3D-Printed electrochemical sensor-integrated transwell systems.

This work presents a 3D-printed, modular, electrochemical sensor-integrated transwell system for monitoring cellular and molecular events in situ without sample extraction or microfluidics-assisted downstream omics. Simple additive manufacturing techniques such as 3D printing, shadow masking, and molding are used to fabricate this modular system, which is autoclavable, biocompatible, and designed to operate following standard operating protocols (SOPs) of cellular biology. Integral to the platform is a flexible porous membrane, which is used as a cell culture substrate similarly to a commercial transwell insert.

Electrochemical measurement of serotonin by Au-CNT electrodes fabricated on microporous cell culture membranes.

Gut-brain axis (GBA) communication relies on serotonin (5-HT) signaling between the gut epithelium and the peripheral nervous system, where 5-HT release patterns from the basolateral (i.e., bottom) side of the epithelium activate nerve afferents.

rAAV Production and Titration at the Microscale for High-Throughput Screening.

In the literature, there are few high-throughput screens or even methods for high-throughput screens of recombinant adeno-associated virus (rAAV) production despite potential benefits to research and production. In this study, a generalizable high-throughput relative rAAV titration method is examined within the context of an siRNA screen as siRNA knockdown is a common means of pathway engineering in bioproduction. Crude samples generated from transfected HEK293T/17 cultures were subjected to quantitative PCR (qPCR) and used to transduce COS7 cells to assess relative differences in genomic and infectious rAAV titer, respectively, at the 384-well scale, evaluating both supernatant and lysed samples.

Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal.

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell-cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2.

An immune magnetic nano-assembly for specifically amplifying intercellular quorum sensing signals.

Quorum sensing (QS) enables intercellular communication after bacterial cells sense the autoinducers have reached or exceeded a critical concentration. Selectively amplifying specific bacterial "quorum" activity at a lower cell density is still a challenge. Here, we propose a novel platform of immune magnetic nano-assembly to amplify specific bacterial QS signaling via improving the bioavailability of autoinducers-2 (AI-2, furanosyl borate) from sender (wide-type, WT cells) to receiver (reporter cells).

Focusing quorum sensing signalling by nano-magnetic assembly.

Quorum sensing (QS) exists widely among bacteria, enabling a transition to multicellular behaviour after bacterial populations reach a particular density. The coordination of multicellularity enables biotechnological application, dissolution of biofilms, coordination of virulence, and so forth. Here, a method to elicit and subsequently disperse multicellular behaviour among QS-negative cells is developed using magnetic nanoparticle assembly.

Biofabricating Functional Soft Matter Using Protein Engineering to Enable Enzymatic Assembly.

Biology often provides the inspiration for functional soft matter, but biology can do more: it can provide the raw materials and mechanisms for hierarchical assembly. Biology uses polymers to perform various functions, and biologically derived polymers can serve as sustainable, self-assembling, and high-performance materials platforms for life-science applications. Biology employs enzymes for site-specific reactions that are used to both disassemble and assemble biopolymers both to and from component parts.

Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies.

Chemoenzymatic glycan remodeling of antibodies using an endoglycosidase and its mutant is emerging as an attractive approach for producing homogeneous antibody glycoforms. We report in this paper a site-specific covalent immobilization of the endoglycosidases (Endo-S2 and its glycosynthase mutant D184M) using a recombinant microbial transglutaminase (MTG) and evaluation of the immobilized enzymes in deglycosylation and glycosylation of a therapeutic antibody. The site-specific covalent immobilization was achieved by introduction of a Q-tag at the C-terminus of the recombinant enzymes followed by conjugation of the enzymes to a primary amine-containing solid support through MTG-catalyzed transglutamination.

A simple and reusable bilayer membrane-based microfluidic device for the study of gradient-mediated bacterial behaviors.

We have developed a user-friendly microfluidic device for the study of gradient-mediated bacterial behaviors, including chemotaxis. This device rapidly establishes linear concentration gradients by exploiting solute diffusion through porous membranes in the absence of convective flows. As such, the gradients are created rapidly and can be sustained for long time periods (e.

Conferring biological activity to native spider silk: A biofunctionalized protein-based microfiber.

Spider silk is an extraordinary material with physical properties comparable to the best scaffolding/structural materials, and as a fiber it can be manipulated with ease into a variety of configurations. Our work here demonstrates that natural spider silk fibers can also be used to organize biological components on and in devices through rapid and simple means. Micron scale spider silk fibers (5-10 μm in diameter) were surface modified with a variety of biological entities engineered with pentaglutamine tags via microbial transglutaminase (mTG).

Enhancing Intercellular Coordination: Rewiring Quorum Sensing Networks for Increased Protein Expression through Autonomous Induction.

While inducing agents are often used to redirect resources from growth and proliferation toward product outputs, they can be prohibitively expensive on the industrial scale. Previously, we developed an autonomously guided protein production system based on the rewiring of E. coli's native quorum sensing (QS) signal transduction cascade.

Quorum Sensing Desynchronization Leads to Bimodality and Patterned Behaviors.

Quorum Sensing (QS) drives coordinated phenotypic outcomes among bacterial populations. Its role in mediating infectious disease has led to the elucidation of numerous autoinducers and their corresponding QS signaling pathways. Among them, the Lsr (LuxS-regulated) QS system is conserved in scores of bacteria, and its signal molecule, autoinducer-2 (AI-2), is synthesized as a product of 1-carbon metabolism.

Directed assembly of a bacterial quorum.

Many reports have elucidated the mechanisms and consequences of bacterial quorum sensing (QS), a molecular communication system by which bacterial cells enumerate their cell density and organize collective behavior. In few cases, however, the numbers of bacteria exhibiting this collective behavior have been reported, either as a number concentration or a fraction of the whole. Not all cells in the population, for example, take on the collective phenotype.

Rational design of 'controller cells' to manipulate protein and phenotype expression.

Coordination between cell populations via prevailing metabolic cues has been noted as a promising approach to connect synthetic devices and drive phenotypic or product outcomes. However, there has been little progress in developing 'controller cells' to modulate metabolic cues and guide these systems. In this work, we developed 'controller cells' that manipulate the molecular connection between cells by modulating the bacterial signal molecule, autoinducer-2, that is secreted as a quorum sensing (QS) signal by many bacterial species.

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.

Distal modulation of bacterial cell-cell signalling in a synthetic ecosystem using partitioned microfluidics.

The human gut is over a meter in length, liquid residence times span several hours. Recapitulating the human gut microbiome "on chip" holds promise to revolutionize therapeutic strategies for a variety of diseases, as well as for maintaining homeostasis in healthy individuals. A more refined understanding of bacterial-bacterial and bacterial-epithelial cell signalling is envisioned and such a device is a key enabler.

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.

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.

Gene network homology in prokaryotes using a similarity search approach: queries of quorum sensing signal transduction.

Bacterial cell-cell communication is mediated by small signaling molecules known as autoinducers. Importantly, autoinducer-2 (AI-2) is synthesized via the enzyme LuxS in over 80 species, some of which mediate their pathogenicity by recognizing and transducing this signal in a cell density dependent manner. AI-2 mediated phenotypes are not well understood however, as the means for signal transduction appears varied among species, while AI-2 synthesis processes appear conserved.