Biology subjects

Keitz, B. K.

Publications and source records attributed to Keitz, B. K..

15 recordsLinked to original sources

bioRxiv2026

Expression and Engineering of Conductive Cytochrome Nanowires

Electrically conductive protein nanowires produced by metal-reducing bacteria have attracted interest as sustainable electronic materials, but their study and engineering have been limited by difficulties in expression, purification, and genetic manipulation. Here we establish Shewanella oneidensis as a heterologous host for production of Geobacter sulfurreducens OmcZ nanowires and progress a complementary in vitro assembly strategy that yields highly pure nanowire preparations. This platform enabled systematic engineering of OmcZ, revealing extensive tolerance to sequence variation and faciliating the design of enhanced-conductivity variants. Guided by comparative analysis of environmental OmcZ homologs, we generated a chimeric nanowire, OmcZ+, that exhibited a [~]3.5-fold increase in bulk conductivity while retaining the overall structure of the parent nanowire. Cryo-electron microscopy revealed unexpected architectural plasticity in OmcZ+ wires, including non-linear dendritic and pentameric assemblies mediated by the solvent-exposed heme VII, suggesting previously unrecognized modes of cytochrome nanowire organization. Incorporation of engineered OmcZ variants into water evaporation-induced electricity generators produced power densities up to [~]25.3 W cm-{superscript 2} and enabled high-performance operation in saline environments, including seawater and human sweat. Together, these results establish a versatile platform for the production, structural analysis, and engineering of cytochrome nanowires, providing a foundation for the development of programmable biological electronic materials.

bioengineering↗

bioRxiv2025

Genome editing of phylogenetically distinct bacteria using portable retron-mediated recombineering

Advanced genome editing technologies have enabled rapid and flexible rewriting of the Escherichia coli genome, benefiting fundamental biology and biomanufacturing. Unfortunately, some of the most useful technologies to advance genome editing in E. coli have not yet been ported into other bacterial species. For instance, the addition of bacterial retrons to the genome editing toolbox has increased the efficiency of recombineering in E. coli by enabling sustained, abundant production of ssDNA recombineering donors by reverse transcription that install flexible, precise edits in the prokaryotic chromosome. To extend the utility of this technology beyond E. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, Bacillota and Actinomycetota) and a total of 15 different species. We found that retron recombineering is functional in all species tested, reaching editing efficiencies above 20% in six of them, above 40% in three of them, and above 90% in two of them. We also tested the extension of the recombitron architecture optimizations and strain backgrounds in a subset of hosts to additionally increase editing rates. The broad recombitron survey carried out in this study forms the basis for widespread use of retron-derived technologies through the whole Bacteria domain.

bioengineering↗

bioRxiv2024

A Linear Mixed Effects Model for Evaluating Synthetic Gene Circuits

A significant advancement in synthetic biology is the development of synthetic gene circuits with predictive Boolean logic. However, there is no universally accepted or applied statistical test to analyze the performance of these circuits. Many basic statistical tests fail to capture the predicted logic (OR, AND, etc.) and most studies neglect statistical analysis entirely. As synthetic gene circuits shift toward advanced applications, primarily in computing, biosensing, and human health, it is critical to standardize the statistical methods used to evaluate gate success. Here, we propose the application of a linear mixed effects model to analyze and quantify genetic Boolean logic gate performance. First, we analyzed 144 currently published Boolean logic gates for trends and used unsupervised machine learning (k-means clustering) to validate the statistical model. Next, we utilized the model to generate estimates for the fixed effect of the ON state, {beta}, as a general descriptor of the Boolean nature of a circuit and used Monte Carlo simulations to recommend sample sizes for evaluating gate performance. Finally, we examined {beta} as a holistic metric for circuit performance using a series of nested repressor OR gates with intentionally degraded performance. We observed a linear correlation between {beta} and the predicted translation rate, highlighting the use of {beta} for the forward design of new Boolean gates. In summary, we utilized a linear mixed effects model to describe synthetic gene circuits and determined that the fixed effect, {beta}, is an appropriate descriptor of gate behavior that can be used to statistically evaluate performance. Significance statementThere is no standard method for statistically evaluating the success of biological Boolean logic gates and common statistical tests are not used or incorrectly applied. We propose the use of a linear mixed effects model to estimate the value for {beta}, a parameter which describes the difference between ON and OFF gate states. A null hypothesis of {beta} = 0 can then be tested to determine if there is a statistically significant difference between the two states.

