Chen-Yu Tsao

• PhD
• Research Assistant Professor at University of Maryland, College Park

Microelectronic devices can directly communicate with biology, as electronic information can be transmitted via redox reactions within biological systems. By engineering biology’s native redox networks, we enable electronic interrogation and control of biological systems at several hierarchical levels: proteins, cells, and cell consortia. First, el...

We enable microelectronic devices to interrogate biology’s molecular communication, perform computations, and in real time control biological systems, including at several hierarchical levels: proteins, cells, and cell consortia. A key driver is establishing electronic access to and from biology’s native redox networks. First, redox-mediated electr...

galactosidase (β‐gal) is one of the most prevalent markers of gene expression. Its activity can be monitored via optical and fluorescence microscopy, electrochemistry, and many other ways after slight modification using protein engineering. Here, we have constructed a chimeric version that incorporates a streptococcal protein G domain at the N‐term...

Chemotaxis is a fundamental bacterial response mechanism to changes in chemical gradients of specific molecules known as chemoattractant or chemorepellent. The advancement of biological platforms for bacterial chemotaxis research is of significant interest for a wide range of biological and environmental studies. Many microfluidic devices have been...

Background Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of cons...

We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is ‘plugged into’ an external electronic system that interrogates and...

Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide cros...

Process conditions established during the development and manufacture of recombinant protein therapeutics dramatically impacts their quality and clinical efficacy. Technologies that enable rapid assessment of product quality are critically important. Here, we describe the development of sensor interfaces that directly connect to electronics and ena...

Microgels of biopolymers such as alginate are widely used to encapsulate cells and other biological payloads. Alginate is an attractive material for cell encapsulation because it is nontoxic and convenient: spherical alginate gels are easily created by contacting aqueous droplets of sodium alginate with divalent cations such as Ca2+. Alginate chain...

Emerging research indicates that biology routinely uses diffusible redox‐active molecules to mediate communication that can span biological systems (e.g., nervous and immune) and even kingdoms (e.g., a microbiome and its plant/animal host). This redox modality also provides new opportunities to create interactive materials that can communicate with...

We developed a bidirectional bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that int...

Biology uses diffusible oxidants to perform functions that range from signaling to matrix assembly, and these oxidation chemistries offer surprising selectivities. Here, it is reported that mediated electrochemistry can access the richness of such oxidation chemistries. Specifically, electrode‐imposed voltage inputs are used to locally generate oxi...

Synthetic biology and metabolic engineering have expanded the possibilities for engineered cell-based systems. The addition of non-native biosynthetic and regulatory components can, however, overburden the reprogrammed cells. In order to avoid metabolic overload, an emerging area of focus is on engineering consortia, wherein cell subpopulations wor...

Synthetic biology is typically exploited to endow bacterial cells with new biosynthetic capabilities. It can also serve to create “smart” bacteria such as probiotics that detect and treat disease. Here, we show how minimally rewiring the genetic regulation of bacterial cells can enable their ability to recognize and report on chemical herbicides, i...

Biomacromolecules often possess information to self-assemble through low energy competing interactions which can make self-assembly responsive to environmental cues and can also confer dynamic properties. Here we coupled self-assembling systems to create biofunctional multilayer films that can be cued to dis-assemble through either molecular or ele...

We report a novel strategy for bridging information transfer between electronics and biological systems within microfluidic devices. This strategy relies on our “electrobiofabrication” toolbox that uses electrode-induced signals to assemble biopolymer films at spatially defined sites and then electrochemically “activates” the films for signal proce...

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 bacte...

Quorum sensing (QS) exists widely among bacteria, enabling a transition to multicellular behavior after bacterial populations reach a particular density. The coordination of multicellularity enables biotechnological application, dissolution of biofilms, coordination of virulence, etc. Here, a method to elicit and subsequently disperse multi‐cellula...

Synthetic biologists construct innovative genetic/biological systems to treat environmental, energy, and health problems. Many systems employ rewired cells for non-native product synthesis, while a few have employed the rewired cells as ‘smart’ devices with programmable function. Building on the latter, we developed a genetic construct to control a...

