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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 developed for its study, but challenges still remain that can obscure analysis. For example, cell migration can be compromised by flow-induced shear stress, and bacterial motility can be impaired by nonspecific cell adhesion to microchannels. Also, devices can be complicated, expensive, and hard to assemble. We address these issues with a three-channel microfluidic platform integrated with natural biopolymer membranes that are assembled in situ. This provides several unique attributes. First, a static, steady and robust chemoattractant gradient was generated and maintained. Second, because the assembly incorporates assembly pillars, the assembled membrane arrays connecting nearby pillars can be created longer than the viewing window, enabling a wide 2D area for study. Third, the in situ assembled biopolymer membranes minimize pressure and/or chemiosmotic gradients that could induce flow and obscure chemotaxis study. Finally, nonspecific cell adhesion is avoided by priming the polydimethylsiloxane (PDMS) microchannel surfaces with Pluronic F-127. We demonstrated chemotactic migration of Escherichia coli as well as Pseudomonas aeruginosa under well-controlled easy-to-assemble glucose gradients. We characterized motility using the chemotaxis partition coefficient (CPC) and chemotaxis migration coefficient (CMC) and found our results consistent with other reports. Further, random walk trajectories of individual cells in simple bright field images were conveniently tracked and presented in rose plots. Velocities were calculated, again in agreement with previous literature. We believe the biopolymer membrane-integrated platform represents a facile and convenient system for robust quantitative assessment of cellular motility in response to various chemical cues.

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 consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. Results We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. Conclusions Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

β-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-terminus of β-gal that binds immunoglobins, namely IgG. This protein G: β-galactosidase fusion enables β-gal-based spectrophotometric and electrochemical measurements of IgG. Moreover, our results show linearity over an industrially relevant range. We demonstrate applicability with rapid spectroelectrochemical detection of IgG in several formats including using an electrochemical sensing interface that is rapidly assembled directly onto electrodes for incorporation into biohybrid devices. The fusion protein enables sensitive, linear, and rapid responses, and in our case, makes IgG measurements quite robust and simple, expanding the molecular diagnostics toolkit for biological measurement.

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 enable near real-time assessment of antibody titer and N-linked galactosylation. We make use of a spatially resolved electroassembled thiolated polyethylene glycol hydrogel that enables electroactivated disulfide linkages. For titer assessment, we constructed a cysteinylated protein G that can be linked to the thiolated hydrogel allowing for robust capture and assessment of antibody concentration. For detecting galactosylation, the hydrogel is linked with thiolated sugars and their corresponding lectins, which enables antibody capture based on glycan pattern. Importantly, we demonstrate linear assessment of total antibody concentration over an industrially relevant range and the selective capture and quantification of antibodies with terminal β-galactose glycans. We also show that the interfaces can be reused after surface regeneration using a low pH buffer. Our functionalized interfaces offer advantages in their simplicity, rapid assembly, connectivity to electronics, and reusability. As they assemble directly onto electrodes that also serve as I/O registers, we envision incorporation into diagnostic platforms including those in manufacturing settings.

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 controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.

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 chains in the gel become cross-linked by Ca2+ cations into a 3-D network. When alginate gels are placed in a buffer, however, the Ca2+ cross-links are eliminated by exchange with Na+, thereby weakening and degrading the gels. With time, encapsulated cells are released into the external solution. Here, we describe a simple solution to the above problem, which involves forming alginate gels enveloped by a thin shell of a covalently cross-linked gel. The shell is formed via free-radical polymerization using conventional monomers such as acrylamide (AAm) or acrylate derivatives, including polyethylene glycol diacrylate (PEGDA). The entire process is performed in a single step at room temperature (or 37 °C) under mild, aqueous conditions. It involves combining the alginate solution with a radical initiator, which is then introduced as droplets into a reservoir containing Ca2+ and monomers. Within minutes of either simple incubation or exposure to ultraviolet (UV) light, the droplets are converted into alginate-polymer microcapsules with a core of alginate and a shell of the polymer (AAm or PEGDA). The microcapsules are mechanically more robust than conventional alginate/Ca2+ microgels, and while the latter swell and degrade when placed in buffers or in chelators like sodium citrate, the former remain stable under all conditions. We encapsulate both bacteria and mammalian cells in these microcapsules and find that the cells remain viable and functional over time. Lastly, a variation of the synthesis technique is shown to generate multilayered microcapsules with a liquid core surrounded by concentric layers of alginate and AAm gels. We anticipate that the approaches presented here will find application in a variety of areas including cell therapies, artificial cells, drug delivery, and tissue engineering.