synthetic biology↗

bioRxiv2024

Single-Cell Phenotyping of Extracellular Electron Transfer via Microdroplet Encapsulation

Electroactive organisms contribute to metal cycling, pollutant removal, and other redox-driven environmental processes. Studying this phenomenon in high-throughput is challenging since extracellular reduction cannot easily be traced back to its cell of origin within a mixed population. Here, we describe the development of a microdroplet emulsion system to enrich EET-capable organisms. We validated our system using the model electroactive organism S. oneidensis and describe the tooling of a benchtop microfluidic system for oxygen-limited processes. We demonstrated enrichment of EET-capable phenotypes from a mixed wild-type and EET-knockout population. As a proof-of-concept application, bacteria were collected from iron sedimentation from Town Lake (Austin, TX) and subjected to microdroplet enrichment. We observed an increase in EET-capable organisms in the sorted population that was distinct when compared to a population enriched in a bulk culture more closely akin to traditional techniques for discovering EET-capable bacteria. Finally, two bacterial species, C. sakazakii and V. fessus not previously shown to be electroactive, were further cultured and characterized for their ability to reduce channel conductance in an organic electrochemical transistor (OECT) and to reduce soluble Fe(III). We characterized two bacterial species not previously shown to exhibit electrogenic behavior. Our results demonstrate the utility of a microdroplet emulsions for identifying putative EET-capable bacteria and how this technology can be leveraged in tandem with existing methods.

bioengineering↗

bioRxiv2023

Rewiring native post-transcriptional carbon regulators to build multi-layered genetic circuits and optimize engineered microbes for bioproduction

As the bioprocessing and synthetic biology spaces rapidly expand, post-transcriptional regulation is emerging as driver for maximizing signal response rate and for minimizing cost per protein within cells. Designing robust post-transcriptional control systems that have precise tunability and can achieve diverse regulatory outcomes are paramount to advance the field. Herein, we develop a new approach for engineered post-transcriptional control in bacteria by rewiring a native regulatory system in Escherichia coli, the Carbon Storage Regulatory (Csr) Network, to create tunable, complex genetic circuits. First, by co-opting native components of the Csr Network to regulate translation of a target mRNA transcript, we establish a Csr-regulated Buffer Gate. Next, by rationally engineering the interactions between our synthetic construct and the native components of the Csr Network, we expand our original design into a genetic toolbox of 12 Buffer Gates that achieve precise tunability across a 10-fold range of target gene expression. Subsequently, to further regulatory capabilities using this approach, we develop a Csr-regulated NOT Gate through engineering a Csr-activated sequence into our synthetic constructs. We then build upon the Csr Buffer and Not Gates to create post-transcriptional dual input Boolean OR, NOR, AND and NAND Logic Gates, as well as a genetic pulse circuit. As a third step, we demonstrate portability of our Csr-regulated Buffer Gates into three industrially relevant bacteria by recapitulating Buffer Gate activity simply by leveraging the conserved homologous Csr Network in each species. Lastly, as a demonstration of downstream application, we apply our system to a proof-of-concept synthetic mevalonate pathway. Using our engineered constructs, we optimize mevalonate production in E. coli resulting in a three-fold increase in production relative to a transcriptionally controlled mevalonate pathway. As a whole, we establish a novel approach to rewire post-transcriptional regulatory networks for complex bacterial computation that can be utilized for efficient bioproduction in engineered microbes.

bioengineering↗

bioRxiv2023

A Hybrid Transistor with Transcriptionally Controlled Computation and Plasticity

Organic electrochemical transistors (OECTs) are ideal devices for translating biological signals into electrical readouts and have applications in bioelectronics, biosensing, and neuromorphic computing. Despite their potential, developing programmable and modular methods for living systems to interface with OECTs has proven challenging. Here we describe hybrid OECTs containing the model electroactive bacterium Shewanella oneidensis that enable the transduction of biological computations to electrical responses. Specifically, we fabricated planar p-type OECTs and demonstrated that channel de-doping is driven by extracellular electron transfer (EET) from S. oneidensis. Leveraging this mechanistic understanding and our ability to control EET flux via transcriptional regulation, we used plasmid-based Boolean logic gates to translate biological computation into current changes within the OECT. Finally, we demonstrated EET-driven changes to OECT synaptic plasticity. This work enables fundamental EET studies and OECT- based biosensing and biocomputing systems with genetically controllable and modular design elements.

