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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.

Combinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers. Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit. / Ph. D.

Sensorische Photorezeptorproteine vermitteln vielfältige Lichtreaktionen in allen Domänen des Lebens. Oftmals dienen verschiedene, durch helikale ‚Linker’ gekoppelte, Module der Lichtperzeption (Sensor) und der Umwandlung in ein biologisches Aktivität (Effektor). Der Zusammenbau chimärer Photorezeptoren aus unterschiedlichen Sensoren und Effektoren ermöglicht die präzise und minimalinvasive Regulation zellulärer Signalwege mit Hilfe von Licht, zu therapeutischen oder analytischen Zwecken. Eine große Herausforderung stellt dabei die korrekte Fusion der Linker beider Module dar, die Kommunikation zwischen Sensor und Effektor erlaubt. Die vorliegende Arbeit nimmt sich diesem Problem an und untersucht Strategien zum effizienten Bau chimärer Photorezeptorproteine. Ein rationaler, auf Sequenz- und Strukturhomologie der parentalen Proteine basierender Ansatz wurde maßgeblich durch unzureichendes Verständnis der funktionellen Mechanismen dieser modularen Proteinen erschwert. Die neuentwickelte und PATCHY-Methode umgeht dieses Hindernis, indem sie eine Bibliothek von Chimären aller Kombinationen der parentalen Linker generiert, welche anschließend mittels bakterieller Testsysteme nach funktionalen Varianten durchsucht wird. Angewendet auf die Fusion eines LOV-Blaulichtsensors und eines Histidinkinase-Effektors fanden sich sowohl lichtaktivierte, als auch zu lichtreprimierte Chimären, deren Linkerlängen jeweils einer Heptadenperiodizität folgten. Dass weniger als 5% aller Linkerkombinationen zu lichtregulierten Chimären führten, deutet zudem auf eine feine Abstimmung von Linkersequenz und Proteinfunktion hin. Die systematische Analyse von Fusionsvarianten mit PATCHY dient daher nicht nur der Entwicklung chimärer Rezeptorproteine zur Manipulation zellulärer Prozesse. Sondern sie zeigt darüber hinaus, komplementär zum rationalen Ansatz, molekulare Faktoren auf, die zur Modulkompatibilität und Signaltransduktion modularer Rezeptorproteine beitragen. / Sensory photoreceptor proteins mediate diverse responses to ambient light in all domains of life. Often distinct modules coupled by helical linkers enable light perception (sensor) and biological output function (effector). Rewiring different sensor and effector modules into photoreceptor chimeras allows using light to control target cellular processes with high spatiotemporal accuracy and minimal invasiveness for therapeutic or analytical purposes. Thereby, a major design challenge is fusing the linkers from both modules in a way that preserves signal transduction within the chimera. The present study tackles this issue and explores strategies for engineering photoreceptor chimeras. An initial rational-design approach guided by sequence and structure homology of the parent proteins was greatly hampered by insufficient knowledge of signaling mechanisms within these modular proteins. A novel and easy-to-use brute-force strategy, termed PATCHY (primer-aided truncation for the creation of hybrid enzymes) circumvents this problem by generating a complete library of fusion variants between target modules harboring all combinations of the parent linkers. Screening fusion libraries of a LOV (light-oxygen-voltage) blue-light sensor coupled to a histidine-kinase effector yielded light-induced and light-repressed chimeras, each group complying with a heptad periodicity of linker lengths. With less than 5% of all possible variants exhibiting light regulation, a delicate fine-tuning of linker sequence and protein function became evident. Thus, systematic testing of fusion variants with PATCHY not only facilitates the development of photoreceptor chimeras for manipulating cellular processes. Complementary to rational design, it also reveals molecular cues determining module compatibility and signal transduction in modular signal receptors.

The natural environments of organisms present a multitude of biotic and abiotic challenges that require both short-term ecological and long-term evolutionary responses. Though most environmental response studies have focused on effects at the ecosystem, community and organismal levels, the ultimate controls of these responses are located in the genome of the organism. Soil nematodes are highly responsive to, and display a wide variety of responses to changing environmental conditions, making them ideal models for the study of organismal interactions with their environment. In an attempt to examine responses to environmental stress (desiccation and freezing), genomic level analyses of gene expression during anhydrobiosis of the Antarctic nematode Plectus murrayi was undertaken. An EST library representative of the desiccation induced transcripts was established and the transcripts differentially expressed during desiccation stress were identified. The expressed genome of P. murrayi showed that desiccation survival in nematodes involves differential expression of a suite of genes from diverse functional areas, and constitutive expression of a number of stress related genes. My study also revealed that exposure to slow desiccation and freezing plays an important role in the transcription of stress related genes, improves desiccation and freezing survival of nematodes. Deterioration of traits essential for biological control has been recognized in diverse biological control agents including insect pathogenic nematodes. I studied the genetic mechanisms behind such deterioration using expression profiling. My results showed that trait deterioration of insect pathogenic nematode induces substantial overall changes in the nematode transcriptome and exhibits a general pattern of metabolic shift causing massive changes in metabolic and other processes. Finally, through field observations and molecular laboratory experiments the validity of the growth rate hypothesis in natural populations of Antarctic nematodes was tested. My results indicated that elemental stoichiometry influences evolutionary adaptations in gene expression and genome evolution. My study, in addition to providing immediate insight into the mechanisms by which multicellular animals respond to their environment, is transformative in its potential to inform other fundamental ecological and evolutionary questions, such as the evolution of life-history patterns and the relationship between community structure and ecological function in ecosystems.