Growth curves, incubation at 37°C. a. Post-induction growth curves. WT-pFZY1 and HCW01-pFZY1 with and without 100 μM hydrogen peroxide were controls. b. Tabulated specific growth rates. HCW01-pHW02 growth with 0–1000 μM hydrogen peroxide induction concentrations. All linear regression analyses for HCW01-pHW02 were compared to 0 μM hydrogen peroxide...

Simulated hydrogen peroxide levels (Comsol). a. Comsol model heat maps. Four points (A-D) were recorded to estimate various hydrogen peroxide concentration gradients over time. Heat gradient maps were constructed for 45 minute (left) and 90 minute (right) gradients. Heat maps were generated using 50, 100, and 200 μM hydrogen peroxide starting conce...

Phenotypic expression of hydrogen peroxide-induced motility. a. Average angle change. The average angle change in degrees per 5 second trajectory of the control bacteria (WT-pFZY1, HCW01-pFZY1) versus the engineered bacteria (HCW01-pHW02; 0–200 μM hydrogen peroxide). These values are inversely proportional to the percent running per trajectory. b....

Strains designs and primer designs of pFZY1-oxyR-poxyS-cheZ. (TIF)

Plasmid designs. a. Plasmid design of pFZY1-oxyR-poxyS-cheZ. b. Plasmid design of pET200-t5-eGFP. (TIF)

Uncut CheZ Western blot images. a. Hydrogen peroxide induction for 5 minutes. b. Hydrogen peroxide induction for 10 minutes. c. Hydrogen peroxide induction for 15 and 60 minutes. His6-CheZ and WT-pFZY1 were controls. (TIF)

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-scienc...

Biology and electronics are both expert at for accessing, analyzing, and responding to information. Biology uses ions, small molecules, and macromolecules to receive, analyze, store, and transmit information, whereas electronic devices receive input in the form of electromagnetic radiation, process the information using electrons, and then transmit...

Antibacterial resistance is an issue of increasing severity as current antibiotics are losing their effectiveness and fewer antibiotics are being developed. New methods for combating bacterial virulence are required. Modulating molecular communication among bacteria can alter phenotype, including attachment to epithelia, biofilm formation and even...

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...

Cover Legend The cover image, by William Bentley et al., is based on the Article Conferring biological activity to native spider silk: A biofunctionalized protein-based microfiber, DOI: 10.1002/bit.26065.

A hydrogel-based dual film coating is electrofabricated for transducing bio-relevant chemical information into electronical output. The outer film has a synthetic biology construct that recognizes an external molecular signal and transduces this input into the expression of an enzyme that converts redox-inactive substrate into a redox-active interm...

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...

Data presented is related to an article entitled “Modular construction of multi-subunit protein complexes using engineered tags and microbial transglutaminase” [1]. In this article, we have presented western blot and flux data associated with assembly of Pfs-LuxS enzyme complexes on beads using uni-tagged and bi-tagged LuxS enzymes. We have also pr...

Motivations for the hierarchical assembly of protein complexes are diverse spanning biosensing, biomedical and bioreactor applications. The assembly processes should be simple, scalable, versatile, and biologically benign to minimize loss of component parts. A “plug and play” methodology comprising a generic linking apparatus may enable rapid desig...

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...

Congruence of solution from finite difference-agent based modeling versus implicit solution of pure ODE’s. A AI2-P trajectory from implicit numerical methods and the average AI2-P concentration from the finite difference-agent based approach. Here, cells from the finite-difference-agent based solution all held the same parameter values as that from...

Supporting information text. (DOCX)

Video of Lsr QS dynamics coupled with gliding during colony growth. Video of a representative simulation for Lsr/AI-2 dynamics coupled with colony growth. QS active cells are in red, whereas inactive cells are in yellow. The complete simulated time of the video is 400 minutes. (MP4)

Measures of the difference between different modes of motility when coupled with Lsr/AI-2 dynamics. Results were derived from finite difference agent based simulations of Lsr activity. (A) The fraction of the population that was QS activated over time, in Lsr simulations of different motility, with average values set in the darker lines and the sta...

Local heterogeneity of Lsr versus LuxIR QS activation in colony growth. Results were derived from finite difference agent based simulations of LuxIR activity or Lsr activity using a median basal of 487.8 and a coefficient of variance of 0.052 (σ = 0.0225) for bacteria growing in a colony. (A) The dark lines represent the average local heterogeneity...