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 living systems. Here, it is reported that the fabrication of a redox-active hydrogel film can autonomously synthesize a H2 O2 signaling molecule for communication with a bacterial population. Specifically, a catechol-conjugated/crosslinked 4-armed thiolated poly(ethylene glycol) hydrogel film is electrochemically fabricated in which the added catechol moieties confer redox activity: the film can accept electrons from biological reductants (e.g., ascorbate) and donate electrons to O2 to generate H2 O2 . Electron-transfer from an Escherichia coli culture poises this film to generate the H2 O2 signaling molecule that can induce bacterial gene expression from a redox-responsive operon. Overall, this work demonstrates that catecholic materials can participate in redox-based interactions that elicit specific biological responses, and also suggests the possibility that natural phenolics may be a ubiquitous biological example of interactive materials.

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 interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic co-culture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and, simultaneously, enables direct electronic feedback from the second strain thus enabling real time electronic verification of biological signal propagation. Overall, we show how this multi-functional bioelectronic platform, termed BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.

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 disassemble through either molecular or electrical signals. To create functional multilayers, we: (i) electrodeposited the pH-responsive self-assembling aminopolysaccharide chitosan, (ii) allowed the lectin Concanavalin A (ConA) to bind to the chitosan-coated electrode (presumably through electrostatic interactions), (iii) performed layer-by-layer self-assembly by sequential contacting with glycogen and ConA, and (iv) conferred biological (i.e., enzymatic) function by assembling glycoprotein (i.e., enzymes) to the ConA-terminated multilayer. Because the ConA tetramer dissociates at low pH, this multilayer can be triggered to disassemble by acidification. We demonstrate two approaches to induce acidification: (i) glucose oxidase can induce multilayer disassembly in response to molecular cues, and (ii) anodic reactions can induce multilayer disassembly in response to electrical cues.

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). Antibody coated magnetic nanoparticle (MNPAB) was fabricated with an average diameter of 12 nm and a specific surface area of 96.5 m2/g. The distribution efficiency of the antibody on the surface was 25.8 μg/m2 of magnetic nanoparticles. It was found that more than 3 × 108 of K12 serotype Escherichia coli (E. coli) reporter or WT cells were collected using 1 mg fabricated MNPAB at a saturated condition. The MNPAB not only captured E. coli WT cells but also brought them into proximity of E. coli (CT104, pCT6+pET-DsRed) reporter cells via magnetic attraction. The amplified QS signaling of the reporter cells by this immune magnetic nano-assembly was approximately 3 times higher than the nature QS signaling in cell suspension at optical density (OD) 0.08. This study foresees potential applications in amplifying specific biological QS signals based on a preprogrammed design.

Rapid and portable detection of viable pathogen is highly desired to minimize the risk of foodborne pathogen outbreaks. Here we report a proof-of-concept fabrication methodology of a multifunctional film that allows established methods from bacterial recognition (antibodies) and nanotechnology (magnetic nanoparticles) to be coupled with electrochemical signal processing methods for detection of viable bacteria. Specifically, we enlist a sequence of externally applied electrical and magnetic signals to: i) guide the self-assembly of stimuli-responsive biopolymer; ii) incorporate magnetic nanoparticles to form a magnetic layer; iii) electro-synthesize a signal processing layer (redox-capacitor). The function of the magnetic layer is collecting and concentrating MMP-bacteria through magnetic attractions between MMP-bacteria and the magnetic layer. The function of the signal processing layer is amplifying electrochemical detection of the collected bacteria by engaging the redox-active mediators with the redox capacitor. Importantly, the fabrication demonstrated here is simple, controllable, and reagentless.

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. We fabricated magnetic nanoparticles (MNPs, ∼5 nm) that electrostatically collect wild-type (WT) Escherichia coli BL21 cells and brings them into proximity of bioengineered E. coli [CT104 (W3110 lsrFG- luxS- pCT6 + pET-DsRed)] reporter cells that exhibit a QS response after receiving autoinducer-2 (AI-2). By shortening the distance between WT and reporter cells (e.g., increasing local available AI-2 concentrations), the QS response signalling was amplified four-fold compared to that in native conditions without assembly. This study suggests potential applications in facilitating intercellular communication and modulating multicellular behaviours based on user-specified designs.

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, including those routinely used to clear weeds from gardens and crops. In so doing, we demonstrate how constructs of synthetic biology, in this case redox-based synthetic biology, can serve as a vector for information flow mediating molecular communication between biochemical systems and microelectronics. We coupled the common genetic reporter, β-galactosidase, with the E. coli superoxide response regulon promoter pSoxS, for detection of the herbicides dicamba and Roundup. Both herbicides activated our genetic construct in a concentration dependent manner. Results indicate robust detection using spectrophotometry, via the Miller assay, and electrochemistry using the enzymatic cleavage of 4-aminophenyl β-d-galactopyranoside into the redox active molecule p-aminophenol. We found that environmental components, in particular, the availability of glucose, are important factors for the cellular detection of dicamba. Importantly, both herbicides were detected at concentrations relevant for aquatic toxicity.

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 design and optimization. One application that desires these qualities is metabolon construction wherein multiple enzymes are organized in defined pathways to mediate biochemical flux. Here, we propose a modular design by incorporation of crosslinking-compliant amino acid tags comprised of lysine or glutamine residues at the N- or C-termini of the to-be-assembled proteins. These amino acid tags enable covalent crosslinking using microbial transglutaminase (mTG). Modularity is demonstrated where stoichiometries and relative positions of enzymes and other functional proteins are altered. Construction of multifunctional complexes is demonstrated by crosslinking domains of different function and origin. Namely, we built a two-subunit quorum sensing (QS) biosynthetic metabolon on solid supports and altered stoichiometries of the limiting constituents to increase the overall rate of reaction. To display functionality beyond biosynthesis, we constructed a molecular communication 'device' (antibody binding Protein G-QS complex) to target bacterial cells and demonstrated tailored QS responses among targeted bacteria. We propose that this approach, solid phase mTG-mediated linkage of biological components, can be used for assembly within many environments including microreactors or lab-on-a-chip systems. Because the methodology is general, we envision construction of multi-functional protein complexes in a 'plug and play' fashion for a variety of biosensing and synthetic biology applications.

We report a novel strategy for bridging information transfer between electronics and biological systems within microdevices. 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 processing capabilities. Compared to conventional electrode surface modification approaches, our signal-guided assembly and activation strategy provides on-demand electrode functionalization, and greatly simplifies microfluidic sensor design and fabrication. Specifically, a chitosan film is selectively localized in a microdevice and is covalently modified with phenolic species. The redox active properties of the phenolic species enable the film to transduce molecular to electronic signals (i.e., “molectronic”). The resulting “molectronic” sensors are shown to facilitate the electrochemical analysis in real time of biomolecules, including small molecules and enzymes, to cell-based measurements such as cytotoxicity. We believe this strategy provides an alternative, simple, and promising avenue for connecting electronics to biological systems within microfluidic platforms, and eventually will enrich our abilities to study biology in a variety of contexts.

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. By exploiting protein engineering methodologies, proteins can be modified to make them more susceptible to biology’s native enzymatic activities. They can be engineered with fusion tags that provide (short sequences of amino acids at the C- and/or N- termini) that provide the accessible residues for the assembling enzymes to recognize and react with. This “biobased” fabrication not only allows biology’s nanoscale components (i.e., proteins) to be engineered, but also provides the means to organize these components into the hierarchical structures that are prevalent in life.

