Browsing by Author "Tabor, Jeffrey J."
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Item A photoconversion model for full spectral programming and multiplexing of optogeneticﾠsystems(EMBO Press, 2017) Olson, Evan J.; Tzouanas, Constantine N.; Tabor, Jeffrey J.Optogenetics combines externally applied light signals and genetically engineered photoreceptors to control cellular processes with unmatched precision. Here, we develop a mathematical model of wavelength‐ and intensity‐dependent photoconversion, signaling, and output gene expression for our two previously engineered light‐sensing Escherichia coli two‐component systems. To parameterize the model, we develop a simple set of spectral and dynamical calibration experiments using our recent open‐source “Light Plate Apparatus” device. In principle, the parameterized model should predict the gene expression response to any time‐varying signal from any mixture of light sources with known spectra. We validate this capability experimentally using a suite of challenging light sources and signals very different from those used during the parameterization process. Furthermore, we use the model to compensate for significant spectral cross‐reactivity inherent to the two sensors in order to develop a new method for programming two simultaneous and independent gene expression signals within the same cell. Our optogenetic multiplexing method will enable powerful new interrogations of how metabolic, signaling, and decision‐making pathways integrate multiple input signals.Item An all-optical biological function generator and oscilloscope framework for characterizing gene circuit dynamics(2014-04-21) Olson, Evan James; Tabor, Jeffrey J.; Igoshin, Oleg A.; Bennett, Matthew R.Gene circuits are dynamical systems that regulate cellular behaviors, often using protein signals as inputs and outputs. Here we have developed an optogenetic ‘function generator’ for programming tailor-made gene expression signals in live E. coli. We designed light sequences with experimentally calibrated models of light-switchable two-component systems and used them to drive intracellular protein levels to match user-defined reference time-courses. This approach enabled generation of accelerated and linearized dynamics, sinusoidal oscillations with desired amplitudes and periods, and a complex waveform, all with unprecedented accuracy and precision. We also combined the function generator with a dual fluorescent protein reporter system, analogous to a dual-channel oscilloscope, to reveal that a synthetic repressible promoter linearly transforms repressor signals with an approximate 7-min delay. Our approach will enable a new generation of dynamic analyses of synthetic and natural gene circuits, providing an essential step toward the predictive design and rigorous understanding of biological systems.Item An Engineered B. subtilis Inducible Promoter System with over 10 000-Fold Dynamic Range(American Chemical Society, 2019) Castillo-Hair, Sebastian M.; Fujita, Masaya; Igoshin, Oleg A.; Tabor, Jeffrey J.; Center for Theoretical Biological PhysicsBacillus subtilis is the leading model Gram-positive bacterium, and a widely used chassis for industrial protein production. However, B. subtilis research is limited by a lack of inducible promoter systems with low leakiness and high dynamic range. Here, we engineer an inducible promoter system based on the T7 RNA Polymerase (T7 RNAP), the lactose repressor LacI, and the chimeric promoter PT7lac, integrated as a single copy in the B. subtilis genome. In the absence of IPTG, LacI strongly represses T7 RNAP and PT7lac and minimizes leakiness. Addition of IPTG derepresses PT7lac and simultaneously induces expression of T7RNAP, which results in very high output expression. Using green fluorescent and β-galactosidase reporter proteins, we estimate that this LacI-T7 system can regulate expression with a dynamic range of over 10 000, by far the largest reported for an inducible B. subtilis promoter system. Furthermore, LacI-T7 responds to similar IPTG concentrations and with similar kinetics as the widely used Phy-spank IPTG-inducible system, which we show has a dynamic range of at most 300 in a similar genetic context. Due to its superior performance, our LacI-T7 system should have broad applications in fundamental B. subtilis biology studies and biotechnology.Item An open-hardware platform for optogenetics and photobiology(Springer Nature, 2016) Gerhardt, Karl P.; Olson, Evan J.; Castillo-Hair, Sebastian M.; Hartsough, Lucas A.; Landry, Brian P.; Ekness, Felix; Yokoo, Rayka; Gomez, Eric J.; Ramakrishnan, Prabha; Suh, Junghae; Savage, David F.; Tabor, Jeffrey J.