Enhanced electrochemical measurement of ß-galactosidase activity in whole cells by coexpression of lactose permease, LacY.

Whole-cell biosensing links the sensing and computing capabilities of microbes to the generation of a detectable reporter. Whole cells enable dynamic biological computation (filtered noise, amplified signals, logic gating etc.). Enzymatic reporters enable in situ signal amplification. Electrochemical measurements are easily quantified and work in turbid environments. In this work we show how the coexpression of the lactose permease, LacY, dramatically improves electrochemical sensing of ß-galactosidase (LacZ) expressed as a reporter in whole cells. The permease facilitates transport of the LacZ substrate, 4-aminophenyl ß-d-galactopyranoside, which is converted to redox active p-aminophenol, which, in turn, is detected via cyclic voltammetry or chronocoulometry. We show a greater than fourfold improvement enabled by lacY coexpression in cells engineered to respond to bacterial signal molecules, pyocyanin and quorum-sensing autoinducer-2.

Electrogenetic Signal Transmission and Propagation in Coculture to Guide Production of a Small Molecule, Tyrosine.

There are many strategies to actuate and control genetic circuits, including providing stimuli like exogenous chemical inducers, light, magnetic fields, and even applied voltage, that are orthogonal to metabolic activity. Their use enables actuation of gene expression for the production of small molecules and proteins in many contexts. Additionally, there are a growing number of reports wherein cocultures, consortia, or even complex microbiomes are employed for the production of biologics, taking advantage of an expanded array of biological function. Combining stimuli-responsive engineered cell populations enhances design space but increases complexity. In this work, we co-opt nature's redox networks and electrogenetically route control signals into a consortium of microbial cells engineered to produce a model small molecule, tyrosine. In particular, we show how electronically programmed short-lived signals (i.e., hydrogen peroxide) can be transformed by one population and propagated into sustained longer-distance signals that, in turn, guide tyrosine production in a second population building on bacterial quorum sensing that coordinates their collective behavior. Two design methodologies are demonstrated. First, we use electrogenetics to transform redox signals into the quorum sensing autoinducer, AI-1, that, in turn, induces a tyrosine biosynthesis pathway transformed into a second population. Second, we use the electrogenetically stimulated AI-1 to actuate expression of ptsH, boosting the growth rate of tyrosine-producing cells, augmenting both their number and metabolic activity. In both cases, we show how signal propagation within the coculture helps to ensure tyrosine production. We suggest that this work lays a foundation for employing electrochemical stimuli and engineered cocultures for production of molecular products in biomanufacturing environments.

Redox Electrochemistry to Interrogate and Control Biomolecular Communication.

Cells often communicate by the secretion, transport, and perception of molecules. Information conveyed by molecules is encoded, transmitted, and decoded by cells within the context of the prevailing microenvironments. Conversely, in electronics, transmission reliability and message validation are predictable, robust, and less context dependent. In turn, many transformative advances have resulted by the formal consideration of information transfer. One way to explore this potential for biological systems is to create bio-device interfaces that facilitate bidirectional information transfer between biology and electronics. Redox reactions enable this linkage because reduction and oxidation mediate communication within biology and can be coupled with electronics. By manipulating redox reactions, one is able to combine the programmable features of electronics with the ability to interrogate and modulate biological function. In this review, we examine methods to electrochemically interrogate the various components of molecular communication using redox chemistry and to electronically control cell communication using redox electrogenetics.

A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks.

There is a growing interest in mediating information transfer between biology and electronics. By the addition of redox mediators to various samples and cells, one can both electronically obtain a redox "portrait" of a biological system and, conversely, program gene expression. Here, we have created a cell-based synthetic biology-electrochemical axis in which engineered cells process molecular cues, producing an output that can be directly recorded via electronics-but without the need for added redox mediators. The process is robust; two key components must act together to provide a valid signal. The system builds on the tyrosinase-mediated conversion of tyrosine to L-DOPA and L-DOPAquinone, which are both redox active. "Catalytic" transducer cells provide for signal-mediated surface expression of tyrosinase. Additionally, "reagent" transducer cells synthesize and export tyrosine, a substrate for tyrosinase. In cocultures, this system enables real-time electrochemical transduction of cell activating molecular cues. To demonstrate, we eavesdrop on quorum sensing signaling molecules that are secreted by Pseudomonas aeruginosa, N-(3-oxododecanoyl)-l-homoserine lactone and pyocyanin.