Browsing by Author "Tabor, Jeff"

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    Development and Characterization of a UV-Violet/Green Photoreversible Transcriptional Regulator in E.coli
    (2016-08-31) Ramakrishnan, Prabha; Tabor, Jeff
    Genetically encoded photoreceptors, or optogenetic tools, have been used to leverage the tractability of light as an input signal to control biological processes in vivo with unrivaled spatiotemporal precision. The Tabor lab has previously engineered green/red and red/far-red photoreversible two-component systems (TCS) for quantitative, programmable control of transcription dynamics. A photoreversible optogenetic tool with spectral sensitivity in the UV-blue region of the visible spectrum would enable new optogenetic applications in single-cell microscopy, and could be combined with existing tools for dynamic control of multiple genes. In this work, we engineer a photoreversible UV-violet/Green transcriptional regulator in E.coli by repurposing the light-switchable cyanobacteriochrome TCS UirS-UirR from Synechocystis PCC 6803. We demonstrate that UirS-UirR regulates the promoter of the low carbon stress-induced small RNA csiR1 (PcsiR1) ~6-fold in response to UV-light. Using a combination of mutations, dose-response experiments, and in vivo phosphorylation measurements, we show that UirS phosphorylates UirR to activate transcription from PcsiR1 in a UV-light dependent manner. This result is in contrast to a proposed sequestration model for UirS-UirR. By measuring the action spectra of UirS-UirR, we show that it responds specifically in the narrow (380-420nm) UV-violet region of the visible spectrum. We show that the gene expression response to input light occurs in minutes and exploit these rapid dynamics to predictably program gene expression signals. By truncating N-terminal protein domains in UirS, we identify an improved sensor with >15-fold light-induced dynamic range but similar response characteristics as UirS. UirS-UirR is the first photoreversible optogenetic tool that responds in the UV-violet region of the visible spectrum, and could be combined with our previously engineered green/red and red/far-red sensors for precise three-input, three-output optogenetic control of transcription, enabling new modes of characterization and control in systems and synthetic biology and metabolic engineering studies. UirS contains a functional ethylene-binding domain similar to the Arabidopsis thaliana ethylene receptors, but is not known have an ethylene response. We have used the E. coli UirS-UirR system for studying the putative ethylene response of UirS heterologously, free of the cross-regulatory networks present in Synechocystis PCC6803. This work provides preliminary evidence that at least in E.coli, UirS does not have a signalling response to ethylene, or that the ethylene response is not transduced through UirR. The UirS-UirR E.coli system provides a test-bed to study the remarkable spectral diversity of the ‘DXCF cyanobacteriochrome’ family of photoreceptors, and the signaling properties of TCSs containing AraC-family DNA binding domains, which are involved in pathogenesis but are poorly understood.
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    Hardware, wetware, and methods for precision control of gene expression
    (2019-04-17) Gerhardt, Karl P; Tabor, Jeff
    The ability to control the biochemical processes of the cell is fundamental to the goals of synthetic biology. This control implies the ability to specify the spatial, temporal, and amount of gene expression and protein activity in individual cells and populations. Hardware, wetware, and methods which advance the ability to control these processes then increase the ability and precision of researchers to perturb and study natural biological systems and of engineers to build useful biological systems and products. In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. In the first portion of this work, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under \$400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments. In the second portion of this work, we describe a general method for independently controlling population mean and noise of cellular protein copy number by convolution of gene expression distributions. In this method, a gene is simultaneously expressed from both a low- and high-noise source, resulting in summation of the gene products within cells and convolution of the gene expression distributions within the population. By tuning the amount and ratio of expression from each source, the mean and noise, respectively, of the convolution can be independently controlled. In principle, this method can be applied in any host using any two sources of low- and high-noise gene expression. We demonstrate this method in \textit{Escherichia coli} by engineering and co-expressing a high-noise, LuxR-based, positive feedback transcriptional unit, and a low-noise, TetR-based, transcriptional unit without feedback. By exposing cells to different amounts and ratios of inducer we can independently control mean and noise in gene expression over a “dynamic area” of 1.1 with a maximum fold-change in CV of 6.9; performance metrics we propose and describe in this study. A mathematical model for mean and noise of a two-gene convolution accurately captures the observed behavior and can be used to predict mean and noise from inducer concentrations. Finally, we use our method to measure the effect of variability in the expression of a toxin on bacterial growth dynamics. We predict that the fraction of cells in the toxin-induced dormant state is a function of mean, while sensitivity to the mean depends on noise. Under some conditions we observe behavior consistent with this prediction, but our results generally suggest more complex and potentially interesting underlying dynamics.
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    High-Throughput Discovery of Stimuli of Bacterial Two-Component Systems from the Human Gut Microbiome
    (2024-04-18) Lorch, Kevin; Tabor, Jeff
    The human gut microbiota is a dense and diverse community of bacteria that interact with the host and have major impacts on health and disease. Two-component systems (TCS) are the primary means by which bacteria, including those in the gut microbiome, sense and adapt to their environment. The prototypical TCS consists of a membrane-bound sensor histidine kinase (SHK) and a cytoplasmic response regulator (RR). In the presence of a specific stimulus, the SHK phosphorylates the RR, which then binds to DNA and activates a transcriptional response. One example of a gut-relevant TCS is the major virulence regulator PhoPQ, which recognizes antimicrobial peptides (AMPs) during Salmonella infection. Here, we identify altered sensitivity to surface-displayed AMPs in E. coli PhoPQ compared to Salmonella Typhimurium PhoPQ. In unpublished work, the Tabor lab has computationally identified over 1,600 uncharacterized TCSs in the genomes of 450 common human gut bacteria. We hypothesize that these gut microbiome TCSs sense a diverse array of small molecules, macromolecules, and other signals associated with gut function and gut-linked diseases. Our goal is to elucidate stimuli detected by gut microbiome TCSs and repurpose them to engineer gut bacteria that diagnose and treat disease. In particular, our lab recently developed a technique to rewire TCSs to well-characterized output promoters by modularly swapping the response regulator DNA-binding domain. Previous work in the Tabor Lab computationally selected 543 diverse TCSs from the human microbiome, synthesized the genes, and transformed the library into E. coli. Each TCS controls the expression of a barcoded mRNA and a GFP reporter gene. Here, we develop a simple computational tool to infer TCS inputs based on nearby genes and predicted regulatory relationships. Using this approach, we have identified two novel divalent metal sensing TCSs, and a candidate sensor of the microbial metabolite cadaverine. We characterize both divalent metal sensing TCSs as homologs of bacterial copper sensors with relaxed metal specificity and amino acid substitutions in metal-binding sites. We characterize specificity of our candidate cadaverine sensor and demonstrate sensitivity of the TetR-inducible gene expression system to polyamines, exposing a critical failure mode in synthetic biological circuits. We also develop a next-generation sequencing (NGS) approach to high-throughput screening of this library in response to discrete chemical ligands. We demonstrate detection of transcriptional signals from TCSs on a 5-minute time scale. We identify low dynamic range in transcriptional signals, and propose design modifications to improve signal strength for high-throughput screens using this method. This unique TCS discovery platform has implications in understanding the function of the microbiome and should enable the engineering of diagnostic and therapeutic gut bacteria for a wide range of diseases.