Biology subjects

Wintenberg, M. E.

Publications and source records attributed to Wintenberg, M. E..

3 recordsLinked to original sources

bioRxiv2025

Genetically encoded RNA strand exchange circuits for programmable protein expression and computation in cells

Programmable cellular information processing could advance biomanufacturing of chemicals and medicine, and enable smart, living therapeutics and diagnostics1,2. Nucleic acids circuits based on toehold mediated strand exchange (TMSE) show tremendous potential for cellular programming due to their scalable, composable, and biocompatible parts3-6. However, these circuits are constrained primarily to in vitro applications because genetically encoding them is challenging and the principles of TMSE in cells remain unknown. Here we show the first demonstration of genetically encoded RNA strand exchange circuits, designed analogously to state-of-the-art TMSE circuits, in living cells. To elucidate the design principles of TMSE in cells, we develop toehold exchange riboregulators, which convert TMSE to protein expression, enabling precise control of protein translation rate. We find many of the design principles and parts used for TMSE in vitro transfer to Escherichia coli, allowing construction of multi-layer cascades and logic elements. We also identify caveats where strand exchange in cells differ substantially from cell-free systems, even in lysate from the same bacterial strain7, suggesting active cellular processes are involved. Our results further highlight bounds on strand exchange circuit architectures feasible in cells. We anticipate this study will lay the groundwork for developing advanced cellular circuits, bringing nucleic acid computing from the test tube to the cell8-10 and enabling new applications by connecting TMSE to gene expression. More broadly, our results have implications for RNA:RNA interactions and gene regulation in bacteria and provide a synthetic system for exploring such phenomena.

synthetic biology↗

bioRxiv2025

Comparative Transcriptomic Analysis of Perfluoroalkyl Substances-Induced Responses of Exponential and Stationary Phase Escherichia coli

Per- and polyfluoroalkyl substances (PFAS) are highly stable chemical contaminants of emerging concern for human and environmental health due to their non-natural chemistry, widespread use, and environmental persistence. Despite conventional metrology, mitigation strategies, and removal technologies, the complexity of this growing problem necessitates the need for alternative approaches to tackle the immense challenges associated with complex environmental PFAS contamination. Recently, biology has emerged as an alternative approach to detect and mitigate PFAS and understand the molecular-level responses of living organisms, including microorganisms, to these compounds. However, further study is needed to understand how microorganisms in different environments and growth phases respond to PFAS. In this study, we performed RNA sequencing at mid-exponential, early stationary phase, and late stationary phase of bacterial growth to determine the global transcriptional response of a model chassis, Escherichia coli MG1655, induced by two PFAS, perfluorooctanoic acid (PFOA) and perfluorododecanoic acid (PFDoA), and equivalent non-fluorinated carboxylic acids (NFCA), octanoic acid and dodecanoic acid. Differential gene expression analysis revealed PFOA and PFDoA induced distinct changes in gene expression throughout cultivation. Specifically, we identified significant changes in expression of the formate regulon and sulfate assimilation at mid-exponential phase and ferrous iron transport, central metabolism, the molecular chaperone network, and motility processes during stationary phase. Importantly, many of these changes are not induced by NFCAs. In summary, we found PFAS induced a system-level change in gene expression, and our results expand the understanding of bacterial-PFAS interactions that could enable the development of future real-time environmental monitoring and mitigation technologies. ImportanceThe prevalence and persistence of PFAS in the environment is a growing area of concern. However, little is understood of the impacts of PFAS on the environment, particularly impacts on microorganisms that play pivotal roles in nearly every ecosystem. Thus, comprehensive measurements that provide systems-level insight into how microorganisms respond and adapt to PFAS in the environment are paramount. Here, we use RNA sequencing to study the global transcriptional response of E. coli MG1655 to two PFAS and non-fluorinated equivalent compounds across growth phases. We find that PFAS induce system-level changes in metabolic, transport, and gene regulatory pathways, providing insight into how these non-natural chemicals interact with a model bacterium. Additionally, the transcriptomic dataset associated with this work provides the community with PFSA-specific gene expression patterns and possible PFAS degradation pathways for the development of future whole-cell biosensors and mitigation efforts.

genomics↗

bioRxiv2023

Design approaches to expand the toolkit for building cotranscriptionally encoded RNA strand displacement circuits

Cotranscriptionally encoded RNA strand displacement (ctRSD) circuits are an emerging tool for programmable molecular computation with potential applications spanning in vitro diagnostics to continuous computation inside living cells. In ctRSD circuits, RNA strand displacement components are continuously produced together via transcription. These RNA components can be rationally programmed through base pairing interactions to execute logic and signaling cascades. However, the small number of ctRSD components characterized to date limits circuit size and capabilities. Here, we characterize 220 ctRSD gate sequences, exploring different input, output, and toehold sequences and changes to other design parameters, including domain lengths, ribozyme sequences, and the order in which gate strands are transcribed. This characterization provides a library of sequence domains for engineering ctRSD components, i.e., a toolkit, enabling circuits with up to four-fold more inputs than previously possible. We also identify specific failure modes and systematically develop design approaches that reduce the likelihood of failure across different gate sequences. Lastly, we show ctRSD gate design is robust to changes in transcriptional encoding, opening a broad design space for applications in more complex environments. Together, these results deliver an expanded toolkit and design approaches for building ctRSD circuits that will dramatically extend capabilities and potential applications.

synthetic biology↗