Antibodies are common recognition elements for molecular detection but often the signals generated by their stoichiometric binding must be amplified to enhance sensitivity. Here, we report that an electrode coated with a catechol-chitosan redox capacitor can amplify the electrochemical signal generated from an alkaline phosphatase (AP) linked immunoassay. Specifically, the AP product p-aminophenol (PAP) undergoes redox-cycling in the redox capacitor to generate amplified oxidation currents. We estimate an 8-fold amplification associated with this redox-cycling in the capacitor (compared to detection by a bare electrode). Importantly, this capacitor-based amplification is generic and can be coupled to existing amplification approaches based on enzyme-linked catalysis or magnetic nanoparticle-based collection/concentration. Thus, the capacitor should enhance sensitivities in conventional immunoassays and also provide chemical to electrical signal transduction for emerging applications in molecular communication.

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. Specifically, we have engineered Escherichia coli to overexpress components responsible for autoinducer uptake ( lsrACDB ), phosphorylation ( lsrK ), and degradation ( lsrFG ), thereby attenuating cell–cell communication among populations. Further, we developed a simple mathematical model that recapitulates experimental data and characterizes the dynamic balance among the various uptake mechanisms. This study revealed two controller ‘knobs’ that serve to increase AI-2 uptake: overexpression of the AI-2 transporter, LsrACDB, which controls removal of extracellular AI-2, and overexpression of the AI-2 kinase, LsrK, which increases the net uptake rate by limiting secretion of AI-2 back into the extracellular environment. We find that the overexpression of lsrACDBFG results in an extraordinarily high AI-2 uptake rate that is capable of completely silencing QS-mediated gene expression among wild-type cells. We demonstrate utility by modulating naturally occurring processes of chemotaxis and biofilm formation. We envision that ‘controller cells’ that modulate bacterial behavior by manipulating molecular communication, will find use in a variety of applications, particularly those employing natural or synthetic bacterial consortia.

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. Thus, the specific attribution of the postulated benefit can remain obscure. This is partly due to our inability to independently assemble a defined quorum, for natural and most artificial systems the quorum itself is a consequence of the biological context (niche and signaling mechanisms). Here, we describe the intentional assembly of quantized quorums. These are made possible by independently engineering the autoinducer signal transduction cascade of Escherichia coli (E. coli) and the sensitivity of detector cells so that upon encountering a particular autoinducer level, a discretized sub-population of cells emerges with the desired phenotype. In our case, the emergent cells all express an equivalent amount of marker protein, DsRed, as an indicator of a specific QS-mediated activity. The process is robust, as detector cells are engineered to target both large and small quorums. The process takes about 6 h, irrespective of quorum level. We demonstrate sensitive detection of autoinducer-2 (AI-2) as an application stemming from quantized quorums. We then demonstrate sub-population partitioning in that AI-2-secreting cells can 'call' groups neighboring cells that 'travel' and establish a QS-mediated phenotype upon reaching the new locale.

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 biological world. Here, we use biological materials and methods to create components and fabricate devices to perform simple molecular communication functions based on bacterial quorum sensing (QS). Components were created by protein engineering to generate a multidomain fusion protein capable of sending a molecular QS signal, and by synthetic biology to engineer E. coli to receive and report this QS signal. The device matrix was formed using stimuli-responsive hydrogel-forming biopolymers (alginate and gelatin). Assembly of the components within the device matrix was achieved by physically entrapping the cell-based components, and covalently conjugating the protein-based components using the enzyme microbial transglutaminase. We demonstrate simple devices that can send or receive a molecular QS signal to/from the surrounding medium, and a two-component device in which one component generates the signal (i.e., issues a command) that is acted upon by the second component. These studies illustrate the broad potential of biofabrication to generate molecular communication devices.

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 toxin production. Intercepting and modulating communication networks provide a means to attenuate virulence without directly interacting with the bacteria of interest. In this work, we target communication mediated by the quorum sensing (QS) bacterial autoinducer-2, AI-2. We have assembled a capsule of biological polymers alginate and chitosan, attached an AI-2 processing kinase, LsrK, and provided substrate, ATP, for enzymatic alteration of AI-2 in culture fluids. Correspondingly, AI-2 mediated QS activity is diminished. All components of this system are “biofabricated” – they are biologically derived and their assembly is accomplished using biological means. Initially, component quantities and kinetics were tested as assembled in microtiter plates. Subsequently, the identical components and assembly means were used to create the “artificial cell” capsules. The functionalized capsules, when introduced into populations of bacteria, alter the dynamics of the AI-2 bacterial communication, attenuating QS activated phenotypes. We envision the assembly of these and other capsules or similar materials, as means to alter QS activity in a biologically compatible manner and in many environments, including in humans. This article is protected by copyright. All rights reserved

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 intermediate, which is detected through an amplification mechanism of the inner redox-capacitor film.

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. Lsr signal transduction processes, therefore, may help organize population scale activities in numerous bacterial consortia. Conceptions of how Lsr QS organizes population scale behaviors remain limited, however. Using mathematical simulations, we examined how desynchronized Lsr QS activation, arising from cell-to-cell population heterogeneity, could lead to bimodal Lsr signaling and fractional activation. This has been previously observed experimentally. Governing these processes are an asynchronous AI-2 uptake, where positive intracellular feedback in Lsr expression is combined with negative feedback between cells. The resulting activation patterns differ from that of the more widely studied LuxIR system, the topology of which consists of only positive feedback. To elucidate differences, both QS systems were simulated in 2D, where cell populations grow and signal each other via traditional growth and diffusion equations. Our results demonstrate that the LuxIR QS system produces an ‘outward wave’ of autoinduction, and the Lsr QS system yields dispersed autoinduction from spatially-localized secretion and uptake profiles. In both cases, our simulations mirror previously demonstrated experimental results. As a whole, these models inform QS observations and synthetic biology designs., Author Summary Bacterial behavior is responsive to a multitude of soluble molecular cues. Among them are self-secreted autoinducers that control quorum sensing (QS) processes. While new quorum sensing systems are constantly being discovered, several systems have been well defined in terms of their molecular and genetic topologies, each influencing a variety of resultant phenotypes. These quorum sensing systems include LuxIR homologs that use an array of species specific autoinducers and Lsr system homologs that share a single autoinducer among numerous species. Here we suggest that the regulatory topology of these two systems mark them as opposites of a sort. Whereas the LuxIR system bears a strong positive intercellular feedback mechanism, the Lsr system bears strong negative intercellular feedback. In our simulations these differences are manifested in distinct patterns of signaling. This was readily visualized in the outward spread of autogenous LuxIR expression in a growing bacterial 2D ‘colony’ whereas a dispersed activity was produced by autogenous Lsr expression in an otherwise identical colony. Here, this dispersed activity is a reflection of bimodal Lsr expression. We show that this bimodality could arise from desynchronized Lsr driven autoinducer import (intercellular negative feedback). This may have consequences on the arrangement of downstream phenotypes.

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 at high yield. Also, the theoretical formalisms of metabolic engineering provided a basis for optimally routing its biochemical flux. These activities focused largely on the cell's intracellular biochemical network and relied less on molecular cues from its immediate surroundings. Quorum sensing (QS) has enabled a reexamination of metabolic flux and regulation by hardwiring population-scale biological function to extracellular cues. Regulatory and fabrication modules are feasible owing to a few relatively simple QS signal transduction cascades. QS now provides a means to create entirely new products and processes that leverage community coordinated behaviors. Indeed, the intersection of QS and synthetic biology has expanded the biotechnology toolbox and is just beginning to yield new promising outcomes.

Signaling between cells guides biological phenotype. Communications between individual cells, clusters of cells and populations exist in complex networks that, in sum, guide behavior. There are few experimental approaches that enable high content interrogation of individual and multicellular behaviors at length and time scales commensurate with the signal molecules and cells themselves. Here we present “biofabrication” in microfluidics as one approach that enables in-situ organization of living cells in microenvironments with spatiotemporal control and programmability. We construct bacterial biofilm mimics that offer detailed understanding and subsequent control of population-based quorum sensing (QS) behaviors in a manner decoupled from cell number. Our approach reveals signaling patterns among bacterial cells within a single biofilm as well as behaviors that are coordinated between two communicating biofilms. We envision versatile use of this biofabrication strategy for cell–cell interaction studies and small molecule drug discovery.

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. Triggered self-assembly relies on electrodeposition of the pH-responsive hydrogelator, 9-fluorenylmethoxycarbonyl-phenylalanine (Fmoc-Phe), in response to electrical inputs that generate a localized pH-gradient. Warm solutions of Fmoc-Phe and gelatin are co-deposited and, after cooling to room temperature, a physical gelatin network forms. Enzymatic assembly employs the cofactor-independent enzyme microbial transglutaminase (mTG) to perform two functions: crosslink the gelatin matrix to generate a thermally stable chemical gel and conjugate proteins to the matrix. To conjugate globular proteins to gelatin these proteins are engineered to have short lysine-rich or glutamine-rich fusion tags to provide accessible residues for mTG-catalysis. Viable bacteria can be co-deposited and entrapped within the crosslinked gelatin matrix and can proliferate upon subsequent incubation. These results demonstrate the potential for enlisting biological materials and mechanisms to biofabricate multifunctional soft matter.

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 technology (fouling). Despite the importance of bacterial biofilms, few experimental platforms and systems are available to assemble complex populations and monitor their activities. Here, a functionalized alginate composite material for creating in vitro model biofilms suitable for cell-cell signaling studies by entrapping bacterial cells in situ is reported. Biofilm assembly is achieved using device-imposed electrical signals to electrodeposit the stimuli-responsive polysaccharide alginate. This electrodeposition mechanism is versatile in that it allows control of the bacterial population density and distribution. For instance, it is demonstrated that a mixed population can be homogeneously distributed throughout the biofilm or can be assembled as spatially segregated populations within a stratified biofilm. The “electroaddressable” biofilms are visualized using both a planar 2D chip with patterned electrodes and a microfluidic bioMEMS device with sidewall electrodes. Specifically, it is observed that bacteria entrapped within the model biofilm recognize and respond to chemical stimuli imposed from the fluidic environment. Finally, reporter cells are used to demonstrate that bacteria entrapped within this model biofilm engage in intercellular quorum sensing. This work demonstrates the functionality of the stimuli-responsive polysaccharide by biofabricating pseudo-3D cell-gel biocomposites, mimicking the formation of biofilms, for interrogating phenotypes of E. coli bacterial populations. In addition to controlling assembly, the microfluidic device allows the biofilm to be monitored through the fluorescence methods commonly used in biological research. This platform technology should be able to be exploited for monitoring biofilm development, as well as for extending the understanding of the interactions between various bacterial species arranged in controlled patterns.

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 signaling among Escherichia coli cells grown over the surface of human epithelial (Caco-2) cells through site-directed attachment of biological nanofactories. These "factories" comprise a fusion protein expressed and purified from E. coli containing two AI-2 bacterial synthases (Pfs and LuxS), a protein G IgG binding domain, and affinity ligands for purification. The final factory is fabricated ex vivo by incubating with an anti-CD26 antibody that binds the fusion protein and specifically targets the CD26 dipeptidyl peptidase found on the outer surface of Caco-2 cells. This is the first report of the intentional "in vitro" synthesis of bacterial autoinducers at the surface of epithelial cells for the redirection of quorum sensing behaviors of bacteria. We envision tools such as this will be useful for interrogating, interpreting, and disrupting signaling events associated with the microbiome localized in human intestine and other environments.

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-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and β-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions.

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 anode to locally solubilize calcium ions from insoluble calcium carbonate. The solubilized Ca 2+ can then bind alginate to induce this polysaccharide to undergo a localized sol-gel transition. Calcium alginate gel formation is shown to be spatially controlled in the normal and lateral dimensions. The deposition method is sufficiently benign that it can be used to entrap the bacteria E. coli. The entrapped cells are able to grow and respond to chemical inducers in their environment. Also, the entrapped cells can be liberated from the gel network by adding sodium citrate that can compete with alginate for Ca 2+ binding. The capabilities of calcium alginate electrodeposition is illustrated by entrapping reporter cells that can recognize the quorum sensing autoinducer 2 (Al-2) signaling molecule. These reporter cells were observed to recognize and respond to Al-2 generated from an external bacterial population. Thus, calcium alginate electrodeposition provides a programmable method for the spatiotemporally controllable assembly of cell populations for cell-based biosensing and for studying cell-cell signaling.