Slower induced import results in less negative intercellular feedback and fewer induced cells. Results were drawn from finite difference agent based simulations of Lsr activity using a reduced rate of induced AI-2 import. The fraction of the population that was QS activated over time given changes to the variation for the distribution of the basal...

Comparison of results from single versus multiple finite difference elements to define environment. The average trajectory of AI2-P for cells with the same parameter sets in simulations where the environment was defined as either a single finite difference element or by the standard array of elements as defined in the methods. Modeling with a singl...

Video of LuxIR QS dynamics coupled with gliding during colony growth. Video of a representative simulation for LuxIR/AHL dynamics coupled with colony growth. QS active cells are in red, whereas inactive cells are in yellow. The complete simulated time of the video is 400 minutes. (MP4)

System sensitivity to parameter changes that manifested in shifting time to Lsr autoinduction. Solutions to ODE’s modeling Lsr activity, where rapidly increasing phosphorylated AI-2 concentration indicates Lsr autoinduction. Shifts in the time to activation were associated with reported parameter value changes. Changes to k2 (transcription Hill par...

Comparison of solution for population with a single basal value versus a population with a unimodally distributed value of basal. Juxtaposition of the solution for extracellular AI-2 for a simulation of cells with a single basal value versus the average solution of extracellular AI-2 for a simulation of cells with a log normal distribution of the p...

Excel file of data used for figures. (XLSX)

Table of parameters and parameter values. (DOCX)

SBML format XML file of Lsr system model. (XML)

In order to match our ability to conceive of and construct cells with enhanced function, we must concomitantly develop facile, real-time methods for elucidating performance. With these, new designs can be tested in silico and steps in construction incrementally validated. Electrochemical monitoring offers the above advantages largely because signal...

Advances in nanotechnology have provided unprecedented physical means to sample molecular space. Living cells provide additional capability in that they identify molecules within complex environments and actuate function. We have merged cells with nanotechnology for an integrated molecular processing network. Here we show that an engineered cell co...

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 concentratio...

The information age was enabled by advances in microfabrication and communication theory that allowed information to be processed by electrons and transmitted by electromagnetic radiation. Despite immense capabilities, microelectronics has limited abilities to access and participate in the molecular-based communication that characterizes our biolog...

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' t...

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-epithe...

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 sen...

We describe an innovation in the immobilization, culture, and imaging of cells in calcium alginate within microfluidic devices. This technique allows unprecedented optical access to the entirety of the calcium alginate hydrogel, enabling observation of growth and behavior in a chemical and mechanical environment favored by many kinds of cells.

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)...

Supplementary Figures S1–6

Synthetic biology provides a means for articulating concepts into new products [1]. Its toolbox is extensive, including the ability to create synthetic genomes and tailor their regulation. Early successes augmented cells' biosynthetic capacities and rewired their regulation, transforming our ability to produce fully functional therapeutic proteins...

Within bacterial communities, signaling molecules interspersed among individual cells cue population-scale behavior, including pathogenesis. While denoted “quorum sensing” (QS) because the multicellular phenotype is often mapped onto and coincides with high cell number, the molecular basis for multicellularity remains somewhat obscure. In part, thi...

Bacterial multicellularity is primarily coordinated by a communicative process known as quorum sensing (QS). Its signal-based and “switch-like” regulation of phenotypic changes provides an inspiring framework for interaction-driven expression among distinct populations within a complex bacterial community. Accordingly, we seek to emulate the QS “sw...

Methods that allow soft matter to be fabricated with controlled structure and function would be beneficial for applications ranging from flexible electronics to regenerative medicine. Here, the assembly of a multifunctional gelatin matrix is demonstrated by triggering its self-assembly and then enzymatically assembling biological functionality. Tri...

Microbially Induced Corrosion (MIC) occurs at metal surfaces and is associated with microorganisms and their metabolic activities. These microbes can coexist as biofilms, growing as synergistic communities (consortia) that are able to affect electrochemical processes, both cathodic and anodic, often through co-operative metabolism. Recent research...

Cell-cell signaling is essential to biological functions. Communications between individual cells, clusters of cells and populations exist in complex networks that, in sum, guide behavior. However, there are few experimental approaches that enable high content interrogation of individual and multicellular behaviors at length and time scales commens...

