Catalytic Activity of Carbon Materials in the Oxidation of Minerals

This study aims to advance the knowledge of using carbon materials as catalysts in the oxidation of chalcopyrite. For this, two different materials (a commercial activated carbon (CC) and commercial biochar (BC)) were added to chalcopyrite ore (CPY) at three weight ratios (1:1, 1:0.5, and 1:0.25). Mixtures were treated with sulfuric/ferric solution for 96 h at 90 °C. Experimental results showed that extraction of copper from CPY was around 36%, increasing to higher than 90% with the addition of CC or BC at the proper ratio. The best result (99.1% Cu extraction) was obtained using a 1:1 ratio of CPY:CC. Analysis of solid residues shows that CC, with a high surface area, adsorbs sulfur onto its surface, limiting elemental sulfur formation. Additionally, the treatment of CPY in the CC’s presence transforms the chalcopyrite into CuS. Sulfur adsorption or CuS formation was not observed after the leaching of chalcopyrite with BC. However, the addition of BC to CPY at a ratio of 1:0.25 also increased the extraction of copper to 91.1%. Two carbon materials were oxidized after treatment with a sulfuric/ferric solution, and BC probably displayed catalytic properties in the leaching medium.Fil: Burbano Patiño, Aura Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Gascó, Gabriel. Universidad Politécnica de Madrid; EspañaFil: Paz Ferreiro, Jorge. Rmit University; AustraliaFil: Méndez, Ana. Universidad Politécnica de Madrid; Españ

Genetically stable CRISPR-based kill switches for engineered microbes

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Genetically stable CRISPR-based kill switches for engineered microbes

Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development

Genomic characterization of antibiotic resistant Escherichia coli isolated from domestic chickens in Pakistan

Poultry husbandry is important for the economic health of Pakistan, but the Pakistani poultry industry is negatively impacted by infections fro

Identification of Candidate Microbial Biomarkers of Disease and Design of Engineered Microbial Therapeutics for the Gut Microbiome