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. Here, 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.Item Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation(EMBO Press, 2017) Daeffler, Kristina N-M; Galley, Jeffrey D.; Sheth, Ravi U.; Ortiz-Velez, Laura C.; Bibb, Christopher O.; Shroyer, Noah F.; Britton, Robert A.; Tabor, Jeffrey J.There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two?component systems from marine Shewanella species, and validate them in laboratory Escherichiaﾠcoli. Then, we port these sensors into a gut?adapted probiotic E.ﾠcoli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.Item FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units(American Chemical Society, 2016) Castillo-Hair, Sebastian M.; Sexton, John T.; Landry, Brian P.; Olson, Evan J.; Igoshin, Oleg A.; Tabor, Jeffrey J.; Center for Theoretical Biological PhysicsFlow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, nonproprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality plots. We validate FlowCal by calibrating a.u. measurements of E. coli expressing superfolder GFP (sfGFP) collected at 10 different detector sensitivity (gain) settings to a single MEF value. Additionally, we reduce day-to-day variability in replicate E. coli sfGFP expression measurements due to instrument drift by 33%, and calibrate S. cerevisiae Venus expression data to MEF units. Finally, we demonstrate a simple method for using FlowCal to calibrate fluorescence units across different cytometers. FlowCal should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond.Item High-Throughput Discovery of Input Stimuli of Bacterial Two-Component Systems(2021-07-22) Brink, Kathryn Renee; Tabor, Jeffrey J.; Martí, Angel A.Two-component systems (TCSs) are the largest class of biological signal transduction pathways and an important class of bacterial sensors. In their native contexts, TCSs enable bacteria to sense and respond to changes in their environment. TCSs are also a major source of novel biosensors for medical, environmental, and industrial applications. In the first portion of this work, we use TCS engineering and chemical input screens to characterize TCSs of unknown function from Shewanella oneidensis. Through these screens, we discover a pH-responsive TCS, which we apply to detect acidification of intestinal tissues in a mouse model of inflammatory bowel disease. In the second portion of this work, we develop a high-throughput screening approach for characterizing peptide-TCS interactions. We screen PhoPQ, a virulence-regulating TCS from Salmonella Typhimurium, against 117 human antimicrobial peptides (AMPs). We discover 13 novel activators of PhoPQ comprising diverse sequences, structures, and biological functions and identify subdomains and peptide biophysical features responsible for PhoPQ activation. Finally, we find that PhoPQ homologs exhibit distinct AMP response profiles, suggesting a role for evolutionary adaptation in AMP sensing. The engineering approaches developed here enable high-throughput discovery and characterization of TCS inputs, which could provide important insights into bacterial stimulus response and reveal novel biosensors with applications across a wide range of sectors.Item How to train your microbe: methods for dynamically characterizing gene networks(Elsevier, 2015) Castillo-Hair, Sebastian M.; Igoshin, Oleg A.; Tabor, Jeffrey J.; Center for Theoretical Biological PhysicsGene networks regulate biological processes dynamically. However, researchers have largely relied upon static perturbations, such as growth media variations and gene knockouts, to elucidate gene network structure and function. Thus, much of the regulation on the path from DNA to phenotype remains poorly understood. Recent studies have utilized improved genetic tools, hardware, and computational control strategies to generate precise temporal perturbations outside and inside of live cells. These experiments have, in turn, provided new insights into the organizing principles of biology. Here, we introduce the major classes of dynamical perturbations that can be used to study gene networks, and discuss technologies available for creating them in a wide range of microbial pathways.Item Identifying ligands for bacterial sensors(2020-10-06) Tabor, Jeffrey J.; Schmidl, Sebastian; Sheth, Ravi; Ekness, Felix; Landry, Brian; Dyuglyarov, Nikola; Rice University; United States Patent and Trademark OfficeMethods to create two component signal transduction systems by replace the DNA binding domains and output promoters in bacteria are described.Item Independent control of mean and noise by convolution of gene expression distributions(Springer Nature, 2021) Gerhardt, Karl P.; Rao, Satyajit D.; Olson, Evan J.; Igoshin, Oleg A.; Tabor, Jeffrey J.; Center for Theoretical BiophysicsGene expression noise can reduce cellular fitness or facilitate processes such as alternative metabolism, antibiotic resistance, and differentiation. Unfortunately, efforts to study the impacts of noise have been hampered by a scaling relationship between noise and expression level from individual promoters. Here, we use theory to demonstrate that mean and noise can be controlled independently by