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Title: Identification and characterization of cold shock loci in Sinorhizobium meliloti 1021
Creator: Gustafson, Ann M. (Ann Marie)
Date: 1999
Collection: Electronic Theses & Dissertations

Title: Mutants of the E. coli dnaA gene : genetic and biochemical analysis of its replication activity
Creator: Sutton, Mark David
Date: 1996
Collection: Electronic Theses & Dissertations

Title: Response of model microbial communities to increased productivity
Creator: Bohannan, Brendan James Marc
Date: 1997
Collection: Electronic Theses & Dissertations

Title: The gol site : a bacteriophage T4 regulatory region that can affect expression of all the T4 late genes
Creator: Champness, Wendy Cooley
Date: 1982
Collection: Electronic Theses & Dissertations

Title: Conversion of the mitochondrial gene for mammalian cytochrome oxidase subunit II to a universal equivalent and expression in E. coli, in vitro, and in Xenopus oocytes
Creator: Cao, Jianli
Date: 1990
Collection: Electronic Theses & Dissertations

Title: The interaction of Escherichia coli gene products with mutant forms of dnaA protein
Creator: Hupp, Theodore Robert
Date: 1990
Collection: Electronic Theses & Dissertations

Title: Bioavailability of tetracycline in water and soil to Escherichia coli for expression of antibiotic resistance
Creator: Zhang, Yingjie
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Tetracyclines are a class of antimicrobials extensively used as human and veterinary medicine, and in livestock production since they were discovered in the 1940s. A large portion of tetracyclines administered to humans and animals are excreted and subsequently released into the environment, where they pose potential risks to ecosystem and human health. There is a growing concern that the presence of antibiotics such as tetracycline at trace levels in the environment is related to the...
Show moreTetracyclines are a class of antimicrobials extensively used as human and veterinary medicine, and in livestock production since they were discovered in the 1940s. A large portion of tetracyclines administered to humans and animals are excreted and subsequently released into the environment, where they pose potential risks to ecosystem and human health. There is a growing concern that the presence of antibiotics such as tetracycline at trace levels in the environment is related to the emergence and ever-increasing abundance of antibiotic resistance genes in natural and engineered microbial populations. However, basic knowledge at the molecular scale of bacterial access to tetracyclines present in environmental matrices and expression of antibiotic resistance genes remain nearly unknown. In this study, we used the E. coli MC4100/pTGM whole-cell bioreporter as an effective tool to investigate bioavailability of tetracycline in water and soil to bacteria for expression of antibiotic resistance genes. Our hypothesis was that the speciation of tetracycline dissolved in water and sorption by soil minerals controls the bioavailabilities for bacterial uptake and subsequent activation of antibiotic resistance genes. The results revealed that activation of antibiotic resistance in the E. coli bioreporter responded linearly to intracellular tetracycline concentration. The extent of tetracycline uptake by E. coli was modulated by tetracycline speciation. We have identified that zwitterionic tetracycline as the primary species favorable for bacterial uptake. Geochemical factors such as pH, salt composition and concentration influenced the fractional distributions of tetracycline species in aqueous solution and hence altered uptake by E. coli. In addition, the presence of organic ligands could also alter tetracycline speciation by releasing tetracycline from its metal complexes in aqueous solution. For tetracycline associated with Mg-smectite, desorption of tetracycline from clay to solution was the major exposure pathway for bacterial uptake and subsequent activation of antibiotic resistance in the diluted clay suspensions. In clay film cultivation, clay-sorbed tetracycline was still bioaccessible to E. coli evoking strong expression of antibiotic resistance. Direct contact of the E. coli bioreporters with clay surfaces and further formation of biofilms plausibly facilitated tetracycline transfer to bacteria. Overall, this study greatly advances the fundamental understanding of bioavailability of tetracycline in the environment to bacteria for expression of antibiotic resistance genes.
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Title: Deconstructing the correlated nature of ancient and emergent traits : an evolutionary investigation of metabolism, morphology, and mortality
Creator: Grant, Nkrumah Alions
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Phenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have...