bioengineering↗

bioRxiv2021

Transcriptional Regulation of Synthetic Polymer Networks

Individual cells direct non-equilibrium processes through coordinated signal transduction and gene expression, allowing for dynamic control over multicellular, system-wide behavior. This behavior extends to remodeling the extracellular polymer matrix that encases biofilms and tissues, where constituent cells dictate spatiotemporal network properties including stiffness, pattern formation, and transport properties. The majority of synthetic polymer networks cannot recreate these phenomena due to their lack of autonomous centralized actuators (i.e., cells). In addition, non-living polymer networks that perform computation are generally restricted to a few inputs (e.g., light, pH, enzymes), limiting the logical complexity available to a single network chemistry. Toward synergizing the advantages of living and synthetic systems, engineered living materials leverage genetic and metabolic programming to establish control over material-wide properties. Here we demonstrate that a bacterial metal respiration mechanism, extracellular electron transfer (EET), can control metal-catalyzed radical cross-linking of polymer networks. Linking metabolic electron flux to a synthetic redox catalyst allows dynamic, tunable, and predictable control over material formation and bulk polymer network mechanics using genetic circuits. By programming key EET genes with transcriptional Boolean logic, we rationally design computational networks that sense-and-respond to multiple inputs in biological contexts. Finally, we capitalize on the wide reactivity of EET and redox catalyses to predictably control another class of living synthetic materials using copper(I) alkyne-azide cycloaddition click chemistry. Our results demonstrate the utility of EET as a bridge for controlling abiotic materials and how the design rules of synthetic biology can be applied to emulate physiological behavior in polymer networks.

synthetic biology↗

bioRxiv2021

Cross-seeding Controls Aβ Fibril Populations and Resulting Function

Amyloid peptides nucleate from monomers to aggregate into fibrils through primary nucleation; pre-existing fibrils can then act as seeds for additional monomers to fibrillize through secondary nucleation. Both nucleation processes can occur simultaneously, yielding a distribution of fibril polymorphs that can generate a spectrum of neurodegenerative effects. Understanding the mechanisms driving polymorph structural distribution during both nucleation processes is important for uncovering fibril structure-function relationships, as well creating polymorph distributions in vitro that better match distributions found in vivo. Here, we explore how cross-seeding WT A{beta}1-40 with A{beta}1-40 mutants E22G (Arctic) and E22{Delta} (Osaka), as well as with WT A{beta}1-42 affects the distribution of fibril structural polymorphs, and how changes in structural distribution impact toxicity. Transmission electron microscopy analysis reveals that fibril seeds derived from mutants of A{beta}1-40 impart their structure to WT A{beta}1-40 monomer during secondary nucleation, but WT A{beta}1-40 fibril seeds do not affect the structure of fibrils assembled from mutant A{beta}1-40 monomers, despite kinetics data indicating accelerated aggregation when cross-seeding of any combination of mutants. Additionally, WT A{beta}1-40 fibrils seeded with mutant fibrils to produce similar structural distributions to the mutant seeds also produced similar cytotoxicity on neuroblastoma cell lines. This indicates that mutant fibril seeds not only impart their structure to growing WT A{beta}1-40 aggregates, but they also impart cytotoxic properties. Our findings provide clear evidence that there is a relationship between fibril structure and phenotype on a polymorph population level, and that these properties can be passed on through secondary nucleation of succeeding generations of fibrils.

biochemistry↗

bioRxiv2021

Extracellular Electron Transfer Enables Cellular Control of Cu(I)-catalyzed Alkyne-Azide Cycloaddition

Extracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species. In the presence of a suitable metal catalyst, EET allows for cellular metabolism to control a variety of synthetic transformations. Here, we report the use of EET from the model electroactive bacterium Shewanella oneidensis for metabolic and genetic control over Cu(I)-catalyzed Alkyne-Azide Cycloaddition (CuAAC). CuAAC conversion under anaerobic and aerobic conditions was dependent on live, actively respiring S. oneidensis cells. In addition, reaction progress and kinetics could be further manipulated by tailoring the central carbon metabolism of S. oneidensis. Similarly, CuAAC activity was dependent on specific EET pathways and could be manipulated using inducible genetic circuits controlling the expression of EET-relevant proteins including MtrC, MtrA, and CymA. EET-driven CuAAC also exhibited modularity and robustness in ligand tolerance and substrate scope. Furthermore, the living nature of this system could be exploited to perform multiple reaction cycles without requiring regeneration, something inaccessible to traditional chemical reductants. Finally, S. oneidensis enabled bioorthogonal CuAAC membrane labelling on live mammalian cells without affecting cell viability, suggesting that S. oneidensis can act as a dynamically tunable biocatalyst in complex environments. In summary, our results demonstrate how EET can expand the reaction scope available to living systems by enabling cellular control of CuAAC.

