Project description:Undergraduate laboratory courses are essential to teaching core principles in STEM. This course, Quantitative Biological Methods, provides a unique approach to teaching molecular biology research techniques to students, in a laboratory that is delivered in a sequence that parallels standard biomedical research laboratory protocols. Students attend a lecture where they are taught the essential principles of biomedical research, and a lab where they learn to use laboratory equipment, perform experiments, and purify and quantify DNA and proteins. The course begins with an introduction to laboratory safety, pipetting, centrifugation, spectrophotometry, and other basic laboratory techniques. Next, the lab focuses on the purification and analysis of glutathione S-transferase (GST) fused to green fluorescent protein (GFP) from an Escherichia coli lysate. Students study this GST-GFP fusion protein and perform protein quantification, enzyme assays, chromatography, fluorescent detection, normalization, SDS-PAGE, and western blotting. Students then learn recombinant DNA technology using the GST-GFP vector that was the source of the fusion protein in the prior labs, and perform ligation, transformation of E. coli cells, blue/white screening, DNA purification via a miniprep, PCR, DNA quantification, restriction enzyme digestion, and agarose gel electrophoresis. Students write laboratory reports to demonstrate an understanding of the principles of the laboratory methods, and they must present and critically analyze their data. The lab methods described herein aim to emphasize the core molecular biology principles and techniques, prepare students for work in a biomedical research laboratory, and introduce students to both GST and GFP, two versatile laboratory proteins.
Project description:Rationale: Exogenous supplementation of beneficial intestinal bacteria can alleviate the behavioural symptoms of psychiatric disorders, such as autism spectrum disorder (ASD), through gut-brain interactions. However, the application of beneficial bacteria, such as Lactobacillus reuteri (L. reuteri), for treating ASD is hindered by limited gut colonization. Methods: Utilizing Spirulina platensis (SP) as a natural microcarrier for intestinal bacteria, a safer and more natural binding approach was employed to bind intestinal bacteria to the surface of SP to produce SP-intestinal bacteria. Due to the high adhesion efficiency and long residence time of SP in the intestines, SP-intestinal bacteria exhibit stronger intestinal colonization ability and better therapeutic effects on ASD. Results: SP is an effective carrier that can bind and deliver bacteria of different shapes and with different gram-staining properties. SP-intestinal bacteria exhibited enhanced colonization capabilities both ex vivo and in vivo. Further research showed that SP-L. reuteri had greater intestinal colonization efficiency than L. reuteri. SP-L. reuteri showed a stronger therapeutic effect on alleviating social deficits in an ASD mouse model by modulating the gut microbiota, enhancing intestinal barrier integrity, reducing lipopolysaccharide entry into the blood and mediating the neuroinflammatory response in the paraventricular thalamic nucleus. Conclusions: This study reports a microalgae-assisted intestinal bacterial delivery system for enhancing intestinal bacterial transplantation for gut-brain axis- or other intestinal-related diseases.
Project description:Modifications made during metabolic engineering for overproduction of chemicals have network-wide effects on cellular function due to ubiquitous metabolic interactions. These interactions, that make metabolic network structures robust and optimized for cell growth, act to constrain the capability of the cell factory. To overcome these challenges, we explore the idea of an orthogonal network structure that is designed to operate with minimal interaction between chemical production pathways and the components of the network that produce biomass. We show that this orthogonal pathway design approach has significant advantages over contemporary growth-coupled approaches using a case study on succinate production. We find that natural pathways, fundamentally linked to biomass synthesis, are less orthogonal in comparison to synthetic pathways. We suggest that the use of such orthogonal pathways can be highly amenable for dynamic control of metabolism and have other implications for metabolic engineering.
