Gene regulation in C. elegans germ cells depend on transgenerational chromatin modification and small RNA pathways. Germline silencing mechanisms evolved to repress foreign DNA from compromising the transfer of genetic information to progeny. Effective genetic tools that circumvent the silencing machinery will facilitate studies using this model organism. Specifically, translation of heat-shock inducible transgenes is inhibited in the germline making it challenging to transiently express enzymes to modify the genome. Here, we describe a genetic screen design that can be used to identify pathways that prevent germline expression of heat-shock induced transgenes. We use split-GFP (GFP1-10 and GFP11) to confine a genetic screen to germ cells. Stable transgenic lines with germline expression of single-copy integrated GFP11 were produced using MosSCI. The insertion lines will be used in RNAi or chemical mutagenesis screens for the germline de-repression of GFP1-10 expressed under heat-shock promoters. The screen is likely to identify candidate RNAi or chromatin factors involved in repressing heat-shock expression in the germline, particularly from extrachromosomal arrays. Inducible high-level expression in the germline from extrachromosomal arrays would be a valuable tool for large-scale genome engineering.

Modern synthetic gene regulatory networks emerge from iterative design-build-test cycles that encompass the decisions and actions necessary to design, build, and test target genetic systems. Historically, such cycles have been performed manually, with limited formal problem-definition and progress-tracking. In recent years, researchers have devoted substantial effort to define and automate many sub-problems of these cycles and create systems for data management and documentation that result in useful tools for solving portions of certain workflows. However, biologists generally must still manually transfer information between tools, a process that frequently results in information loss. Furthermore, since each tool applies to a different workflow, tools often will not fit together in a closed-loop and, typically, additional outstanding sub-problems still require manual solutions. This thesis describes an attempt to create a tool that harnesses many smaller tools to automate a fully closed-loop decision-making process to design, build, and test synthetic biology networks and use the outcomes to inform redesigns. This tool, called Phoenix, inputs a performance-constrained signal-temporal-logic (STL) equation and an abstract genetic-element structural description to specify a design and then returns iterative sets of building and testing instructions. The user executes the instructions and returns the data to Phoenix, which then processes it and uses it to parameterize models for simulation of the behavior of compositional designs. A model-checking algorithm then evaluates these simulations, and returns to the user a new set of instructions for building and testing the next set of constructs. In cases where experimental results disagree with simulations, Phoenix uses grammars to determine where likely points of design failure might have occurred and instructs the building and testing of an intermediate composition to test where failures occurred. A design tree represents the design hierarchy displayed in the user interface where progress can be tracked and electronic datasheets generated to review results. Users can validate the computations performed by Phoenix by using them to create sets of classic and novel temporal synthetic genetic regulatory functions in E. coli. / 2016-12-31T00:00:00Z

Antibiotics and their corresponding resistance genes act as a tool to control bacterial survival. Antibiotic resistance is used to select for desired engineered cells, and study how pathogens acquire resistance to continue infection. Here, we develop tools to control the expression of antibiotic resistance genes using light. To accomplish this, we use optogenetics, the regulation of cellular behavior using light as a direct and programmable input for gene expression. We develop an optogenetic recombinase in Escherichia coli through split-protein engineering techniques, and characterize the behavior of our best candidate in detail: a split Cre recombinase that responds to blue light. We apply this optogenetic system to control the expression of resistance genes for four antibiotics: ampicillin/carbenicillin, kanamycin, chloramphenicol, and tetracycline. By varying the expression levels of these genes, we tune the concentrations at which bacteria can survive before and after light exposure. We then apply this system to improve production of fatty acids. Finally, we make progress toward characterizing the impact of resistance activation timing on bacterial survival. This work creates tools that are broadly useful for spatiotemporal control of bacterial survival, and enables precise studies on how bacterial resistance spreads at the single-cell level. / 2024-08-29T00:00:00Z

No description available.

Synthetic biology is an emerging interdisciplinary research field, which leverages the maturation of DNA synthesis technologies. By introducing engineering principles to synthetic biological systems design, synthetic biology shows great potential to shed new lights on biology and benefit human beings. Computer assisted design (CAD) tools will play an important role in the rational design of synthetic genetic systems. This dissertation presents the first CAD tool for synthetic biology — GenoCAD, a linguistic-based web application. By viewing DNA sequences as a language, we developed the first syntactic model to design and verify synthetic genetic constructs. Then we conducted a careful curation of the terminal set in the grammar - the first comprehensive analysis of the Registry of standard biological parts. The implementation and major features of GenoCAD are discussed, and in particular we showed how to develop a domain-specific grammar for BioBrick-based construct design and make GenoCAD a useful tool for the iGEM students. Finally, we went beyond the syntactic level to explore the semantics of synthetic DNA sequences: by associating attributes with biological parts and coupling semantic actions with grammar rules, we developed the first semantic models to relate the genotype to the phenotype of synthetic genetic constructs. The theories and techniques presented in this dissertation, along with the informative results presented, will serve as a foundation for the future developments of GenoCAD. / Ph. D.