Many recent advances in bioprocessing have been enabled by developments in miniaturization and microfluidics. A continuing challenge, however, is integrating multiple unit operations that require distinct spatial boundaries, especially with included labile biological components. We have suggested "biofabrication" as a means for organizing cells and...

Bacteria often reside at surfaces as complex biofilms in which an exopolysaccharide matrix entraps the population while allowing access to its chemical environment. There is a growing awareness that the biofilm structure and activity are integral to a wide array of properties important to health (the microbiome), disease (drug resistance) and techn...

Programmable 3D cell assembly under physiological pH conditions is achieved using electrodeposited stimuli-responsive alginate gels in a microfluidic device, with parallel sidewall electrodes enabling direct observation of the cell assembly. Electrically triggered assembly and subsequent viability of mammalian cells is demonstrated, along with spat...

Cell-to-cell communication, or quorum sensing (QS), enables cell density-dependent regulation of bacterial gene expression which can be exploited for the autonomous-signal-guided expression of recombinant proteins (C. Y. Tsao, S. Hooshangi, H. C. Wu, J. J. Valdes, and W. E. Bentley, Metab. Eng. 12:291-297, 2010). Earlier observations that the metab...

In order to control the behavior of bacteria present at the surface of human epithelial cells, we have created a biological "nanofactory" construct that "coats" the epithelial cells and "activates" the surface to produce the bacterial quorum sensing signaling molecule, autoinducer-2 (AI-2). Specifically, we demonstrate directed modulation of signal...

Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-en...

The emergence of bacteria that evade antibiotics has accelerated research on alternative approaches that do not target cell viability. One such approach targets cell-cell communication networks mediated by small molecule signaling. In this report, we assemble biological nanofactories within a bioMEMS device to capture and manipulate the behavior of...

We demonstrate the biofabrication of three dimensional (3D) biopolymer membranes and the assembly of two cell lines in a micro-sandwich structure in microfluidics. A freestanding chitosan membrane was first fabricated by using pH gradients generated at the flow interface of two converging flows. The micro-sandwich was then fabricated by cross-linki...

Bacterial quorum sensing (QS) is a cell-cell communication process, mediated by signaling molecules, that alters various phenotypes including pathogenicity. Methods to interrupt these communication networks are being pursued as next generation antimicrobials. We present a technique for interrupting communication among bacteria that exploits their n...

Electroaddressing of biological components at specific device addresses is attractive because it enlists the capabilities of electronics to provide spatiotemporally controlled electrical signals. Here, the electrodeposition of calcium alginate hydrogels at specific electrode addresses is reported. The method employs the low pH generated at the anod...

We report the assembly of seven different antibodies (and two antigens) into functional supramolecular structures that are specifically designed to facilitate integration into devices using entirely biologically based bottom-up fabrication. This is enabled by the creation of an engineered IgG-binding domain (HG3T) with an N-terminal hexahistidine t...

Fibers are a versatile platform because standard methods are available for the hierarchical assembly of individual fibers into controllable patterns (e.g., fabrics). Here, we report a method to biofunctionalize individual fibers by the reversible binding of proteins, and we suggest the potential of fiber assemblies by generating simple multifiber s...

Magnetic 'nanofactories', for localized manufacture and signal-guided delivery of small molecules to targeted cell surfaces, are demonstrated. They recruit nearby raw materials for synthesis, employ magnetic mobility for capture and localization of target cells, and deliver molecules to cells triggering their native phenotypic response, but with us...

Supplementary Information

Supplementary Figures

XML file for base case

Quorum sensing (QS) is an important determinant of bacterial phenotype. Many cell functions are regulated by intricate and multimodal QS signal transduction processes. The LuxS/AI-2 QS system is highly conserved among Eubacteria and AI-2 is reported as a 'universal' signal molecule. To understand the hierarchical organization of AI-2 circuitry, a c...

Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake...

A setup with two dissolved oxygen sensors for on-line measurement of the oxygen uptake rate in an aerobic microbial fermentation system was developed. An external loop with two dissolved oxygen sensors was linked to the fermentor. A gas-liquid separator was designed and installed in front of the dissolved oxygen sensors. The oxygen uptake rate was...