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

This thesis is built upon two areas of research concerning metal-organic frameworks (MOFs). The first focuses on the functionalisation of MOFs via post-synthetic modification (PSM). The second involves the investigation on the potential of MOFs as hosts for insect pheromones. Chapter 1 introduces the field of MOF chemistry, and covers their properties along with a brief description of their applications. The concept of PSM is introduced and a review of recent literature given. The aims of the thesis are also detailed at the end of this chapter. Chapter 2 describes the PSM of [Zr6O4(OH)4(BDC-NH2)6], UiO-66-NH2, via Aza-Michael reactions. Different functionalities were successfully introduced into its pores and the degrees of conversion were determined via 1H NMR spectroscopy. Gas sorption measurements (CO2 and N2) of the PSM products were carried out and compared. In particular, two PSM products were shown to exhibit higher CO2 over N2 selectivity than that for the starting MOF, UiO-66-NH2. Chapter 3 describes a new PSM route in obtaining azole-functionalised MOFs via Mannich reactions. The amino groups in three different MOFs were converted into a range of azole-functionalised MOFs with conversions up to 100%. In particular, one of the PSM reactions afforded a new material, formulated as [Zn3(BDC-NH2)1.32(BDC-NHCH2N2C3H3)1.68(C6H12N2)], based on single crystal X-ray crystallography, 1H NMR and TGA analyses. Gas sorption studies demonstrate increased selectivity for CO2 over N2 for the PSM products. One of the modified MOFs was shown to exhibit a high Hg(II) uptake from aqueous solutions. Chapter 4 introduces the concept of using MOFs as hosts for ant pheromones. The factors which influenced the pheromone loading in zinc and zirconium based MOFs were investigated. The MOFs containing the linker BDC-NHPr (2-(propylamino)benzene-1,4-dicarboxylate) were found to be effective at hosting two types of ant pheromones, 3-octanone and (S)-4-methyl-3-heptanone.

The goal of this project was to clone and express the antimicrobial peptide protegrin 1 (PG-1). Initially a yeast system was chosen but was discarded due to technical difficulties. Invitrogen's bacterial T7 expression system was chosen next to express the peptide. PG-1 expression was verified by anti-his immunoblot and then the peptide was purified by IMAC. Its activity was verified using a Bacillus subtillis radial diffusion assay.

The cement industry contributes around 5-7% of man-made CO2 emissions globally because of the Portland Cement (PC) production. Therefore, innovative reactive magnesia cement, with significant sustainable and technical advantages, has been proposed by blending reactive MgO and hydraulic binders in various proportions. MgO is currently produced from the calcination of magnesite (MgCO3), emitting more CO2 than the production of PC, or from seawater/brine which is also extremely energy intensive. Hence this research aims to investigate an innovative method to produce MgO from reject brine, a waste Mg source, through carbon sequestration, by its reaction with CO2, to provide a comparable low carbon manufacturing process due to the recycling of CO2. The produced deposits are then calcined to oxides with potential usage in construction industry. The entire system is a closed loop to achieve both environmental optimisation and good productivity. This research focuses on the chemical manufacturing process, integrated with material science knowledge and advancements, instead of concentrating purely on chemistry evaluations. Six series of studies were conducted, utilising MgCl2, CaCl2, MgCl2-CaCl2, MgCl2-CaCl2-NaCl, and MgCl2-CaCl2-NaCl-KCl to react with CO2 under alkaline conditions. The precipitates include hydrated magnesium carbonates, calcium carbonates and magnesian calcite. Generated carbonates were then calcined in a furnace to obtain MgO, CaO or dolime (CaO•MgO). All six series of carbonation processes were carried out under a controlled pH level, to study the constant pH’s effect on the process and resulting precipitates. Other controllable factors include pH, temperature, initial concentration, stirring speed, and CO2 flux rate. In conclusion, the optimum parameters for the production of the carbonated precipitates are: 0.25MgCl2 + 0.05CaCl2 + 2.35NaCl + 0.05KCl, 700rpm stirring speed, 25oC room temperature, pH=10.5, and 500cm3/min CO2 infusion rate. Reaction time is within a day. These parameters are chosen based on the sequestration level, particle performance morphology and the operational convenience. The optimum calcination parameters are at 800oC heating temperature with a 4h retention time.

Thesis. 1976. M.S.--Massachusetts Institute of Technology. Dept. of Mechanical Engineering. / Microfiche copy available in Archives and Engineering. / Includes bibliographical references. / by Julian A. LoRusso. / M.S.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Synthetic chemistry has played a pivotal role in the evolution of modern life. More recently, the emerging field of synthetic biology holds the promise to bring about a paradigm shift with designer microbes to renewably synthesize complex molecules in a fraction of the time and cost. Still, given synthetic chemistry’s superior parsing powers to access a greater number of unnatural end products and nature’s virtuosity at stitching a staggering palette of carbon frameworks with ease, a hybrid approach that leverages the respective strengths of the two fields could prove advantageous for the efficient production of valuable natural molecules and their analogs. In a first demonstration of the hybrid approach where the biosynthesized intermediate is not part of the target molecule’s biosynthetic pathway, we engineered E. coli to produce Z,E-farnesol, which we subsequently transformed into a library of novel analogs of the commercially important amber fragrance Ambrox®, including the first synthetic patchouli scent. In a second demonstration of the hybrid approach, we produced the valuable tocotrienols (vitamin E) from yeast-produced geranylgeraniol in a single step C–C coupling with concomitant regioselective cycloetherification of the most proximal vinyl of the polyene, the first such process of its kind. The novel acid catalyst system that allowed for this unique regioselective cyclization holds promise as an asymmetric proton transfer tool and could open the door to facile asymmetric synthesis of vitamin E and other molecules.

