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Abstract 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. Multimodal electrochemical sensors fabricated on the membrane allow direct access to cells and their products. A pair of gold electrodes on the top side of the membrane measures impedance over the course of cell attachment and growth, characterized by an exponential decrease (~160% at 10 Hz) due to an increase in the double layer capacitance from secreted extracellular matrix (ECM) proteins. Cyclic voltammetry (CV) sensor electrodes, fabricated on the bottom side of the membrane, enable sensing of molecular release at the site of cell culture without the need for downstream fluidics. Real-time detection of ferrocene dimethanol injection across the membrane showed a three order-of-magnitude higher signal at the membrane than in the bulk media after reaching equilibrium. This modular sensor-integrated transwell system allows unprecedented direct, real-time, and noninvasive access to physical and biochemical information, which cannot be obtained in a conventional transwell system. 

Abstract 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 anEscherichia colistrain with enhanced ability to detect and respond to AI‐2. In anE. colistrain that does not produce AI‐2, we monitored AI‐2 uptake and reporter protein expression in a strain that overproduced the AI‐2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI‐2 degradation units LsrF and LsrG, and an “enhanced” strain with both overproduction of AI‐2 uptake and deletion of AI‐2 degradation elements. By adding up to 40 μM AI‐2 to growing cell cultures, we determine that this “enhanced” AI‐2 sensitive strain both uptakes AI‐2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI‐2 quorum sensing processes both in native environments and for synthetic biology applications.