R-3 Repository :: Browsing by Author "Tabor, Jeffrey J"

Browsing by Author "Tabor, Jeffrey J"

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A Synthetic Biology Approach to Engineering Bacterial Two- Component Systems for Sensor Development and Discovery of Anti-Virulence Agents
(2019-12-06) Ekness, Felix; Tabor, Jeffrey J; Matthews, Kathleen
Bacterial two-component systems (TCSs) are the largest family of signal transduction pathways that enable bacteria to sense a diversity of stimuli including small peptides, environmental pollutants, and light. Canonical TCSs are composed of a transmembrane sensor histidine kinase (SK) that converts stimulus detection into phosphorylation of a cognate response regulator (RR). Upon phosphorylation, the cytoplasmic RR binds target output promoters, hereby modulating gene expression. TCSs are valuable sensors for synthetic biology due to their diverse sensing capabilities and straightforward transduction of detected stimulus into transcriptional regulation. TCSs are also emerging targets for novel therapeutic development due to their extensive role in regulating bacterial virulence and antibiotic resistance. Although TCSs are exciting sensors for synthetic biology and targets for therapeutic applications, most TCSs remain difficult to harness for applications and study due to output promoters that are unknown, subject to cross- regulation, or silent in heterologous hosts. In the first portion of my work, I develop a method to overcome the hurdles in characterizing and utilizing TCSs as biosensors. Through the framework of synthetic biology, I demonstrate that the two largest families of RR DNA binding domains (DBDs) can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. In collaboration with Kristina Daeffler, we exploit this plasticity to eliminate cross-regulation and in collaboration with Brian Landry, we un-silence a gram-negative TCS in a gram-positive host and engineer a sensor with over 1,300-fold activation. In collaboration with Kathryn Brink, we also apply DBD swapping to screen uncharacterized Shewanella oneidensis TCSs in Escherichia coli, leading to the discovery of a novel pH sensor. In the second portion of my work, I demonstrate a method for identifying inhibitors of a virulence regulating TCS. This work focuses on the methicillin-resistant Staphylococcus aureus (MRSA) virulence regulating TCS SaeRS. I first heterologously express saeRS in Bacillus subtilis to remove the native, confounding regulation of the TCS and its output promoter. I then screen heterologous SaeRS against a diverse 1,593 small molecule library to identify inhibitors of its signaling. A lead compound emerged from the screen as an effective inhibitor of SaeRS signaling, leading to decreased exoprotein secretion and virulence from treated MRSA similar to MRSA lacking saeRS. My work described herein should accelerate 1) fundamental TCS studies and the engineering of a large family of biosensors with diverse applications and 2) the discovery of new anti-virulence compounds.
Design Principles of Cellular Differentiation Regulatory Networks
(2015-12-01) Narula, Jatin; Igoshin, Oleg A; Tabor, Jeffrey J; Bennett, Matthew R
To understand cellular differentiation programs is to understand the often large and complex gene regulatory networks (GRNs) that control and orchestrate these programs. The work presented here aims to exploit the wealth of newly available experimental information and methods to identify design principles that relate how GRN structures relate to the functional requirements in three model differentiation/stress-response programs: embryonic hematopoiesis, sporulation and σB general stress response. First we used a statistical thermodynamic approach to characterize the biophysical mechanisms of combinatorial regulation by distant enhancers in eukaryotes and demonstrate how the GRN controlling embryonic hematopoiesis acts as an irreversible bistable switch with low-pass noise filtering properties. We further used our model of the hematopoiesis network to reconcile discrepant experimental observations about the regulator Runx1 and explained how it limits HSC emergence in vitro. In the second project we investigated the Bacillus subtilis sporulation network and showed how a cascade of feed-forward loops downstream of the master regulatory Spo0A~P control cell-fate during starvation. We also identified a rate-responsive network module in the Spo0A regulon to explain why accelerated accumulation of Spo0A~P leads to a dramatic reduction in sporulation efficiency. Further we found that the arrangement of two sporulation network genes on opposite ends of the chromosome ties Spo0A~P activation to the DNA replication status. We were also able to show that the slowdown of cell growth is the primary starvation signal that determines sporulation cell-fates by controlling Spo0A~P activation. For the third project we built a detailed model of the σB network in Bacillus subtilis to mechanistically explain the experimentally observed pulsatile response of this network under stress. We further showed that the same network architecture that enables this pulsatile response insulates the σB network from the effects of competition for cellular resources like RNA polymerase. The design principles identified in the studies of these networks are related to their topological structure and function rather than the specific genes and proteins that comprise them. As a result, we expect them to be widely applicable to and help in the study of a diverse array of other differentiation GRNs.