The human gut microbiome is a compositionally and functionally diverse community of microorganisms that profoundly influences the health of the host. Deep characterization of gut microbiomes via high-throughput sequencing has identified associations between the gut microbiome and disease states, and spurred development of engineered microbial therapeutics. Successful translation of these research efforts to the clinic will involve i) consideration of how engineered microbes behave and adapt in the gut, towards the implementation of biosafety mechanisms, and ii) identification and validation of microbial biomarkers of disease. This dissertation describes both investigative (Chapter 2) and engineering (Chapters 3 and 4) approaches to improving the safety and efficacy of microbial therapies, using the commensal Escherichia coli Nissle 1917 (EcN) as a model chassis. This work further investigates the gut microbiome as an early predictor of preclinical Alzheimer Disease (Chapter 5), with corresponding identification of candidate microbial biomarkers. Unlike traditional therapeutics, engineered microbes are subject to selection potentially at the cost of their intended function. In Chapter 2, gnotobiotic and conventional mouse models of colonization were used to identify the major selective forces acting on EcN in the dysbiotic mammalian gut. Functional metagenomic selections and isolate whole-genome sequencing identified access to preferred carbon sources as the main selective pressure on EcN in low-diversity guts. In functional metagenomic selections, EcN populations encoding heterologous glycosyl hydrolases enabling consumption of dietary polysaccharides were strongly enriched. In the absence of this repertoire of heterologous functions, wild-type EcN mutated to better consume host mucins. In addition, EcN was observed to be a reservoir of aminoglycoside resistance-conferring mutations subsequent to a single antibiotic exposure. These findings carry clinical implications for the administration of engineered probiotics in low-diversity, dysbiotic guts. As such, biocontainment of engineered functions and microbes in situ is an important design consideration, with the goal of mitigating potential for pathology and environmental contamination. In Chapter 3, a transcript-barcoding approach enabling measurement of activity of many synthetic constructs in parallel was developed and validated in mice. The relative activities of 30 pooled EcN strains, each harboring a distinct synthetic construct, were ranked in multiple gut sites; this work informed the development of EcN strains engineered for the treatment of phenylketonuria, the efficacy of which were validated in a murine model of the disease (Chapter 2). This transcript barcoding method will facilitate the design and testing of bio-sensing synthetic constructs in vivo, towards the restriction of engineered functions to target sites of interest. In Chapter 4, a CRISPR-based approach to microbial biocontainment was developed and tested in mice. The kill switch design enabled selective removal of EcN from the guts of mice upon chemical induction; inclusion of temperature-responsive element further enabled kill switch induction upon excretion from the gut. Due to the stringent selective pressure imposed by kill switches, such designs are prone to mutational inactivation. Genetic stability was achieved using parallel approaches of engineering and environmental control, the first by inclusion of functional redundancies, a plasmid retention system, and knockouts of SOS response genes, and the latter by the innovative leveraging of inter-strain exclusion behaviors observed in the gut. Specifically, co-administration of kill switch-encoding EcN with a control EcN strain, in tandem with kill switch induction, enabled virtually complete eradication of the kill switch population from the guts of mice. This approach is attractive when the goal is to remove an engineered subpopulation of engineered microbes, and not a probiotic species itself, from the gut. In Chapter 5, the gut microbiome was instead considered as source of candidate microbial biomarkers of preclinical Alzheimer Disease (AD). Identification of microbiome correlates at the preclinical stage has the potential for clinical significance in that canonical biomarkers of preclinical AD require access to PET-CT scanners, or invasive CSF lumbar puncture, whereas stool is a cheaply acquired analyte. Gut microbiome features were correlated with amyloid biomarkers, but not markers of tau or neurodegeneration, highlighting the potential utility of gut microbiome correlates in early screening as abnormal amyloid levels are considered the most antecedent in disease progression. Further, addition of microbiome features to machine learning classifiers for preclinical status improved sensitivity, predominantly when metagenome-assembled genomes were included as features, rather than taxa identified through clade-specific marker sequences. This suggests taxonomic associations with AD status may be strain-specific, and encourages the pursuit of strain-level resolution in microbiome-wide association studies. As we work to translate observations about the gut microbiome to actionable clinical interventions, we must consider the interactions of engineered microbial therapeutics and recipient microbiomes, as well as the resolution necessary to validate gut microbiome-derived biomarkers of disease. This thesis work represents efforts to that end, and will ultimately inform the design of novel gut-directed therapies

Catalytic Activity of Carbon Materials in the Oxidation of Minerals

This study aims to advance the knowledge of using carbon materials as catalysts in the oxidation of chalcopyrite. For this, two different materials (a commercial activated carbon (CC) and commercial biochar (BC)) were added to chalcopyrite ore (CPY) at three weight ratios (1:1, 1:0.5, and 1:0.25). Mixtures were treated with sulfuric/ferric solution for 96 h at 90 °C. Experimental results showed that extraction of copper from CPY was around 36%, increasing to higher than 90% with the addition of CC or BC at the proper ratio. The best result (99.1% Cu extraction) was obtained using a 1:1 ratio of CPY:CC. Analysis of solid residues shows that CC, with a high surface area, adsorbs sulfur onto its surface, limiting elemental sulfur formation. Additionally, the treatment of CPY in the CC’s presence transforms the chalcopyrite into CuS. Sulfur adsorption or CuS formation was not observed after the leaching of chalcopyrite with BC. However, the addition of BC to CPY at a ratio of 1:0.25 also increased the extraction of copper to 91.1%. Two carbon materials were oxidized after treatment with a sulfuric/ferric solution, and BC probably displayed catalytic properties in the leaching medium

RiboTALE: A modular, inducible system for accurate gene expression control.

A limiting factor in synthetic gene circuit design is the number of independent control elements that can be combined together in a single system. Here, we present RiboTALEs, a new class of inducible repressors that combine the specificity of TALEs with the ability of riboswitches to recognize exogenous signals and differentially control protein abundance. We demonstrate the capacity of RiboTALEs, constructed through different combinations of TALE proteins and riboswitches, to rapidly and reproducibly control the expression of downstream targets with a dynamic range of 243.7 ± 17.6-fold, which is adequate for many biotechnological applications