Show morePhenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have maintained - and even improved - the capacity of E. coli to grow in an anoxic environment despite 50,000 generations of relaxed selection for anaerobic growth. I present genomic evidence illustrating substantially more mutations have accumulated in anaerobic-specific genes and show parallel evolution at two genetic loci whose protein products regulate the aerobic-to-anaerobic metabolic switch. My findings reject the "if you don't use it, you lose it" notion underpinning relaxed selection and show modules with deep evolutionary roots can overlap more, hence making them harder to break. In chapter 2, I revisit previous work in the LTEE showing that the fitness increases measured for the 12 populations positively correlated with an increase in cell size. This finding was contrary to theory predicting smaller cells should have evolved. Sixty thousand generations have surpassed since that initial study, and new fitness data collected for the 12 populations show fitness has continued to increase over this period. Here, I asked whether cell size also continued to increase. To this end, I measured the size of cells for each of the 12 populations spanning 50,000 generations of evolution using a particle counter, microscopy, and machine learning. I show cell size has continued to increase and that it remains positively correlated with fitness. I also present several other observations including heterogeneity in cell shape and size, parallel mutations in cell-shape determining genes, and elevated cell death in the single LTEE population that evolved a novel metabolism - namely the ability to grow aerobically on citrate. This last observation formed the basis of my chapter 3 research where my collaborators and I fully examine the cell death finding and the associated genotypic and phenotypic consequences of the citrate metabolic innovation.
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Title: Coevolution of bacterial-phage interactions
Creator: Meyer, Justin R.
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Bacteria and their viruses, phage, are the most abundant and genetically diverse group of organisms on earth. Given their prevalence, it is no wonder that recent studies have found their interactions important for ecosystem function, as well as the health of humans. Unfortunately, because of technical challenges with studying microbes, some of the most basic questions on their interactions, such as who infects whom, and how their relationships evolved in the first place, remain unanswered....
Show moreBacteria and their viruses, phage, are the most abundant and genetically diverse group of organisms on earth. Given their prevalence, it is no wonder that recent studies have found their interactions important for ecosystem function, as well as the health of humans. Unfortunately, because of technical challenges with studying microbes, some of the most basic questions on their interactions, such as who infects whom, and how their relationships evolved in the first place, remain unanswered. Here I report six studies on bacterial-phage interactions, each focused on understanding their pattern and the underlying biophysical, ecological, and evolutionary processes that shape them. To do this, I tested a number of hypotheses using laboratory experiments and analyses of natural microbial diversity. First, I tested whether Esherichia coli cultured without phage would counter-intuitively evolve new interactions with phage. Typically bacterial traits responsible for phage resistance have pleiotropic consequences on growth, therefore as a side-effect of adapting to an abiotic environment, bacteria may also evolve to become more or less vulnerable to their parasites. After 45,000 generations of laboratory culturing without phage, E. coli gained resistance to lambda phage, gained sensitivity to a mutant T6 phage, and remained resistant to wild type T6. Each response was explained by understanding the pleiotropic costs or benefits of mutations that confer resistance. Because of pleiotropy, interactions may even evolve in the absence of one player.For the rest of my studies I examined how interactions evolve when host and parasite co-occur. First, I found that when E. coli and phage lambda are cocultured, E. coli evolves resistance by reducing the number of phage genotypes that can infect it, whereas, lambda evolves to increase the number of bacterial genotypes it can infect. This antagonism produces a interaction matrix with a nested form where less derived host-ranges fall one within another. To determine whether this nested pattern is an artifact of the labratory environment, or if the pattern is general to natural communities, I performed a metaanalysis on already published phage-bacterial interaction matrices. The majority of networks were significantly nested (28 of 38). Lastly, I examined the molecular basis of E. coli resistance to lambda and found that resistance often evolves through mutations in E. coli's lamB, the gene for the phage receptor. Also, the strength of resistance is correlated with how the mutation perturbs the orientation specific features of the protein structure, primarily loop four which extends out of the cell membrane. For the final two chapters, I studied whether lambda could evolve to target a novel receptor and the evolutionary consequences of such an innovation. Under particular laboratory conditions, E. coli evolves resistance by down-regulating LamB, which sets the stage for lambda to evolve the necessary mutations to exploit a new protein receptor. When allowed to coevolve under this condition, lambda evolved to exploit another outer-membrane protein, OmpF. This new function is the result of a particular combination of four mutations in J, the gene for the protein ligand lambda uses to bind to its host. Once lambda evolves this novel interaction, an evolutionary arms-race begins that drives rapid diversification of the bacteria and phage. Overall, my studies show that coevolution between bacteria and phage, whether it be in the lab or in nature, produces nested interactions matrices. Secondly, antagonistic coevolution is a creative process able to generate new genotypes of host and parasite and promote the evolution of novel function. Lastly, costs for resistance have many important effects, from determining whether resistance will evolve or be lost, to the generation of diversity.
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Title: X-ray crystallographic studies of Escherichia coli branching enzyme in complex with maltooctaose and rice branching enzyme I in complex with dodecaose
Creator: Fawaz, Remie
Date: 2016
Collection: Electronic Theses & Dissertations
Description: ABSTRACTX-RAY CRYSTALLOGRAPHIC STUDIES OF ESCHERICHIA COLI BRANCHING ENZYME IN COMPLEX WITH MALTOOCTAOSE AND RICE BRANCHING ENZYME I IN COMPLEX WITH DODECAOSEByRemie FawazBranching enzyme plays a key role in determining the final structure of glycogen or starch; this outcome structure is unique to every species, therefore the diversity of branching enzymes structures. While the biosynthesis of the polymers constitutes of three major steps, the last reaction of the pathway catalyzed by...
Show moreABSTRACTX-RAY CRYSTALLOGRAPHIC STUDIES OF ESCHERICHIA COLI BRANCHING ENZYME IN COMPLEX WITH MALTOOCTAOSE AND RICE BRANCHING ENZYME I IN COMPLEX WITH DODECAOSEByRemie FawazBranching enzyme plays a key role in determining the final structure of glycogen or starch; this outcome structure is unique to every species, therefore the diversity of branching enzymes structures. While the biosynthesis of the polymers constitutes of three major steps, the last reaction of the pathway catalyzed by branching enzyme embodies the cleavage of the α-1,4 glycosidic bond and the transfer of the produced oligosaccharide to the specific α-1,6 position creating an α-1,6 branch point. This process results in highly branched polymeric structures, which represent major carbon sources and carbohydrate storage compounds in living organisms, and are essential both in nature and industry. The structure of Escherichia coli glycogen branching enzyme has been solved both in the apo and holo forms. Binding to linear and cyclic oligosaccharides has been studied and showed seven external binding sites, but binding in the active site was not seen. Oligomers longer than previously used were investigated in an attempt to see binding in the catalytic center, only to discover more peripheral binding sites seemingly independent of each other. Examining the binding sites’ locations and sugars’ orientations indicates that these sites probably cooperate together in order to hold the long sugar polymer on the surface of the protein during the branching reaction. Hypotheses regarding the mode of binding between E. coli branching enzyme and the sugar during the reaction, and the roles for key binding sites and residues in the mechanism are proposed.Rice branching enzyme I, a starch branching enzyme, was also previously crystallized and the structure of the truncated version was solved. Binding attempts on this protein showed surface binding sites as well. In an effort to better understand the mechanism of action of this enzyme, we crystallized the protein and soaked it in a 12-unit oligomer, which bound to the enzyme hanging over the active site without reaching into the groove. Two new binding sites were discovered for this protein and the residues involved were identified. There are several hydrogen bonds and aromatic stacking interactions within the binding sties. Hypotheses for the binding mode and reaction mechanism are proposed.In addition, we have worked on ADP-glucose pyrophosphorylases that catalyze the first step in the biosynthesis pathway. The small subunit of the protein expresses as a homotetramer that is highly active. Although the potato tuber form was previously expressed in a collaborating laboratory at extremely small levels, and the protein was crystallized in the inactive form in the Geiger lab, our intensive attempts only succeeded in purifying the protein of interest, but the enzyme would precipitate instead of concentrating.
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Title: Breaking biofilms : regulation of Type II secretion system in V. cholerae and the formation of the hyper-pseudopilus
Creator: Sloup, Rudolph E.
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is the causative agent of the human disease cholera, it resides in aquatic resevoirs and forms biofilms, which are closely associated communities of bacteria embedded in polysaccharides, DNA, and proteins. In V. cholerae biofilm formation is regulated by the second messenger molecule cyclic di-GMP (c-di-GMP). A genetic screen for promoters regulated by the c-di-GMP revealed a novel promoter (PepsG) in the eps operon encoding the V. cholerae Type 2 secretion system (T2SS). The...