Project description:The widespread agricultural use of the phenylurea herbicide Diuron (DCMU) requires the investigation of ecotoxicological risk in freshwater and soil ecosystems in light of potential effects on non-target primary producers and a heavier effect on higher trophic levels. We used microalgae-based fluorimetric bioassays for studying the interferences on the photosynthesis of a freshwater and soil model green microalga (Chlamydomonas reinhardtii) induced by environmentally relevant concentrations of the herbicide DCMU. Measurements of steady-state chlorophyll a (Chl-a) fluorescence emission spectra were performed; as well, the kinetics of the Chl-a fluorescence transient were recorded. Percentage indexes of interference on photosynthesis were calculated after comparison of steady-state and kinetic Chl-a fluorescence measurements of DCMU-exposed and control C. reinhardtii cell suspensions. The results obtained after 30 min exposure to the herbicide DCMU confirmed a significant inhibitory effect of DCMU 2 μg/L, and no significant differences between %ι values for DCMU 0.2 μg/L and 0.02 μg/L exposures. Positive %ε values from kinetic measurements of the Chl-a fluorescence transient confirmed the same interfering effect of 2 μg/L DCMU on PSII photochemistry in the exposed C. reinhardtii cell suspensions. Negative values of %ε observed for 0.2 and 0.02 μg/L DCMU exposures could be attributable to a presumptive 'stimulatory-like' effect in the photochemistry of photosynthesis. Short-term exposure to sub-μg/L DCMU concentration (≤0.2 μg/L) affects the photosynthetic process of the model microalga C. reinhardtii. Similar environmental exposures could affect natural communities of unicellular autotrophs, with hardly predictable cascading secondary effects on higher trophic levels.
Project description:Here, we present surface-enhanced Raman data for the calculation of signal uniformity and enhancement factor in SENSERS (surface-exposed nanoparticle sheet enhanced Raman spectroscopy). SEM was used to characterize the microstructure of the solid sample. The interaction between the solid sample and surface-exposed nanoparticle sheet was characterized using SERS and SEM. Based on these data a "skin" versus "sheet" type calculation method was used to calculate the magnitude of Raman signal enhancement within SENSERS. The data presented in this article is related to the research article entitled "Pressing Solids Directly Into Sheets of Plasmonic Nanojunctions Enables Solvent-Free Surface-Enhanced Raman Spectroscopy" (Xu et al., 2018).
Project description:In the last decade, Amaranthus tuberculatus has evolved resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-hydroxyphenylpyruvate dioxygenase inhibitors in multiple states across the midwestern United States. Two populations resistant to both mode-of-action groups, one from Nebraska (NEB) and one from Illinois (CHR), were studied using an RNA-seq approach on F2 mapping populations to identify the genes responsible for resistance. Using both an A. tuberculatus transcriptome assembly and a high-quality grain amaranth (A. hypochondriacus) genome as references, differential transcript and gene expression analyses were conducted to identify genes that were significantly over- or underexpressed in resistant plants. When these differentially expressed genes (DEGs) were mapped on the A. hypochondriacus genome, physical clustering of the DEGs was apparent along several of the 16 A. hypochondriacus scaffolds. Furthermore, single-nucleotide polymorphism calling to look for resistant-specific (R) variants, and subsequent mapping of these variants, also found similar patterns of clustering. Specifically, regions biased toward R alleles overlapped with the DEG clusters. Within one of these clusters, allele-specific expression of cytochrome P450 81E8 was observed for 2,4-D resistance in both the CHR and NEB populations, and phylogenetic analysis indicated a common evolutionary origin of this R allele in the two populations.
Project description:Glycans comprise ubiquitous and essential biopolymers, which usually occur as highly diverse mixtures. The myriad different structures are generated by a limited number of carbohydrate-active enzymes (CAZymes), which are unusual in that they catalyze multiple reactions by being relatively unspecific with respect to substrate size. Existing experimental and theoretical descriptions of CAZyme-mediated reaction systems neither comprehensively explain observed action patterns nor suggest biological functions of polydisperse pools in metabolism. Here, we overcome these limitations with a novel theoretical description of this important class of biological systems in which the mixing entropy of polydisperse pools emerges as an important system variable. In vitro assays of three CAZymes essential for central carbon metabolism confirm the power of our approach to predict equilibrium distributions and non-equilibrium dynamics. A computational study of the turnover of the soluble heteroglycan pool exemplifies how entropy-driven reactions establish a metabolic buffer in vivo that attenuates fluctuations in carbohydrate availability. We argue that this interplay between energy- and entropy-driven processes represents an important regulatory design principle of metabolic systems.