The field of synthetic biology has leveraged engineering tools such as molecular cloning to create new biological components, networks, and processes. While many of these components and networks have been deployed in the cytosol, there is a shortage of systems that utilize the surface of the cell. In order to address this shortcoming, we have created a synthetic, surface-displayed substrate anchor for bacteria. This approach allows us to engineer surface-based synthetic biological systems as a complement to existing intracellular approaches. We leveraged the tools of synthetic biology to display a catalytically active enzyme that covalently bonds itself to benzylguanine (BG) groups. We created a fusion protein allowing us to place human O6-alkylguanine DNA alkyltransferase (hAGT), also known as SNAP, on the surface of a bacterial cell. Initially, we used this synthetic component as a tool for spatially segregating orthogonal synthetic gene outputs by visualizing an extracellular synthetic green fluorescent reporter, SNAP-Cell® 505-Star, simultaneously with an intracellular red fluorescent protein, mCherry. Moreover, we have shown that our construct enables cells to selectively bond to BG-conjugated magnetic beads. As a result, we have demonstrated that surface displayed SNAP facilitates engineering a direct channel between intracellular gene expression and extracellular material capture. In the near future, we believe this magnetic capture can be expanded as a sortable reporter for synthetic biology as a direct extension of this work. Moreover, our work serves as an enabling technology, paving the way for extracellular synthetic biological systems that may coexist orthogonally to intracellular processes. / Master of Science

Microrobotics is an emerging discipline with the potential to radically affect fields ranging from medicine to environmental stewardship. Already, there have been remarkable breakthroughs; small scale robots have been made that can selectively traverse the gastrointestinal tract, and others have been built that can fly in a manner inspired from bees. However, there are still significant challenges in microrobotics, and it remains difficult to engineer reliable power sources, actuators, and sensors to create robust, modular designs at the microscale. The miniaturization of the robotic system makes design and efficiency of these components particularly difficult. However, biological systems demonstrate the key features of robotics " sensing, actuation, processing" and are remarkably complex at the microscale. As such, many researchers have turned to biology for inspiration and living robotic components. In our laboratory we have engineered an Escherichia coli (E. coli) capable of producing surface display proteins to either anchor the cells, bind to functionalized nanoparticles, or capture small molecules from the environment, all complex actuation features. Additionally, we have created a processing unit that can create signals based on biological components, yet is non-living. This thesis focuses on the characterization of the surface display E. Coli system and the creation of programmable microparticle scaffolds that may be controlled by biological circuitry. In particular, by leveraging the strong interaction between biotin and streptavidin, I have created programmable microparticle scaffolds capable of attenuating the intensity of a fluorescent response in response to perturbations in the local environmental conditions. We believe this is an excellent enabling technology to facilitate the creation of complex behaviors at the microscale and can be used as a processing unit for simple decision making on microrobots. We foresee this technology impacting disciplines from medical microrobotics to environmental sensing and remediation. / Master of Science

The demand for economical, efficient protein production, reuse, and recovery has never been greater due to their versatility in a large variety of applications ranging from industrial chemical manufacturing to pharmaceutical drug production. The applications for naturally and artificially produced proteins include protein drugs and other pharmaceutical products, as biocatalysts in environmentally friendly chemical manufacturing, as enzymes for food processing purposes, and as an essential component in many biomedical devices. However, protein production suffers from many challenges, which include the cost of production, protein stability especially under harsh conditions, and recoverability and reusability of the proteins. The combination of two developing technologies, cell-free protein synthesis systems (CFPS) and unnatural amino acid incorporation, provides solutions to these protein production challenges.This dissertation reports on the use of cell-free protein synthesis systems and unnatural amino acid incorporation to develop new proteins and enzyme immobilization techniques that significantly increase activity and stability while simplifying recoverability and reuse.

Microbial metabolism can be tailored to meet human specifications, but the degree to which these living systems can be repurposed is still unknown. Artificial biological control strategies are being developed with the goal of enabling the predictable implementation of novel biological functions (e.g., engineered metabolism). This dissertation project contributes genetic tools useful for modulating gene expression levels (extending promoters with UP elements) and isolating transcription and translation of engineered DNA from the endogenous cellular network (expression by orthogonal cellular machinery), which have been demonstrated in Escherichia coli for the production of lycopene, a 40-carbon tetraterpene carotenoid with antioxidant activity and a number of other desirable properties.

Ebolavirus is responsible for a deadly hemorrhagic fever that has claimed thousands of lives in Africa and could become a global health threat. Because of the danger of infection, novel Ebola research is restricted to BSL-4 laboratories; this slows progress due to both the cost and expertise required to operate these laboratories. The development of a safe surrogate would speed research and reduce risk to researchers. Two highly conserved Ebola gene segments—from the glycoprotein and nucleoprotein genes—were designed with modifications preventing expression while maintaining sequence integrity, spliced into high copy number plasmids, cloned into E.coli, and tested for stability, safety, and potential research applications. The surrogates were stable over 2-3 months, had a negligible mutation rate (<0.165% over the experiment), and were detectable in human blood down to 5.8E3-1.17E4 surrogates/mL. These protocols could be used to safely simulate other pathogens and promote infectious disease treatment and detection research. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection