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Browsing by Author "Thompson, Seth"

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    Data supporting Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins
    (2024-07-15) Khakimzhan, Aset; Izri, Ziane; Thompson, Seth; Dmytrenko, Oleg; Fischer, Patrick; Beisel, Chase; Noireaux, Vincent; noireaux@umn.edu; Noireaux, Vincent; Noireaux Lab
    Integral and interacting membrane proteins (IIMPs) constitute a vast family of biomolecules that perform essential functions in all forms of life. However, characterizing their interactions with lipid bilayers remains limited due to challenges in purifying and reconstituting IIMPs in vitro or labeling IIMPs without disrupting their function in vivo. Here, we report cell-free transcription-translation in a quartz crystal microbalance with dissipation (TXTL-QCMD) to dynamically characterize interactions between diverse IIMPs and membranes without protein purification or labeling. As part of TXTL-QCMD, IIMPs are synthesized using cell-free transcription-translation (TXTL), and their interactions with supported lipid bilayers are measured using a quartz crystal microbalance with dissipation (QCMD). TXTL-QCMD reconstitutes known IIMP-membrane dependencies, including specific association with prokaryotic or eukaryotic membranes, and the multiple-IIMP dynamical pattern-forming association of the E. coli division-coordinating proteins MinCDE. Applying TXTL-QCMD to the recently discovered Zorya anti-phage system that is unamenable to labeling, we discovered that ZorA and ZorB integrate within the lipids found at the poles of bacteria while ZorE diffuses freely on the non-pole membrane. These efforts establish the potential of TXTL-QCMD to broadly characterize the large diversity of IIMPs.
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    Decomposition and Production of Dissolved Organic Matter by Aquatic Bacteria
    (2019-06) Thompson, Seth
    Aquatic ecosystems transport large amounts of organic matter from the landscape to the oceans. Along this pathway, heterotrophic bacteria rapidly cycle these compounds by acting as both degraders and producers of organic compounds. Understanding the ultimate fate of the organic matter and predicting how increased organic matter exports from terres-trial ecosystems will impact its delivery to the ocean, requires a better understanding of the factors that influence organic matter degradation and production in freshwater systems. While many scientists have approached this problem by focusing on microbial modifica-tions of carbon (C), much less attention has been paid to other major elements found in or-ganic molecules (namely nitrogen (N) and phosphorus (P)). A more integrated approach that incorporates microbial processing of both C and major macronutrients such as N and P is needed to describe the biogeochemical transformations of organic matter in freshwaters. In this dissertation I examine the degradation and production of dissolved organic matter by heterotrophic bacteria, specifically focusing on dissolved organic phosphorus (DOP). In chapter 1, I quantify the degradation rates and overall bioavailability of DOP across 27 unique aquatic systems and explore important environmental and chemical regu-lators of these rates. Data from these systems show that DOP degradation rates are spatially variable, but are typically as high or higher than rates of degradation for C. Also, the chem-ical composition of organic matter was an important predictor of DOP bioavailability with DOP bioavailability being lowest when DOP was scarce relative to C. This relationship means that DOP is degraded by bacteria in systems that are more likely to experience P lim-itation, suggesting that DOP may be an important source of P to bacteria in these systems. Chapter 1 concludes by documenting the importance of incorporating estimates of organic matter bioavailability into estimates of resource imbalance experienced by aquatic bacteria. Accounting for the bioavailability of organic matter generally reduces the estimates of nutri-ent imbalance experienced by aquatic bacteria compared to estimates using bulk nutrient concentrations. This reduction in imbalance would result in more efficient C cycling by aquatic bacteria, which has important implications for understanding the composition of organic matter exported downstream and ultimately to the ocean. Chapter 2 goes on to explore the production of organic matter by heterotrophic bac-teria. It is well documented in marine systems that bacteria can produce an immensely di-verse set of organic molecules, even when they are only given a single carbon source to start from. However, the factors that control this production and the extent to which bacteria also produce DOP remains unclear. Previous work has shown that bacteria in freshwaters have different stoichiometric strategies for dealing with nutrient imbalance, with some strains of bacteria capable of changing the chemical composition of their cells to more closely match that of their resources. This flexibility in biomass nutrient composition has important implications for the recycling rates of multiple nutrients and therefore likely im-pacts the production of organic molecules by bacteria as well. Using previously isolated bacterial strains that have had their biomass flexibility quantified, I test the impact of these different stoichiometric strategies on the composition of the organic matter the strains pro-duce. In this chapter, I show that bacteria produce measurable amounts of dissolved organic phosphorus, even under strongly phosphorus limited conditions. Overall, bacteria convert-ed ~0.01%-10% of the phosphate in the original media to dissolved organic phosphorus, with the highest conversion efficiencies under carbon limited growth conditions. Interest-ingly, the conversion efficiency was higher under extreme phosphorus limitation than mod-erate phosphorus limitation. This pattern was driven primarily by relatively high conversion efficiencies by bacteria with flexible biomass stoichiometry in the most phosphorus limited conditions demonstrating the importance of physiological responses to nutrient imbalance. This chapter also explores the impact of bacterial biomass flexibility on the optical proper-ties of the organic matter produced by bacteria. I show that biomass flexibility is signifi-cantly and positively related to the specific ultraviolet absorbance at a wavelength of 254 nm, a measure of the aromaticity of the organic matter, when grown under extreme phos-phorus limitation. This suggested that bacteria with more flexible biomass stoichiometry produce more complex carbon molecules under strong phosphorus limitation than less flex-ible strains do. While more work is needed to fully understand how the physiological growth strategies of different microbial taxa impact the production of DOM, this chapter provides some important first insights into this question. In the final chapter, I transition away from research on aquatic ecology into what I consider to be another fundamental aspect of being a scientist: training the next generation of scientific thinkers. Over the last decade, there has been a clear call to shift the instruc-tional methods used for teaching undergraduate biology courses. We now know that active learning approaches to teaching science lead to better science outcomes for students. Fur-thermore, engaging undergraduate science students in undergraduate research experiences has been shown to have a number of important benefits for students such as increased stu-dent engagement, interest in science careers, and understanding of the scientific process. To offer the benefits of research experiences to a broader set of students, many institutions have started offering Course-based Undergraduate Research Experiences (CUREs) in la-boratory classes for students. It is common for these laboratory sections to be primarily fa-cilitated by undergraduate or graduate teaching assistants (TAs) rather than full-time faculty members. For these TAs to efficiently achieve the goals of these CUREs they must under-stand both (a) the philosophical underpinnings of discovery-based inquiry, and (b) strate-gies for facilitating inquiry, based on evidence-based practices, in the teaching laboratory. However, TAs are rarely trained in pedagogy, which likely limits their abilities to effective-ly facilitate inquiry in the laboratory. Chapter 3 is a case study documenting the results of a theoretically grounded professional development pilot program. This pilot program revealed that novice TAs are initially concerned primarily about the logistical aspects of teaching: classroom management, content preparation, grading assignments, etc. These concerns limit their readiness for engaging with the more complex pedagogical concepts of evidence-based instruction or inclusive teaching. This means that TA professional development needs to be designed to parallel the dynamic nature of TA concerns and that programing focused on advanced teaching techniques is only effective after TAs have established a sense of comfort and confidence in their own teaching.