Biology subjects

Bennett, G. N.

Publications and source records attributed to Bennett, G. N..

6 recordsLinked to original sources

bioRxiv2024

Gender, interdisciplinary graduate training, and confidence working in teams

Teamwork is recognized as critical to solving complex societal challenges related to energy, health, and sustainability. With graduate education, students often gain teamwork experience through a problem-focused approach where they are brought into existing collaborations to pursue research that is focused on studying questions that have already been identified. Here, we describe an interdisciplinary educational program where graduate students were tasked with leading team formation, problem identification, and research formulation. This "team-first" training approach used a two-year curriculum to bring together students enrolled in diverse engineering and science graduate programs and provided students with a pedagogical understanding of interdisciplinarity, nurtured the development of student communication skills across disciplines, fostered student-led team formation and idea development, and empowered students to forge new connections between research groups. Assessment of three cohorts immediately following curriculum completion (n = 36) revealed significant gains in confidence in teamwork (p < 0.001) when compared to a control group of academic peers (n = 74). These gains varied across demographic groups, with women in science, technology, engineering, and mathematics presenting the strongest gains. This finding illustrates the importance of exploring how interdisciplinary team curricula in graduate school could support overcoming the gender gap in confidence. SignificancePedagogical models for graduate education often neglect the importance of teamwork training. Here, we describe an interdisciplinary training program that was developed to bring together doctoral students from diverse science, technology, engineering, and mathematics disciplines for a two-year curriculum that focused on teamwork training through student-led team formation, problem identification, and research formulation. Following program participation, we measured participant confidence in teamwork relative to a peer group. Our findings reveal gains with confidence in teamwork, with women presenting the strongest increases without negative effects on other groups. This pedagogical approach represents a strategy to close gender gaps in professional role confidence while complementing the benefits of traditional disciplinary training approaches.

scientific communication and education↗

bioRxiv2021

Real-time environmental monitoring of contaminants using living electronic sensors

Real-time chemical sensing is needed to counter the global threats posed by pollution. We combine synthetic biology and materials engineering to develop a living bioelectronic sensor platform with minute detection times. Escherichia coli was programmed to reduce an electrode in a chemical-dependent manner using a modular, eight-component, synthetic electron transport chain. This strain produced significantly more current upon exposure to thiosulfate, an anion that causes microbial blooms. Incorporating a protein switch into the synthetic pathway and encapsulation of microbes with electrodes and conductive nanomaterials yielded a living bioelectronic sensor that could detect an endocrine disruptor within two minutes in riverine water, implicating the signal as mass transfer limited. These findings provide a new platform for miniature, low-power sensors that safeguard ecological and human health. One Sentence SummaryChemicals are detected electrically using an allosterically-regulated electron transfer pathway in designer microbes.

synthetic biology↗

bioRxiv2020

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

ABSTRACTHomologous recombination can be used to create enzymes that exhibit distinct activities and stabilities from proteins in nature, allowing researchers to overcome component limitations in synthetic biology. To investigate how recombination affects the physical properties of an oxidoreductase that transfers electrons, we created ferredoxin (Fd) chimeras by recombining distantly-related cyanobacterial and cyanomyophage Fds that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also be used to build synthetic pathways that support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required to support similar levels of cellular electron transfer. These results show how recombination can be used to rapidly diversify the physical properties of protein electron carriers and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic electron transfer pathway.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Prochlorococcus phage ferredoxin: structural characterization and interactions with cyanobacterial sulfite reductases

Marine cyanobacteria are infected by phage whose genomes encode ferredoxin (Fd) electron carriers. While these Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, it is not clear how the biophysical properties and partner specificities of phage Fds relate to those in photosynthetic organisms. Bioinformatic analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects Prochlorococcus marinus, revealed high similarity to cyanobacterial Fds ([≤]0.5 [A] RMSD). Additionally, pssm2-Fd exhibits a low midpoint reduction potential (-336 mV vs. SHE) similar to other photosynthetic Fds, albeit lower thermostability (Tm = 28{degrees}C) than many Fds. When expressed in an Escherichia coli strain with a sulfite assimilation defect, pssm2-Fd complemented growth when coexpressed with a Prochlorococcus marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterial-encoded oxidoreductases.

biochemistry↗

bioRxiv2019

Combinatorial design of chemical-dependent protein switches for controlling intracellular electron transfer

One challenge with controlling electron flow in cells is the lack of biomolecules that directly couple the sensing of environmental conditions to electron transfer efficiency. To overcome this protein component limitation, we randomly inserted the ligand binding domain (LBD) from the human estrogen receptor (ER) into a thermostable 2Fe-2S ferredoxin (Fd) from Mastigocladus laminosus and used a bacterial selection to identify Fd-LBD fusion proteins that support electron transfer from a Fd-NADP reductase (FNR) to a Fd-dependent sulfite reductase (SIR). Mapping LBD insertion sites onto structure revealed that Fd tolerates domain insertion adjacent to or within the tetracysteine motif that coordinates the 2Fe-2S metallocluster. With both classes of the fusion proteins, cellular ET was enhanced by the ER antagonist 4-hydroxytamoxifen. In addition, one of Fds arising from ER-LBD insertion within the tetracysteine motif acquires an oxygen-tolerant 2Fe-2S cluster, suggesting that ET is regulated through post-translational ligand binding.

synthetic biology↗

bioRxiv2018

Novel Method for Viability Assessment of Bifidobacterium longum ATCC 15707 on Non-dairy Foods during drying

This study demonstrates a new technique for separating and purifying viable microbes from samples that interfere with viability staining. The viability of Bifidobacterium longum ATCC 15707 was assessed using PBDC to separate bacteria from complex non-dairy food matrices and Quantitative Fluorescence Microscopy (QFM) to determine individual cells using LIVE/DEAD BacLight bacterial viability staining. Water agar (3%) was used to retain cells of B. longum and offered a lower fluorescence background with BacLight viability staining, compared with fixation on polycarbonate (PC) black membrane. The effect of drying temperatures and non-dairy foods on viability of B. longum was assessed. B. longum coated on oat, peanut or raisin was separated by filtration, low- and high-speed centrifugation, flotation and sedimentation buoyant density centrifugation. Purified cells were subsequently deposited on water agar for rehydration followed by LIVE/DEAD BacLight viability staining and enumeration. Conventional plate counting was also conducted to compare viability results. Finally, the applicability of this novel method for viability assessment was demonstrated and informative information of cell membrane damages of B. longum incorporated onto non-dairy foods during 24 h drying was observed. Viability assessment of B. longum coated onto oat, peanut, or raisin was much lower by plate counting compared to viability staining. Drying appeared to have a greater impact when viability was assessed by plate counting compared to viability staining.\n\nIMPORTANCEEnumeration of viable beneficial bacteria from function foods presents a significant bottle neck for product development and quality control. Interference with microscopic and/or fluorescent techniques by ingredients, time required to incubate plated microbes, and the transient nature of the colony forming unit make rapid assessment of viable bacteria difficult. Viability assessment of Bifidobacterium longum ATCC 15707 by Percoll Buoyant Density Gradient Centrifugation with LIVE/DEAD BacLight viability staining on water agar (3%) was in agreement with serial dilution enumeration. Without the need for incubation viability assessment by staining provided a more rapid means to assess the impact of drying on the viability of B. longum coated onto oat, peanut or raisin.

microbiology↗