Discovering and Calibrating Design Rules for Programming Adeno-Associated Virus Nanoparticles
(2015-12-03) Ho, Michelle Liane; Suh, Junghae; Silberg, Jonathan J; Tabor, Jeffrey J
Effective gene therapy must deliver therapeutic genes to disease sites while avoiding healthy tissue. However, engineering targeted gene delivery vectors to ensure exclusive delivery to diseased sites remains a challenge. Adeno-associated virus (AAV) is receiving increasing attention for its potential as a gene delivery vehicle because it offers several advantages: it is considered the safest viral vector, it infects human cells efficiently, and it can be genetically altered to improve therapeutic efficacy. However, even slight modifications to the virus capsid (the outer protein shell covering its genome) lead to unpredictable outcomes. Thus, a governing set of design rules for virus capsid assembly and function is needed to improve future engineering efforts. To this end, this thesis uncovers some of these rules by applying a computational model, often used in protein engineering, to the AAV capsid. A new strategy to improve AAV targeting was also explored by engineering AAVs to sense and become activated by extracellular proteases found in diseased tissues. The specificity of these protease-activatable viruses can be tuned to recognize a variety of protease profiles to treat a multitude of diseases. Design rules for these platform technologies are unveiled through their development and in-depth characterization. We also explore new motifs in the AAV capsid to further our understanding of AAV basic biology. Ultimately, these studies advance our ability to program virus nanoparticles for many biomedical applications.
Dynamic characterization of bacterial optogenetic sensors and their use in manipulating the gut microbiome to extend host longevity
(2018-04-11) Hartsough, Lucas; Tabor, Jeffrey J
The development and characterization of novel sensor elements are critical to synthetic biology, as they enable engineered organisms to respond intelligently and predictably to their environment. Bacterial two component systems (TCSs) are a particularly attractive family of sensors by virtue of their ubiquity and sensing diversity. We have previously engineered multiple TCSs that have the unique advantage of sensing specific wavelengths of light. The precision of light enables such optogenetic sensors to remotely manipulate biological systems with unprecedented spatiotemporal control. However, in order to leverage these advantages, we must accurately model optogenetic TCS dynamics. In this work, we demonstrate the application of a simple characterization method from control theory & electrical engineering to two optogenetic TCSs. We then compare its performance to previously published models and identify regimes in which it is reasonably accurate. In the second portion of this work, we leverage the spatiotemporal precision of one such optogenetic TCS, CcaSR, to to remotely control E. coli expression dynamics in the gastrointestinal tract of live C. elegans nematodes. Next, we engineer a synthetic genetic system that places biosynthesis of the exopolysaccharide colanic acid (CA), which has been recently demonstrated to enhance C. elegans longevity, under control of CcaSR. Finally, we demonstrate that light-mediated activation of CA production in gut bacteria elicits a protective effect on a host mitochondrial phenotype. Our method can be used to control the expression of virtually any gene in gut-resident E. coli and should enable a new of era of mechanistic studies of gut microbe-host interactions. Finally, we describe the development of a software package designed to facilitate the high-throughput identification of arbitrary protein clusters, including TCSs and other sensors, from bacterial genomes. We validate this package by correctly identifying 24/27 TCSs in E. coli, and then extract 45654 putative, nonredundant TCSs from 5550 RefSeq genomes, including 115 optogenetic TCSs, as well as over 500 novel TCSs from the human gut microbiome. Characterizing TCSs from these libraries could yield new optogenetic sensors with diverse input spectra, new models for studying alternative TCS signaling architectures, and novel sensors of disease in the gut microbiome.
