Bacteria naturally contain a wide variety of multi-component molecular machines that perform complex functions such as chemotaxis, photosynthesis, chemical fixation, and protein secretion, all of which are potentially valuable to synthetic biologists for use in engineered organisms. However, in most cases, the gene clusters encoding these functions are complex and intricately regulated by the bacteria, making them difficult to reuse for new applications.
One potential method to address these difficulties is refactoring: a process of recoding the genes for the purpose of simplification and re-use. Here, we present the methods, techniques and results of refactoring the Salmonella Pathogenicity Island I (SPI-1) Type III Secretion System (T3SS). The SPI-1 T3SS is used naturally by Salmonella to inject pathogenic effector agents into its host cell and, unlike the most commonly used secretion mechanisms in bacteria, is capable of transporting functional proteins through both membranes of the bacteria directly into the culture media. This function may enable such biotech applications as targeted peptide/drug delivery and continuous protein expression. In addition, the methods developed here should be broadly applicable to a wide range of bacterial gene-clusters.
Computation underlies the organization of cells into higher-order structures; for example, during development or the spatial association of bacteria in a biofilm. Each cell performs a simple computational operation, but when combined with cell-cell communication, intricate patterns emerge. Here, we study this process by combining a simple genetic circuit with quorum sensing in order to produce more complex computations in space. A simple NOR gate is constructed by arranging two tandem promoters that function as inputs to drive the transcription of a repressor. The repressor inactivates a promoter that serves as the output. Individual colonies of E. coli carry the same NOR gate, but the inputs and outputs are wired to different orthogonal quorum sensing "sender" and "receiver" devices. The quorum molecules form the wires between gates. By arranging the colonies in different spatial configurations, all possible 2-input gates are produced, including the difficult XOR and EQUALS functions. The response is strong and robust, with 5- to >300-fold changes between the ON and OFF states. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calculations by rewiring communication between cells.
The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 (SPI-1) to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting an array of spider silk monomers representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg L-1 hour-1 are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in gram negative bacteria, which will be broadly applicable to problems in biotechnology.
Two-component systems are a highly conserved signal transduction pathway that enable bacteria to sense changes in their environment and adjust gene expression to adapt to nutrients, stresses, and other signals. The body of this work seeks to determine the extent to which E. coli uses these sensors as a network to process their environment. This is broken into two parts: (1) Whether cross talk can occur at the phosphorelay level and (2) whether the sensors are able to function as a combinatorial sensor. A combinatorial sensor is made up of a set of sensors, each of which is activated to different degrees by many inputs such that the pattern of their activation defines the signal. Using promoter reporters and flow cytometry, we measured the response of three two-component osmosensors in E. coli (envZ/ompR, cpxA/cpxR, and rcsC/rcsD/rcsB) to 38 chemicals including known inducers of the systems, membrane perturbing agents, alcohols and chemicals of industrial relevance. We found that each system responded to a wide spectrum of conditions and that the three systems are uncorrelated, meaning that unique patterns of gene expression are generated by even closely related chemical compounds. Of the eight possible patterns generated by a three sensor system, we observe five. This data show that bacteria are able to use a limited set of sensory components to identify a diverse set of compounds and environmental conditions.
Living organisms exhibit many fascinating behaviors that profoundly impact the world surrounding us. Some of these functions have been harnessed within biotechnology and redirected towards solving problems of industrial relevance, e.g. the microbial synthesis of pharmaceuticals and biofuels. However, these behaviors tend to be encoded by relatively simple genetics and constrained to a handful of lab-friendly organisms. To gain access to diverse, more complex behaviors, I have developed a number of methods for understanding and reprogramming sophisticated genetic networks inside cells. First, I reverse engineered and predictively modified the control of the Type III secretion system in Salmonella using mathematical modeling and high-throughput gene expression measurements in single cells. Next, a "refactoring" methodology was developed to entirely reconstruct and specify the genetics of a complex behavior using synthetic parts. I applied this strategy to reprogram and modularize the agriculturally relevant behavior of nitrogen fixation from Klebsiella. Then, classical engineering methods for nonlinear systems optimization were adapted to guide the selection of parts and optimize performance in well-specified genetic systems. Finally, orthogonal genetic wires were developed to engineer multiple behaviors in a single cell. I utilized these wires to construct orthogonal networks for sensing environmental conditions, performing computation, and synthesizing red and green pigments in E. coli. Together, these techniques and engineering principles represent a major step towards the design and synthesis of entire genomes based solely on genetic information found in sequence databases.
Genetically-encodable optical reporters, such as Green Fluorescent Protein, have revolutionized the observation and measurement of cellular states. However, the inverse challenge of using light to precisely control cellular behavior has only recently begun to be addressed; in recent years, semi-synthetic chromophore-tethered receptors and naturally-occurring channel rhodopsins have been used to directly perturb neuronal networks. The difficulty of engineering light sensitive proteins remains a significant impediment to the optical control to most cell-biological processes. I have focused my work over the last five years on the production of genetically-encoded light-sensitive reagents for the control of both bacterial and eukaryotic signalling networks. I have demonstrated minute-timescale control of bacterial transcriptional networks with engineered light-sensitive histidine kinases. I have also demonstrated the use of a new genetically encoded light-control system based on an optimized reversible protein-protein interaction from the phytochrome signaling network of Arabidopsis thaliana. Because protein-protein interactions are one of the most general currencies of cellular information, this latter system can in principal be generically used to control diverse functions. I show that this system can be used to precisely and reversibly translocate target proteins to the membrane with micrometer spatial resolution and second time resolution. I show that light-gated
translocation of the upstream activators of rho-family GTPases, which control the actin cytoskeleton, can be used to precisely reshape and direct the cell morphology of mammalian cells. The light-gated protein-protein interaction that has been optimized in this latter work should be useful for the design of diverse light-programmable reagents, potentially enabling a new generation of perturbative, quantitative experiments in cell biology.
Cells use a variety of different mechanisms to sense and respond to the constantly changing environment. Single celled organisms use signaling pathways to find food, escape toxins, and protect themselves in dangerous or overcrowded environments. The failure of cell signaling in these organisms usually leads to the death of the cell. In a multi-celled organism, individual cells rely on signaling pathways to cooperate with the rest of the organism. A failure in cell to cell signaling in higher organisms may lead to the brief survival of the cell but the eventual failure and death of the organism as a whole. We chose to apply various methods to study cell signaling and communication in both prokaryotic and eukaryotic organisms. The first two approaches we took to this problem were entirely computational. We developed parameters for phosphorylated amino acids and used these parameters to predict structural changes as a function of phosphorylation. In addition, we showed that both phosphate charge and the geometry by which this phosphate interacts with other residues determine the energy gained or lost as a result of this interaction. Next, we joined computation and experiment to successfully predict agonists and antagonists for a G-protein coupled receptor. In a follow up study, we used the information gained on the G-protein coupled receptor to investigate selectivity among a set of similar receptors from different mammals. In the final section of this work, we use a mixture of computation and experiment to show that two component signaling pathways do not interfere with one another in vivo. We then apply this knowledge to deconstruct the bacterial chemotaxis pathway into two separate, orthogonal signaling systems.
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