selection of informative studies that set the stage for these experiments are presented in chapter one. Chapters two and three detail how engineered codon tables may bias evolution under two different types of selection. These chapters speak to the rigidity of the existing codon table and the broad adaptive potential that exists within the current set of 20 amino acids. Chapters four and five restrict their focus to engineer the genomes of single stranded viral individuals rather than amino acid repertoires, and then lay out how these synthetic genomes adapt in response to designed architectures. Lastly, chapter six describes new tools to amalgamate ‘bottom-up’ chemical information and then uses an algorithm embedding that information to guide the engineering of improved protein phenotypes. Taken together, these approaches form an anthology of strategies that use biological engineering to prospectively bias the adaptation of populations.