bioengineering↗

bioRxiv2020

In Situ Optical Quantification of Extracellular Electron Transfer from Shewanella oneidensis using Plasmonic Metal Oxide Nanocrystals

Extracellular electron transfer (EET) is a critical form of microbial metabolism that enables respiration on a variety of inorganic substrates, including metal oxides. However, quantifying current generated by electroactive bacteria has been predominately limited to biofilms formed on electrodes. To address this, we developed a platform for quantifying EET flux from cell suspensions using aqueous dispersions of infrared plasmonic tin-doped indium oxide nanocrystals. Tracking the change in optical extinction during electron transfer enabled quantification of current generated by planktonic Shewanella oneidensis cultures. Using this method, we differentiated between starved and actively respiring cells, between cells of varying genotype, and between cells engineered to differentially express a key EET gene using an inducible genetic circuit. Overall, our results validate the utility of colloidally stable plasmonic metal oxide nanocrystals as quantitative biosensors in native biological environments and contribute to a fundamental understanding of planktonic S. oneidensis electrophysiology using simple in situ spectroscopy.

microbiology↗

bioRxiv2020

Functionalized Mesoporous Silicas Direct Structural Polymorphism of Amyloid-β Fibrils

The aggregation of Amyloid-{beta} (A{beta}) is associated with the onset of Alzheimers Disease (AD) and involves a complex kinetic pathway as monomers self-assemble into fibrils. A central feature of amyloid fibrils is the existence of multiple structural polymorphs, which complicates the development of disease-relevant structure-function relationships. Developing these relationships requires new methods to control fibril structure. In this work, we demonstrate that mesoporous silicas (SBA-15) functionalized with hydrophobic (SBA-PFDTS) and hydrophilic groups (SBA-PEG) direct the aggregation kinetics and resulting structure of A{beta}1-40 fibrils. The hydrophilic SBA-PEG had little effect on amyloid kinetics while as-synthesized and hydrophobic SBA-PFDTS accelerated aggregation kinetics. Subsequently, we quantified the relative population of fibril structures formed in the presence of each material using electron microscopy. Fibrils formed from A{beta}1-40 exposed to SBA-PEG were structurally similar to control fibrils. In contrast, A{beta}1-40 incubated with SBA-15 or SBA-PFDTS formed fibrils with shorter cross-over distances that were more structurally representative of fibrils found in AD patient-derived samples. Overall, these results suggest that mesoporous silicas and other exogenous materials are promising scaffolds for the de novo production of specific fibril polymorphs of A{beta}1-40 and other amyloidogenic proteins. Significance StatementA major challenge in understanding the progression of Alzheimers Disease lies in the various fibril structures, or polymorphs, adopted by Amyloid-{beta} (A{beta}). Heterogenous fibril populations may be responsible for different disease phenotypes and growing evidence suggests that A{beta} fibrils formed in vitro are structurally distinct from patient-derived fibrils. To help bridge this gap, we used surface-functionalized mesoporous silicas to influence the formation of A{beta}1-40 fibrils and evaluated the distribution of resulting fibril polymorphs using electron microscopy (EM). We found that silicas modified with hydrophobic surfaces resulted in fibril populations with shorter cross-over distances that are more representative of A{beta} fibrils observed ex vivo. Overall, our results indicate that mesoporous silicas may be leveraged for the production of specific A{beta} polymorphs.

biochemistry↗

bioRxiv2019

Tuning Extracellular Electron Transfer by Shewanella oneidensis Using Transcriptional Logic Gates

Extracellular electron transfer pathways, such as those in the bacterium Shewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux in S. oneidensis has proven challenging since a functional understanding of its EET pathway proteins and their effect on engineering parameterizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway of S. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriate S. oneidensis knockout strains, we pinpointed construct/strain pairings that expressed cymA, mtrA, and mtrC with maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures, that predictably turn on and turn off EET flux, respectively, in response to IPTG. Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs. inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.