Engineering and application of two component system biosensors
(2018-03-16) Landry, Brian P; Tabor, Jeffrey J
Bacterial sensors are a critical component of the sense-compute-respond framework used by synthetic biologists to engineer bacteria, and better tools are needed to implement and optimize these sensors. Two-component systems (TCSs) are the largest family of signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which these biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the strength of the phosphatase activity of the sensor histidine kinase. We experimentally validate this result by using known phosphatase-altering mutations to rationally tune the detection thresholds of engineered Bacillus subtilis nitrate and E. coli aspartate sensors up to two orders of magnitude. We go on to demonstrate that a widely-conserved residue previously shown to impact phosphatase activity can be mutated to apply our “TCS tuning” method to sensors for which no well-characterized mutations exist. In the second portion of this work, we engineer a soil bacterium to measure levels of fertilizer in soil. We utilize synthetic promoters and engineered transcription factors to transfer a nitrate sensor from the model organism E. coli to the soil bacterium B. subtilis to create a high performing nitrate sensor. We next develop a protocol to use this strain as a biosensor of levels of nitrate in soil, and use this capability to report levels of soil fertilization. Lastly, the detection threshold tuning technique developed in the first half of this work is utilized to expand the range of fertilizer concentrations that can be monitored. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.
Engineering Bacterial Optogenetic Sensors
(2017-10-02) Ong, Nicholas Ting Xun; Tabor, Jeffrey J
Optogenetic tools use genetically encoded photoreceptors to transduce light signals and control biological processes in the cell with unprecedented spatio-temporal precision. Our lab and other groups have previously developed UV/green, blue, green/red, red/far-red and near-infrared (NIR) photosensors in E. coli to regulate gene expression. The existing NIR photosensor can be activated by shorter red wavelengths (< 700 nm), has slow response dynamics and relies on secondary messenger signaling that can affect vital cell functions. A new optogenetic tool that has a rapid photoreversible response to longer NIR wavelengths would facilitate multiplexing with existing photoreceptors to provide dynamic control of multiple genes, and enable the remote control of bacterial gene expression in the gut microbiome as NIR light has superior penetration of tissue. Here, we engineer R. palustris BphP1-PpsR2 as a photoreversible NIR/red transcriptional regulatory tool in E. coli. We also explore other photoreceptor candidates and approaches for engineering new NIR optogenetic tools, namely the R. palustris BphP4 two-component system, and swapping a NIR-absorbing cyanobacteriochrome (CBCR) minimal photosensory domain into our existing CBCR CcaS green sensor (v2.0) in E. coli. We demonstrate that BphP1-PpsR2 PBr_crtE transcriptional output can be precisely tuned by varying NIR/red light intensities. BphP1-PpsR2 has rapid photoreversible dynamics and shows changes in gene expression within minutes. BphP1-PpsR2 is the most red-shifted bacterial optogenetic tool yet reported and is strongly activated by NIR wavelengths up to 780 nm. Unlike the previously reported NIR sensor, BphP1-PpsR2 has much quicker response dynamics and does not rely on secondary messenger signaling. Additionally, based on recent literature, we apply domain truncations to our v2.0 CcaS sensor to engineer a miniaturized v3.0 CcaS sensor that retains its green/red response. We show that our miniaturized v3.0 CcaS sensor in E. coli has an enhanced dynamic range (593-fold vs. 110-fold) and lower transcriptional output when deactivated, but shares similar light sensitivity and rapid response dynamics to those of the v2.0 sensor. In sum, BphP1-PpsR2 expands the spectral boundaries of the existing bacterial optogenetic toolkit further into the NIR region, while the v3.0 CcaS sensor provides greater utility and impact with its enhanced performance characteristics.
Engineering multi-input gene regulation for applications in Synthetic Biology
(2015-04-17) Shis, David Liu; Bennett, Matthew R; Shamoo, Yousif; Silberg, Jonathan J; Tabor, Jeffrey J
Synthetic biology offers insight into molecular biology through the design and implementation of synthetic gene networks. One challenge in this effort is implementing transcriptional logic gates that enable synthetic gene networks to make decisions based on multiple inputs. However, the ability to implement transcriptional logic gates is inhibited by a lack of parts available to build them. In this work, we explore strategies for facilitating multi-input gene regulation in prokaryotes. That is, we develop methods for making the expression of a reporter gene dependent on two or more inputs in Escherichia coli. We first demonstrate how fragmentation of T7 RNA Polymerase (T7 RNAP) creates a multi-fragment transcription complex that facilitates AND transcriptional logic. We find split T7 RNAP to be functional in vivo and that both fragments of the split protein must be present for transcription from the T7 Promoter, PT7, to occur. We also find that the specificity of the split protein can be modified to create split protein mutants with orthogonal specificity. In addition to split T7 RNAP, we test the AND transcriptional logic made possible by co-expressing multiple chimeric LacI/GalR transcriptional repressors. We find that each chimeric repressor regulates the operator site of its DNA binding domain (DBD) according to the ligand sensed by its ligand binding domain(LBD). By co-expressing multiple chimeric repressors, we find each repressor independently regulates its DBD's operator. As a result, the number of inputs at a promoter relates directly to the number of species of chimeric repressors with the same DBD. Further, by modifying the DBD we find that we can create chimeras with orthogonal specificities that facilitate an orthogonal open reading frame. We find expression of our chimeric repressors en mass facilitates regulation such as a four-input transcriptional AND gate or two orthogonal transcriptional AND gates. Split T7 RNAP and the coexpression of chimeric LacI/GalR repressors both demonstrate strategies for multi-input gene regulation in prokaryotes. This work also suggest strategies for the engineering of additional components for use in synthetic gene networks.