Show moreVibrio cholerae is the causative agent of the human disease cholera, it resides in aquatic resevoirs and forms biofilms, which are closely associated communities of bacteria embedded in polysaccharides, DNA, and proteins. In V. cholerae biofilm formation is regulated by the second messenger molecule cyclic di-GMP (c-di-GMP). A genetic screen for promoters regulated by the c-di-GMP revealed a novel promoter (PepsG) in the eps operon encoding the V. cholerae Type 2 secretion system (T2SS). The T2SS, which exports proteins from the periplasm to the extracellular space, is phylogenetically related to Type 4 pili. The major pseudopilin is encoded by epsG which forms a short piston like structure necessary for secretion. I hypothesized that differential regulation of the eps operon extends the pseudopilin forming a structure called a hyper-pseudopilus outside the cell where it promotes biofilm development. In Chapter 2, I determined that the promoter upstream of the operon (PepsC1) is induced four fold by c-di-GMP and this induction is mediated by the c-di-GMP binding transcription factor VpsR directly. High levels of c-di-GMP were found to decrease the activity of extra cellular proteases secreted by the T2SS, however this effect was not a direct result of regulation of the T2SS as determined by mutation of the VpsR binding site in PepsC1. I was unable to establish a phenotype for the transcriptional control of the eps operon. This work establishes T2S as a new phenotype which is transcriptionally controlled by c-di-GMP and the biofilm associated transcription factor VpsR. In Chapter 3, I show that overexpression of epsG in a continuous flow cell system increased V. cholerae biofilms while a ΔepsG strain showed no biofilm formation. However, there was no change in activity of T2S dependent serine proteases while epsG was over expressed indicating increased biofilms is not likely due to increased secretion. Polyclonal antibody stained EpsG was also detectable on the surface of WT cells and long pseudopili were visualized with over expression of epsG. This evidence suggests the T2SS forms a hyper-pseudopilus important for biofilm formation. In Chapter 4, I present my work identifying novel anti-biofilm compounds. In 2011 Escherichia coli O104:H4 caused the deadliest E. coli outbreak in modern times resulting in 54 deaths and the highest rate of hemolytic uremic syndrome ever recorded. Subsequently, we showed a correlation between biofilm gene expression and virulence factor expression. I sought to identify small molecule compounds effective at inhibiting O104:H4 biofilms. I discovered at a concentration of 0.01% the nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to inhibit biofilm formation by 90% and 91% respectively. These compounds were able to disperse preformed biofilms. Treatment of mice infected with E. coli O104:H4 resulted in high bacterial loads and inflammation. While addition of PS80 in the drinking water of the mice did not reduce bacterial loads, it completely abolished inflammation symptoms. PS80 is an FDA approved compound, well studied and effective at low nanomolar concentrations that reduces symptoms of infection in mice. which establishes it as an excellent candidate for further study as an anti-infective agent with anti-biofilm capabilities
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Title: Development of a resistance based biosensor utilizing conducting microfibers for microbial pathogen detection
Creator: McGraw, Shannon Katie
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Escherichia coli O157:H7 (E. coli O157:H7) is one of the U.S. military's top pathogens of interest for the development of rapid diagnostic systems. The enteric pathogen can cause severe gastroenteritis and is spread through the consumption of contaminated food and water. This is of concern to the U.S. military and warfighter because an outbreak of diarrheal disease in the field has the ability to rapidly render a large number of warfighters ineffective in...