synthetic biology↗

bioRxiv2019

Genetic Control of Radical Crosslinking in a Semi-Synthetic Hydrogel

Enhancing materials with the qualities of living systems, including sensing, computation, and adaptation, is an important challenge in designing next-generation technologies. Living materials seek to address this challenge by incorporating live cells as actuating components that control material function. For abiotic materials, this requires new methods that couple genetic and metabolic processes to material properties. Toward this goal, we demonstrate that extracellular electron transfer (EET) from Shewanella oneidensis can be leveraged to control radical crosslinking of a methacrylate-functionalized hyaluronic acid hydrogel. Crosslinking rates and hydrogel mechanics, specifically storage modulus, were dependent on a variety of chemical and biological factors, including S. oneidensis genotype. Bacteria remained viable and metabolically active in the crosslinked network for a least one week, while cell tracking revealed that EET genes also encode control over hydrogel microstructure. Moreover, construction of an inducible gene circuit allowed transcriptional control of storage modulus and crosslinking rate via the tailored expression of a key electron transfer protein, MtrC. Finally, we quantitatively modeled dependence of hydrogel stiffness on steady-state gene expression, and generalized this result by demonstrating the strong relationship between relative gene expression and material properties. This general mechanism for radical crosslinking provides a foundation for programming the form and function of synthetic materials through genetic control over extracellular electron transfer.\n\nSignificance StatementNext-generation materials will require coupling the advantages of engineered and natural systems to solve complex challenges in energy, health, and the environment. Living cells, such as bacteria, naturally possess many of the qualities essential to addressing these challenges, including sensing, computation, and actuation, using their genetic and metabolic machinery. In addition, bacteria are attractive for incorporation into materials due to their durability, ease-of-use, and programmability. Here, we develop a platform for controlling hydrogel properties (e.g., stiffness, crosslinking rate) using extracellular electron transfer from the bacterium Shewanella oneidensis. In our system, metabolic electron flux from S. oneidensis to a metal catalyst generates radical species that crosslink an acrylate-based macromer to form the gel. This synthetic reaction is under direct control of bacterial genetics and metabolism, which we demonstrate through inducible circuits and quantitative modeling of gene expression and resultant hydrogel properties. Developing methods that capitalize on the programmability of biological systems to control synthetic material properties will enable hybrid material designs with unprecedented functions.

synthetic biology↗

bioRxiv2018

Extracellular Electron Transfer by Shewanella oneidensis Controls Pd Nanoparticle Phenotype

AbstarctBiological production of inorganic materials is impeded by relatively few organisms possessing genetic and metabolic linkage to material properties. The physiology of electroactive bacteria is intimately tied to inorganic transformations, which makes genetically tractable and well-studied electrogens, such as Shewanella oneidensis, attractive hosts for material synthesis. Notably, this species is capable of reducing a variety of transition-metal ions into functional nanoparticles, but exact mechanisms of nanoparticle biosynthesis remain ill-defined. We report two key factors of extracellular electron transfer by S. oneidensis, the outer membrane cytochrome, MtrC, and soluble redox shuttles (flavins), that affect Pd nanoparticle formation. Changes in the expression and availability of these electron transfer components drastically modulated particle phenotype, including particle synthesis rate, structure, and cellular localization. These relationships may serve as the basis for biologically tailoring Pd nanoparticle catalysts and could potentially be used to direct the biogenesis of other metal nanomaterials.

synthetic biology↗

bioRxiv2018

Microbial Reduction of Metal-Organic Frameworks Enables Synergistic Chromium Removal

Microbe-material redox interactions underpin many emerging technologies, including bioelectrochemical cells and bioremediation. However, commonly utilized material substrates, such as metal oxides, suffer from a lack of tunability and can be challenging to characterize. In contrast, metal-organic frameworks, a class of porous materials, exhibit well-defined structures, high crystallinity, large surface areas, and extensive chemical tunability. Here, we report that metal-organic frameworks can support the growth of the electroactive bacterium Shewanella oneidensis. Specifically, we demonstrate that Fe(III)-containing frameworks, MIL-100 and Fe-BTC, can be reduced by the bacterium via its extracellular electron transfer pathways and that reduction rate/extent is tied to framework structure, surface area, and particle morphology. In a practical application, we show that cultures containing S. oneidensis and reduced frameworks can remediate lethal concentrations of Cr(VI), and that pollutant removal exceeds the performance of either component in isolation or bioreduced iron oxides. Repeated cycles of Cr(VI) dosing had little effect on bacterial viability or Cr(VI) adsorption capacity, demonstrating that the framework confers protection to the bacteria and that no regenerative step is needed for continued bioremediation. In sum, our results show that metal-organic frameworks can serve as microbial respiratory substrates and suggest that they may offer a promising alternative to metal oxides in applications seeking to combine the advantages of bacterial metabolism and synthetic materials.

microbiology↗