Mechanistic understanding of gene regulation is crucial for rational engineering of new genetic systems through synthetic biology. Genetic engineering efforts in new organisms are often hampered by a lack of knowledge about how regulatory components function in new host contexts. This dissertation focuses on efforts to overcome these challenges through the development of generalizable experimental methods for studying the behavior of DNA regulatory sequences in diverse species at large-scale. Chapter 2 describes experimental approaches for quantitatively assessing the functions of thousands of diverse natural regulatory sequences through a combination of metagenomic mining, high-throughput DNA synthesis and deep sequencing. By employing these methods in three distinct bacterial species, we revealed striking functional differences in gene regulatory capacity. We identified regulatory sequences with activity levels with activity levels spanning several orders of magnitude, which will aid in efforts to engineer diverse bacterial species. We also demonstrate functional species-selective gene circuits with programmable host behaviors that may be useful for microbial community engineering. In Chapter 3 we provide evidence for the evolution of altered stringency in σ70-mediated transcriptional activation based on patterns of initiation and activity from promoters of diverse compositions. We show that the contrast in GC content between a regulatory element and the host genome dictates both the likelihood and the magnitude of expression. We also discuss the potential implications of this proposed mechanism on horizontal gene transfer. The next two chapters focus on efforts aimed at extending the high-throughput methods described in earlier chapters to new organisms. Chapter 4 presents an in vitro approach for multiplexed gene expression profiling. Through the development and use of cell-free expression systems made from diverse bacteria, it was possible to rapidly acquire thousands of transcriptional measurements in small volume reactions, enabling functional comparisons of regulatory sequence function across multiple species. In Chapter 5 we characterize the restriction-modification system repertoires of several commensal bacterial species. We also describe ongoing efforts to develop methods for bypassing these systems in order to increase transformation efficiencies in species that are difficult or impossible to transform using current approaches.

Burkholderia pseudomallei is the aetiological agent of melioidosis and a potential bioterror threat. Infections are difficult to treat due to extensive antibiotic resistance and there is no prophylactic vaccine available. Studies have shown that the capsular polysaccharide (CPS) of B. pseudomallei is a virulence factor, immunogen and candidate antigen for a glycoconjugate vaccine. However, polysaccharides are complex to synthesise. One approach is to genetically engineer Escherichia coli to express the CPS; however, previous attempts at cloning the CPS coding locus from B. pseudomallei into E. coli were unsuccessful. This project proposes to clone only the essential genes from B. pseudomallei and to use native E. coli mechanisms to complete CPS synthesis. This would contribute to development of a new platform for the expression of any bespoke polysaccharide in E. coli. Six biosynthetic genes for the nucleotide sugar precursor were successfully expressed in E. coli. The structure of the precursor was verified by mass spectrometry. Precursor synthesis was also performed in an in vitro microfluidics system. This minimised the quantity of substrates and enzymes required, in preparation for the characterisation of glycosyltransferases required for CPS assembly. A novel assay for characterising glycosyltransferase activity was also developed, as current available options are prohibitively expensive and require significant quantities of glycosyltransferase which are difficult to purify. Finally, plasmids for the expression of additional glycosyltransferases to link the nascent B. pseudomallei CPS to truncated polysaccharides in E. coli were constructed. The aim of this project was to contribute to the development of a platform for the expression of bespoke polysaccharides in E. coli. The CPS of B. pseudomallei was chosen as the model polysaccharide as it has a simple structure and its manufacture is desirable for use in a vaccine against melioidosis.

The object of inquiry of this thesis is synthetic biology. In this thesis I ask what is this ‘thing’ that is synthetic biology (Latour, 2005) and what might it mean for synthetic biology to inhabit the world and to inhabit it well? Synthetic biology’s coming into being has been accompanied by a considerable amount of ‘hype’ and ‘hyperbole’ (Marris and Rose, 2012) – by what the philosopher Annemarie Mol (1999) would describe as a noisy ‘perspectivalism.’ My aim in this thesis is to contribute to the telling of different kinds of less-perspectival and less-technologically-deterministic stories about the development of this burgeoning approach to biological engineering. In drawing on a combination of empirical material from over 30 1-2 hour interviews with leading synthetic biologists and ethnographic materials generated from working alongside the UK’s Health and Safety Executive (the UK regulatory authority responsible for overseeing the development of synthetic biology), I aim to multiply outwards registers for understanding what synthetic biology is and what it might become. I highlight, for example, how synthetic biology is not simply a hubristic endeavour (Lewens, 2013) but is also about processes of learning and apprehension. What’s more, depending on how synthetic biology takes shape(s) in different practices, ‘time’ also becomes aleatory and freed from its modernist shackles (Serres, 2008). I use the lens of regulation as a means of addressing the question of what it might mean for synthetic biology to inhabit the world well. Synthetic biology’s regulatory provocations have been largely underexplored within STS and human geography literatures. The thesis is informed by and builds upon, theoretical notions of multiplicity (Mol, 2002) and of syncretisms (Law and Mol, 2013). The thesis contributes to a broader shift in social theory from critique towards compositionism and concludes by arguing for the development of a modest geography of novel biologies.