Show moreEscherichia coli O157:H7 (E. coli O157:H7) is one of the U.S. military's top pathogens of interest for the development of rapid diagnostic systems. The enteric pathogen can cause severe gastroenteritis and is spread through the consumption of contaminated food and water. This is of concern to the U.S. military and warfighter because an outbreak of diarrheal disease in the field has the ability to rapidly render a large number of warfighters ineffective in performing their duties. Current field &ldquoportable&rdquo detection technologies can be cumbersome and require generous quantities of chemicals to operate. In addition, the current FDA gold standard for identification of this pathogen from food matrices takes up to 3 days to generate a confirmed positive result. The objective of this dissertation research was to develop a rapid, novel electrochemical biosensor based on the use of polypropylene microfiber membranes coated with a conductive polypyrrole and antibody functionalized for the biological capture and detection of E. coli O157:H7. In this dissertation research, an electrotextile composed of conductive polymer coated microfibers containing functional attachment sites for biorecognition elements was developed. The electrotextiles were optically and electrically assessed based on the polymerization chemicals and reaction time to determine how these factors affected the resistance of the fibers. Based on these experiments, a mathematical model was developed, optimized, and validated. Various methods of antibody immobilization and surface blocking on the fibers were also assessed. Using glutaraldehyde, pathogen specific antibodies were covalently attached to the conductive microfiber electrotextiles which were then blocked using a 5% bovine serum albumin solution. The functionalized membranes were exposed to E. coli O157:H7 cells, washed in Butterfield's phosphate buffer and added to a phosphate buffer electrolyte solution. When a voltage was applied to the system, the presence of the captured pathogen on the fiber surface resulted in an increase in resistance at the electrotextile electrode surface, indicating a positive result. It was found that the conductivity of the components of the system, other than the electrotextile fibers, was not statistically significant. Proof&ndashof&ndashconcept experiments were conducted and it was determined that the electrotextile electrode was able to differentiate between positive and negative samples using the pathogen E. coli O157:H7 cells as the target over a concentration range of 100 &ndash 109 colony forming units per milliliter (CFU/mL). The reproducibility of the sensor results was tested and it was found that the trends in the biosensor results were reproducible. By testing the significance of the biosensor response it was determined that the biosensor can successfully function as a yes / no screening system. The results show that the biosensor has an experimental lower limit of detection of 3.23 x 100 CFU/mL for the detection of E. coli O157:H7 in pure culture.
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Title: Quantitative transfer of Escherichia coli O157:H7 during pilot-plant production of fresh-cut leafy greens
Creator: Buchholz, Annemarie Lucia
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Cross-contamination/multi-directional transfer of Escherichia coli O157:H7 during commercial shredding, conveying, flume-washing and drying of fresh-cut leafy greens has become a major public health concern. However, the extent of bacterial transfer during the various unit operations involved in post-harvest processing of leafy greens is poorly understood. Consequently, a pilot-scale leafy green processing line consisting of a commercial shredder, conveyor, flume tank,...
Show moreCross-contamination/multi-directional transfer of Escherichia coli O157:H7 during commercial shredding, conveying, flume-washing and drying of fresh-cut leafy greens has become a major public health concern. However, the extent of bacterial transfer during the various unit operations involved in post-harvest processing of leafy greens is poorly understood. Consequently, a pilot-scale leafy green processing line consisting of a commercial shredder, conveyor, flume tank, shaker table, and centrifugal dryer was assembled to E. coli O157:H7 transfer during processing of iceberg lettuce, Romaine lettuce, and baby spinach.Using 22.7 kg of leafy greens inoculated with a 4-strain avirulent, GFP-labeled, ampicillin-resistant E. coli O157:H7 cocktail at 6, 4 and 2 log CFU/g, bacterial transfer coefficients were determined between the product, the water, and the equipment surfaces during processing, with the effect of post-inoculation hold time and shred size on transfer also evaluated. During leafy green processing, ~90% of the E. coli O157:H7 inoculum transferred to the wash water. After processing, E. coli O157:H7 populations on equipment were highest on the shredder and conveyor. Shred size did not affect the numbers of E. coli O157:H7 transferred from contaminated leafy greens during processing. However, extending the time between inoculation and processing decreased the removal and transfer of this pathogen during processing. When 22.7 kg of product containing 10 6 and 10 4 E. coli O157:H7 CFU/g were processed followed by 22.7 kg uninoculated product, E. coli O157:H7 was quantifiable in all iceberg and romaine lettuce samples. At an inoculation level of 2 log CFU/g, E. coli