Project description:Background: There is an unmet demand for community-scale fecal sludge treatment units (FSTUs) that serve communities of between 1,000 and 50,000 people and are able to operate in non-sewered and off-grid environments. An emerging industry standard for FSTUs includes as a key criteria energy independence in steady-state. Theoretically, there is sufficient thermal energy available in fecal sludge to provide the electrical power needed to run the FSTU. However, such a system had never been implemented. Methods: Biomass Controls has previously demonstrated the thermal treatment of fecal sludge using the Biogenic Refinery, a thermal FSTU deployed in three sites in India. In this article we describe testing where a Biogenic Refinery was paired with a thermal fluid heat exchanger and organic Rankine cycle generator to generate electrical power. Results: This Biogenic Refinery combined heat and power system generated sufficient electrical power to offset electrical parasitic loads in steady-state operation and produce a surplus of 1.2 kWe. Conclusions: The results of the study demonstrate that there is an excess of energy available and reliable mechanisms to generate electrical energy using an FSTU. Additional steps are necessary to transition to a true off-grid FSTU.
Project description:Microalgal-indigenous bacterial wastewater treatment (MBWT) emerges as a promising approach for the concurrent removal of nitrogen (N) and phosphorus (P). Despite its potential, the prevalent use of MBWT in batch systems limits its broader application. Furthermore, the success of MBWT critically depends on the stable self-adaptation and synergistic interactions between microalgae and indigenous bacteria, yet the underlying biological mechanisms are not fully understood. Here we explore the viability and microbial dynamics of a continuous flow microalgae-indigenous bacteria advanced wastewater treatment system (CFMBAWTS) in processing actual secondary effluent, with a focus on varying hydraulic retention times (HRTs). The research highlights a stable, mutually beneficial relationship between indigenous bacteria and microalgae. Microalgae and indigenous bacteria can create an optimal environment for each other by providing essential cofactors (like iron, vitamins, and indole-3-acetic acid), oxygen, dissolved organic matter, and tryptophan. This collaboration leads to effective microbial growth, enhanced N and P removal, and energy generation. The study also uncovers crucial metabolic pathways, functional genes, and patterns of microbial succession. Significantly, the effluent NH4+-N and P levels complied with the Chinese national Class-II, Class-V, Class-IA, and Class-IB wastewater discharge standards when the HRT was reduced from 15 to 6 h. Optimal results, including the highest rates of CO2 fixation (1.23 g L-1), total energy yield (32.35 kJ L-1), and the maximal lipid (33.91%) and carbohydrate (41.91%) content, were observed at an HRT of 15 h. Overall, this study not only confirms the feasibility of CFMBAWTS but also lays a crucial foundation for enhancing our understanding of this technology and propelling its practical application in wastewater treatment plants.
Project description:1. The metabolism of 2,6-dichlorobenzonitrile was studied in rabbits and rats. Oral administration caused an increased urinary excretion of glucuronides and ethereal sulphates. There was also an indication of mercapturic acid formation. 2,6-Dichloro-3-hydroxybenzonitrile and its 4-hydroxy analogue were identified as metabolites in the urine. A small amount of the unchanged substance was recovered from the faeces. 2. By using 2,6-dichlorobenzo[(14)C]nitrile the phenolic metabolites were determined quantitatively and some other possible metabolic routes were excluded. 3. Incubation of 2,6-dichlorobenzonitrile with enzyme preparations (papain and high-speed supernatant of rat-liver homogenate plus glutathione) gave no indications for a reaction with thiol compounds.