expressing two copies of a gene from separate inducible promoters in the same cell. We engineer low and high noise inducible promoters to validate this result in Escherichia coli, and develop a model that predicts the experimental distributions. Finally, we use our method to reveal that the response of a promoter to a repressor is less sensitive with higher repressor noise and explain this result using a law from probability theory. Our approach can be applied to investigate the effects of noise on diverse biological pathways or program cellular heterogeneity for synthetic biology applications.Item Leveraging synthetic biology for tissue engineering applications(The Japanese Society of Inflammation and Regeneration, 2014) Lee, Esther J.; Tabor, Jeffrey J.; Mikos, Antonios G.Restoration of damaged tissues and organs requires precise control of cellular processes at the molecular level. Synthetic biology offers genetic tools that can be used to program the molecular biology of the cell, thereby potentially overcoming the various challenges hampering contemporary tissue engineering applications.Item Mucosal acidosis elicits a unique molecular signature in epithelia and intestinal tissue mediated by GPR31-induced CREB phosphorylation(National Academy of Sciences, 2021) Cartwright, Ian M.; Dowdell, Alexander S.; Lanis, Jordi M.; Brink, Kathryn R.; Mu, Andrew; Kostelecky, Rachael E.; Schaefer, Rachel E.M.; Welch, Nichole; Onyiah, Joseph C.; Hall, Caroline H.T.; Gerich, Mark E.; Tabor, Jeffrey J.; Colgan, Sean P.; Systems, Synthetic, and Physical Biology ProgramMetabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn’s disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.Item Multiplexing cell-cell communication(EMBO Press, 2020) Sexton, John T.; Tabor, Jeffrey J.The engineering of advanced multicellular behaviors, such as the programmed growth of biofilms or tissues, requires cells to communicate multiple aspects of physiological information. Unfortunately, few cell-cell communication systems have been developed for synthetic biology. Here, we engineer a genetically encoded channel selector device that enables a single communication system to transmit two separate intercellular conversations. Our design comprises multiplexer and demultiplexer sub-circuits constructed from a total of 12 CRISPRi-based transcriptional logic gates, an acyl homoserine lactone-based communication module, and three inducible promoters that enable small molecule control over the conversations. Experimentally parameterized mathematical models of the sub-components predict the steady state and dynamical performance of the full system. Multiplexed cell-cell communication has applications in synthetic development, metabolic engineering, and other areas requiring the coordination of multiple pathways among a community of cells.Item Optogenetic control of gut bacterial metabolism to promote longevity(eLife Sciences Publications, Ltd, 2020) Hartsough, Lucas A.; Park, Mooncheol; Kotlajich, Matthew V.; Lazar, John Tyler; Han, Bing; Lin, Chih-Chun J.; Musteata, Elena; Gambill, Lauren; Wang, Meng C.; Tabor, Jeffrey J.Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.Item Optogenetic control of Bacillus subtilis gene expression(Springer Nature, 2019) Castillo-Hair, Sebastian M.; Baerman, Elliot A.; Fujita, Masaya; Igoshin, Oleg A.; Tabor, Jeffrey J.The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.Item Phosphatase activity tunes two-component system sensor detection threshold(Springer Nature, 2018) Landry, Brian P.; Palanki, Rohan; Dyulgyarov, Nikola; Hartsough, Lucas A.; Tabor, Jeffrey J.Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the phosphatase activity of the sensor histidine kinase. We experimentally validate this result in engineered Bacillus subtilis nitrate and E. coli aspartate TCS sensors by tuning their detection threshold up to two orders of magnitude. We go on to apply our TCS tuning method to recently described tetrathionate and thiosulfate sensors by mutating a widely conserved residue previously shown to impact phosphatase activity. Finally, we apply TCS tuning to engineer B. subtilis to sense and report a wide range of fertilizer concentrations in soil. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.Item Real-time detection of response regulator phosphorylation dynamics in live bacteria(National Academy of Sciences, 2022) Butcher, Ryan J.; Tabor, Jeffrey J.Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct in vivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.Item Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems(American Chemical Society, 2014) Schmidl, Sebastian R.; Sheth, Ravi U.; Wu, Andrew; Tabor, Jeffrey J.Light-switchable proteins enable unparalleled control of molecular biological processes in live organisms. Previously, we have engineered red/far-red and green/red photoreversible two-component signal transduction systems (TCSs) with transcriptional outputs in E. coli and used them to characterize and control synthetic gene circuits with exceptional quantitative, temporal, and spatial precision. However, the broad utility of these light sensors is limited by bulky DNA encoding, incompatibility with commonly used ligand-responsive transcription factors, leaky output in deactivating light, and less than 10-fold dynamic range. Here, we compress the four genes required for each TCS onto two streamlined plasmids and replace all chemically inducible and evolved promoters with constitutive, engineered versions. Additionally, we systematically optimize the expression of each sensor histidine kinase and response regulator, and redesign both pathway output promoters, resulting in low leakiness and 72- and 117-fold dynamic range, respectively. These second-generation light sensors can be used to program the expression of more genes over a wider range and can be more easily combined with additional plasmids or moved to different host strains. This work demonstrates that bacterial TCSs can be optimized to function as high-performance sensors for scientific and engineering applications.Item Regulation of cell number and cell movement in Dictyostelium discoideum(2013-09-16) Phillips, Jonathan; Gomer, Richard H.; Braam, Janet; Beckingham, Kathleen M.; Farach-Carson, Cindy; Tabor, Jeffrey J.Little is known about how the size of a tissue is established during development and maintained subsequently. Proliferation-inhibiting signals secreted by cells within a tissue that act specifically on cells within that tissue can provide negative feedback on cell number, thus regulating tissue size. A better understanding of tissue-specific inhibitors of proliferation could be useful for designing therapies for cancer and other diseases. However, few signals of this sort have been identified, and little is known about how these signals function. Two examples of such signals are the proteins AprA and CfaD, which are secreted by the social amoeba Dictyostelium discoideum and inhibit cell proliferation in a concentration-dependent manner. Cells lacking either AprA or CfaD proliferate rapidly, and adding recombinant AprA or CfaD to cells reduces proliferation. However, little is known about the signal transduction pathways downstream of AprA and CfaD. I identified three proteins that are required for the normal function of AprA and CfaD: the kinase QkgA, the putative transcription factor BzpN, and the putative kinase PakD. Cells lacking any one of these proteins proliferate rapidly, and adding AprA or CfaD to cells lacking these proteins does not cause reduced proliferation, indicating that these proteins are involved in AprA/CfaD signal transduction. I also found that, in addition to its proliferation-inhibiting activity, AprA also functions as an autocrine chemorepellant. Colonies of cells lacking AprA expand less rapidly than wild-type colonies, despite the fact that individual cells lacking AprA show a random motility like that of wild-type cells. Further, two independent assays demonstrate that cells show a biased movement away from a source of AprA. The chemorepellant activity of AprA requires CfaD, QkgA, and PakD, but not BzpN, indicating that AprA affects proliferation and chemorepulsion through distinct but overlapping pathways. These results suggest that AprA functions as a readout of local cell density, to which cells respond by slowing proliferation and chemotaxing to regions of lower cell density, where nutrients are more likely to be present. The study of human AprA, CfaD, QkgA, BzpN, and PakD orthologs may serve to guide therapeutic approaches that modulate chemorepulsive or antiproliferative processes.Item Rewiring bacterial two-component systems by modular DNA-binding domain swapping(Springer Nature, 2019) Schmidl, Sebastian R.; Ekness, Felix; Sofjan, Katri; Daeffler, Kristina N-M; Brink, Kathryn R.; Landry, Brian P.; Gerhardt, Karl P.; Dyulgyarov, Nikola; Sheth, Ravi U.; Tabor, Jeffrey J.Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways and valuable sensors for synthetic biology. However, most TCSs remain uncharacterized or difficult to harness for applications. Major challenges are that many TCS output promoters are unknown, subject to cross-regulation, or silent in heterologous hosts. Here, we demonstrate that the two largest families of response regulator DNA-binding domains can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. We exploit this plasticity to eliminate cross-regulation, un-silence a gram-negative TCS in a gram-positive host, and engineer a system with over 1,300-fold activation. Finally, we apply DNA-binding domain swapping to screen uncharacterizedﾠShewanella oneidensisﾠTCSs inﾠEscherichia coli, leading to the discovery of a previously uncharacterized pH sensor. This work should accelerate fundamental TCS studies and enable the engineering of a large family of genetically encoded sensors with diverse applications.