O157:H7 was sporadically detected after processing 81.6 kg of uninoculated iceberg and found throughout the entire batch of uninoculated romaine lettuce. Using Radicchio as a colored surrogate for iceberg lettuce, this study demonstrated that 9.1 kg of E. coli O157:H7-inoculated Radicchio could contaminate 907 kg of subsequently processed uncontaminated iceberg lettuce. After processing, shreds of Radicchio were found in all but one of the ~38 bags of shredded lettuce. Hundreds of contaminated Radicchio shreds remained on the processing line, with the majority found on the conveyor, followed by the shredder, flume tank, and shaker table, showing that contaminated product can be continually spread during leafy green processing long after a contamination event. The effect bacterial attachment time on cross-contamination was determined by processing 0.5 kg of E. coli O157:H7-inoculated Radicchio immediately and after storage for 1 d at 4 ° C or 5 d at 22 ° C, followed by 45.5 kg of uninoculated iceberg lettuce. After processing, E. coli O157:H7 was found in all uncontaminated iceberg lettuce samples, with mean counts highest for 1 h- followed by 1 d- and 5 d-held product. Scanning electron microscopy images showed greater bacterial attachment for 5 d-held product, with larger cell aggregates enveloped in a dense extracellular matrix. When transfer between inoculated and uninoculated product was assessed during partial dewatering, faster cross-contamination was seen using an E. coli O157:H7-inoculated Radicchio to uninoculated iceberg lettuce ratio of 1:100 than 1:10,000 (w/w) until a maximum population was reached due to the higher number of E. coli O157:H7 cells available for transfer. These findings, which demonstrate E. coli O157:H7 transfer between product, wash water, and equipment surfaces, and to large quantities of uncontaminated fresh-cut leafy greens, are critical to the development of science-based transfer models for risk analysis.
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Title: Development of a nanoparticle DNA based biosensor for the Shiga-like toxin 1 detection using co-polymerization hybridization readout amplification
Creator: Anderson, Michael Jeffrey
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Approximately 3.4 million deaths occur each year due to lack of clean water. The Shigatoxigenic group of Escherichia coli (STEC) bacteria causes approximately 2.2 million of these deaths and nearly a billion cases of illness. Current approved testing methods for Escherichia coli require a 12-24 hours growth of the sample in order to test for an indicator species. This indicator species only suggests possible E. coli O157:H7 presence, but does not confirm it. Direct culture testing cannot...
Show moreApproximately 3.4 million deaths occur each year due to lack of clean water. The Shigatoxigenic group of Escherichia coli (STEC) bacteria causes approximately 2.2 million of these deaths and nearly a billion cases of illness. Current approved testing methods for Escherichia coli require a 12-24 hours growth of the sample in order to test for an indicator species. This indicator species only suggests possible E. coli O157:H7 presence, but does not confirm it. Direct culture testing cannot identify the pathogenic STEC bacteria either. Confirmation of toxicity requires molecular methods of identification leading to specialized equipment needs and higher testing costs. Typical molecular methods use polymerase chain reaction (PCR) to amplify a very specific target DNA sequence, indicating the presence of a specific microorganism. PCR requires a thermocycler and a clean laboratory environment. Recent work has been successfully explored using nanomaterial based systems to identify the same genes used in a PCR reaction in a simpler detection system. The nanomaterials are used for both signal amplification and as the detection molecule. In this dissertation work a biosensor utilizing nanoparticles has been developed for the detection of Shiga-like toxin 1 gene that is present in Escherichia coli O157:H7. The system has been successfully constructed using a novel carbohydrate coated gold nanoparticle for delivery of the self assembling co-polymerization DNA oligonucleotide reporters. A set of self-assembled single-stranded DNA (ssDNA) molecules has been developed to amplify a target sequence without the use of enzymes. The full detection system incorporates the gold nanoparticle and a magnetic microparticle into a DNA recognition step. The gold nanoparticle is used for both co-polymerization detection and electrochemical detection. Input material (genomic DNA) is extracted using a modified commercial extraction method to produce PCR quality DNA from whole bacterial cells. When extraction and recognition elements are combined, a limit of detection for E. coli O157:H7 of 105 cells/mL using co-polymerization and 101 cells/mL with electrochemical detection has been achieved. Sample preparation from spiked culture samples until final detection takes a total of 7 hours. Electrochemical detection provides only a presence or absence indicator, where tethered co-polymerization is able to provide quantitative values of the input bacterial concentration. So a combination of co-polymerization amplification and electrochemical detection can provide the potential for more sensitive and quantitative measurement without the need for enzymes as in PCR applications.
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