Autonomous bacterial localization and gene expression based on nearby cell receptor density.

Escherichia coli were genetically modified to enable programmed motility, sensing, and actuation based on the density of features on nearby surfaces. Then, based on calculated feature density, these cells expressed marker proteins to indicate phenotypic response. Specifically, site-specific synthesis of bacterial quorum sensing autoinducer-2 (AI-2) is used to initiate and recruit motile cells. In our model system, we rewired E. coli's AI-2 signaling pathway to direct bacteria to a squamous cancer cell line of head and neck (SCCHN), where they initiate synthesis of a reporter (drug surrogate) based on a threshold density of epidermal growth factor receptor (EGFR). This represents a new type of controller for targeted drug delivery as actuation (synthesis and delivery) depends on a receptor density marking the diseased cell. The ability to survey local surfaces and initiate gene expression based on feature density represents a new area-based switch in synthetic biology that will find use beyond the proposed cancer model here.

Pathway engineering via quorum sensing and sRNA riboregulators-interconnected networks and controllers.

The advent of genetic engineering has elevated our level of comprehension of cellular processes and functions. A natural progression of these findings is determining not only how these processes function within individual cells but also within a community. Bacterial cells monitor the conditions and microorganisms in their vicinity by producing, releasing and sensing chemical-signaling molecules. When a specific cell-density threshold is reached, a quorum is perceived, gene expression profiles are altered and the community orchestrates activities that are more effective en masse. This communication mechanism, in the language of autoinducers (AI), is referred to as quorum sensing (QS). It has become increasingly evident that while scientists attempt to decipher the intricacies of cellular communication and quorum sensing networks, we must remain conscious of the broader context of how a cell may identify itself in the environment and how this also impacts QS. Importantly, these phenomena span time and length scales by several orders in magnitude. Though the revelation of small RNAs, as both sensing and regulatory elements participating in the quorum sensing cascade, has connected new pieces of the puzzle, it has also added a new tier of uncertainty. The complexity of quorum sensing networks makes resolution of its diverse mechanisms difficult. The ability to design simpler networks with defined, more predictable or even "modular" elements will help elucidate these actions. Because it embraces innovative concepts of biological design accommodating the many length and time scales at play, synthetic biology serves as one of the most promising platforms for describing QS phenomena as well as enabling novel implementation strategies for biotechnological application.

Bacterial secretions of nonpathogenic Escherichia coli elicit inflammatory pathways: a closer investigation of interkingdom signaling.

UNLABELLED: There have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenic Escherichia coli strains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq). E. coli BL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response. IMPORTANCE: The symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to "communicate" with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

Evolved Quorum sensing regulator, LsrR, for altered switching functions.

In order to carry out innovative complex, multistep synthetic biology functions, members of a cell population often must communicate with one another to coordinate processes in a programmed manner. It therefore follows that native microbial communication systems are a conspicuous target for developing engineered populations and networks. Quorum sensing (QS) is a highly conserved mechanism of bacterial cell-cell communication and QS-based synthetic signal transduction pathways represent a new generation of biotechnology toolbox members. Specifically, the E. coli QS master regulator, LsrR, is uniquely positioned to actuate gene expression in response to a QS signal. In order to expand the use of LsrR in synthetic biology, two novel LsrR switches were generated through directed evolution: an "enhanced" repression and derepression eLsrR and a reversed repression/derepression function "activator" aLsrR. Protein modeling and docking studies are presented to gain insight into the QS signal binding to these two evolved proteins and their newly acquired functionality. We demonstrated the use of the aLsrR switch using a coculture system in which a QS signal, produced by one bacterial strain, is used to inhibit gene expression via aLsrR in a different strain. These first ever AI-2 controlled synthetic switches allow gene expression from the lsr promoter to be tuned simultaneously in two distinct cell populations. This work expands the tools available to create engineered microbial populations capable of carrying out complex functions necessary for the development of advanced synthetic products.

Tuning cell cycle of insect cells for enhanced protein production.

The eukaryotic cell cycle consists of many checkpoints during which certain conditions must be met before passing to subsequent stages. These safeguards ensure cells' integrity and survival, but may also limit growth and protein synthesis in protein production processes. In this work, we employ metabolic engineering principles to "tune" the cell cycle to overcome checkpoint processes in order to facilitate faster cell growth, and independently, arrest the cell cycle in gap1 (G1) phase for greater protein productivity. Specifically, we identified the complete cyclin E (cycE) cDNA sequence from industrially relevant, Trichoplusia ni (T. ni) derived High Five™ genomes. We then both knocked down (through RNA interference; RNAi) and overexpressed (on an expression plasmid) cycE gene expression to tune the cell phenotype. We successfully up- and down-regulated cycE transcription, enhancing and hindering cell growth, respectively. We also measured the effects of titrated cycE expression on the cell cycle phase distribution. Finally, we investigated the dose-dependent effects of dsCycE on recombinant protein production using the baculovirus expression system and demonstrated a nearly 2-fold increase in expression of model protein (GFPuv).