Engineering topics

Norton-Baker, Brenna

Publications and source records attributed to Norton-Baker, Brenna.

4 records

DOE OSTI2024

Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

Abstract As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

36 MATERIALS SCIENCE↗

DOE OSTI2024

Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1–2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

09 BIOMASS FUELS↗

DOE OSTI2022

Deamidation of the human eye lens protein $\gamma$S-crystallin accelerates oxidative aging

Cataract, a clouding of the eye lens from protein precipitation, affects millions of people every year. The lens proteins, the crystallins, show extensive post-translational modifications (PTMs) in cataractous lenses. The most common PTMs, deamidation and oxidation, promote crystallin aggregation; however, it is not clear precisely how these PTMs contribute to crystallin insolubilization. Here, we report six crystal structures of the lens protein γS-crystallin (γS): one of the wild-type and five of deamidated γS variants, from three to nine deamidation sites, after sample aging. The deamidation mutations do not change the overall fold of γS; however, increasing deamidation leads to accelerated disulfide-bond formation. Addition of deamidated sites progressively destabilized protein structure, and the deamidated variants display an increased propensity for aggregation. Here these results suggest that the deamidated variants are useful as models for accelerated aging; the structural changes observed provide support for redox activity of γS-crystallin in the lens.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Solution-phase p-type doping of highly enriched semiconducting single-walled carbon nanotubes for thermoelectric thin films

Single-walled carbon nanotubes (SWCNTs) are attractive materials for next-generation energy-harvesting technologies, including thermoelectric generators, due to their tunable opto-electronic properties and high charge carrier mobilities. Controlling the Fermi level within these unique 1D nanomaterials is often afforded by charge transfer interactions between SWCNTs and electron or hole accepting species. Conventional methods to dope SWCNT networks typically involve the diffusion of molecular redox dopant species into solid-state thin films, but solution-phase doping could potentially provide routes and/or benefits for charge carrier transport, scalability, and stability. Here, we develop a methodology for solution-phase doping of polymer-wrapped, highly enriched semiconducting SWCNTs using a p-type charge transfer dopant, F4TCNQ. This allows doped SWCNT inks to be cast into thin films without the need for additional post-deposition doping treatments. We demonstrate that the introduction of the dopant at varying stages of the SWCNT dispersion process impacts the ultimate thermoelectric performance and observe that the dopant alters the polymer selectivity for semiconducting vs metallic SWCNTs. In contrast to dense semiconducting polymer films, where solution-phase doping typically leads to disrupted morphologies and poorer TE performance than solid-state doping, thin films of solution-doped s-SWCNTs perform similarly to their solid-state doped counterparts. Interestingly, our results also suggest that solution-phase F4TCNQ doping leads to fully ionized and dimerized F4TCNQ anions in solid-state films that are not observed in films doped with F4TCNQ after deposition. Our results provide a framework for the application of solution-phase doping to a broad array of high-performance SWCNT-based thermoelectric materials and devices that may require high-throughput deposition techniques.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