Engineering Optogenetic Control of Bacterial Metabolism in Stationary Phase of Growth
(2023-09-06) Lazar, John Tyler; Tabor, Jeffrey J; Thyer, Ross
Genetically-encoded sensors are used to induce metabolite production in bacterial fermentations. However, these sensors are typically optimized for exponential growth phase rather than stationary phase where the majority of metabolite production occurs. In the first portion of this work, we find that our exponential phase-optimized green light-activated E. coli two-component gene regulatory system CcaSR is effectively non-functional in stationary phase. We show that the major causes of failure are stationary-phase specific mutation of the plasmid-borne biosynthetic pathway used to produce the required chromophore phycocyanobilin (PCB) and accumulation of the response regulator CcaR leading to very high leaky target gene expression. To address these problems, we move the PCB biosynthetic pathway into the chromosome and re-optimize expression of the component enzymes, and re-balance CcaR expression for stationary phase . The resulting CcaSRstat system exhibits low levels of leakiness and an 80-fold activation of target gene expression in stationary phase. Notably, our stationary phase-optimized CcaSRstat system is not functional in exponential phase, a feature that may have benefits for metabolic engineering and other applications. In the second portion of this work, we combine CcaSRstat with static and periodic illumination patterns to achieve high levels of production of several industrially-relevant phenylpropanoid metabolites such as p-coumaric acid and bisdemethoxycurcumin. We then proceed to demonstrate that our optimal light signals at the 0.5 mL light plate apparatus (LPA) volume scale to 25 mL optogenetic bioreactors. CcaSRstat is a useful tool for optimizing bacterial metabolite production and could be used to control bacterial behaviors in other non-growth environments such as the gastrointestinal tract or soil. Our work lays the foundation for increasing the exploration space of dynamic control of metabolic pathways while also providing valuable insight into design considerations of biosensors in the stationary phase of growth.
Multiplexing cell-cell communication
(2019-04-18) Sexton, John Thomas; 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. In this work, I engineer a genetically-encoded channel selector device that enables a single communication system to transmit two separate intercellular conversations. My 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 amongst a community of cells.
Optogenetic programming of complex, multiplexed gene expression signals
(2016-04-20) Olson, Evan J; Tabor, Jeffrey J
Optogenetic tools are genetically expressed signaling pathways that transduce extracellular light signals into intracellular, biochemical signals. These biological signals can be used to interface with intracellular biological networks, enabling a perturbative experimental approach that can be used to reverse-engineer the molecular basis underlying cellular behaviors. Light is particularly well suited as an experimentally tunable control signal, because the intensity and wavelength of light sources can be exquisitely controlled in both time and space. Many optogenetic tools have been developed in the past decade; however, the ability to use them to perform the biological function generation required for the interrogation of cellular networks has been limited. Here, I have worked to overcome these limitations by 1) establishing the concept of a biological function generator and identifying a roadmap for the optogenetic characterization of biological systems, 2) developing and demonstrating the first biological function generator used to characterize a cellular circuit in live E. coli cells, 3) implementing a photoconversion-based multispectral model which enables gene expression programming with any light input signal, and 4) developing the first multiplexed optogenetic tool capable of simultaneous, independent generation of two biological functions. This work has produced the world's most precise means of producing arbitrary gene expression signals in live cells. The approach and tools developed here should be generalizable to other optogenetic systems, even in eukaryotic organisms. I describe the technical developments in hardware, software, laboratory protocols, and mathematical models which were required to make this progress. The biological function generator approach and tools developed here are an unprecedented means for characterizing biological systems and controlling cellular behaviors, and will enable novel experimental approaches in both systems and synthetic biology.

Browsing by Author "Tabor, Jeffrey J"

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