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 tag that facilitates purification and a C-terminal enzyme-activatable pentatyrosine “pro-tag” that facilitates covalent coupling to the pH stimuli-responsive polysaccharide, chitosan. Because we confer pH-stimuli responsiveness to the IgG-binding domain, it can be electrodeposited or otherwise assembled into many configurations. Importantly, we demonstrate the loading of both HG3T and antibodies can be achieved in a linear fashion so that quantitative assessment of antibodies and antigens is feasible. Our demonstration formats include: conventional multiwell plates, micropatterned electrodes, and fiber networks. We believe biologically based fabrication (i.e., biofabrication) provides bottom-up hierarchical assembly of a variety of nanoscale components for applications that range from point-of-care diagnostics to smart fabrics. Biotechnol. Bioeng. 2009;103: 231–240. © 2008 Wiley Periodicals, Inc.

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). Enzymes, enzyme pathways, antibodies, and fluorescent proteins were all assembled onto spider silk fibers using this biomolecular engineering/biofabrication process. Additionally, arrangement of biofunctionalized fiber should in of itself generate a secondary level of biomolecular organization. Toward this end, as proofs of principle, spatially defined arrangement of biofunctionalized spider silk fiber was shown to generate effects specific to silk position in two cases. In one instance, arrangement perpendicular to a flow produced selective head and neck carcinoma cell capture on silk with antibodies complexed to conjugated protein G. In a second scenario, asymmetric bacterial chemotaxis arose from asymmetric conjugation of enzymes to arranged silk. Overall, the biofabrication processes used here were rapid, required no complex chemistries, were biologically benign, and also the resulting engineered silk microfibers were flexible, readily manipulated and functionally active. Deployed here in microfluidic environments, biofunctional spider silk fiber provides a means to convey complex biological functions over a range of scales, further extending its potential as a biomaterial in biotechnological settings. Biotechnol. Bioeng. 2017;114: 83-95. © 2016 Wiley Periodicals, Inc.

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 user-specified control. Our nanofactories, which synthesize and deliver the “universal” bacterial quorum-sensing signal molecule, autoinducer AI-2, to the surface of Escherichia coli , are assembled by first co-precipitating nanoparticles of iron salts and the biopolymer chitosan. E. coli AI-2 synthases, Pfs and LuxS, constructed with enzymatically activatable “pro-tags”, are then covalently tethered onto the chitosan. These enzymes synthesize AI-2 from metabolite S-adenosylhomocysteine. Chitosan serves as a molecular scaffold and provides cell capture ability; magnetite provides stimuli responsiveness. These magnetic nanofactories are shown to modulate the natural progression of quorum-sensing activity. New prospects for small molecule delivery, based on localized synthesis, are envisioned.

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 transduction stems from direct electron transfer, allowing for potentially quicker and more integrated measurements. One of the most common genetic reporters, β-galactosidase, can be measured both spectrophotometrically (Miller assay) and electrochemically. However, since the relationship between the two is not well understood, the electrochemical methods have not yet garnered the attention of biologists. With the aim of demonstrating the utility of an electrochemical measurement to the synthetic biology community, we created a genetic construct that interprets and reports (with β-galactosidase) on the concentration of the bacterial quorum sensing molecule autoinducer-2. In this work, we provide a correlation between electrochemical measurements and Miller Units. We show that the electrochemical assay works with both lysed and whole cells, allowing for the prediction of one from the other, and for continuous monitoring of cell response. We further present a conceptually simple and generalized mathematical model for cell-based β-galactosidase reporter systems that could aid in building and predicting a variety of synthetic biology constructs. This first-ever in-depth comparison and analysis aims to facilitate the use of electrochemical real-time monitoring in the field of synthetic biology as well as to facilitate the creation of constructs that can more easily communicate information to electronic systems.

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 consortium autonomously generates feedback to chemical cues. Moreover, abiotic components are readily assembled onto cells, enabling amplified and ‘binned' responses. Specifically, engineered cell populations are triggered by a quorum sensing (QS) signal molecule, autoinducer-2, to express surface-displayed fusions consisting of a fluorescent marker and an affinity peptide. The latter provides means for attaching magnetic nanoparticles to fluorescently activated subpopulations for coalescence into colour-indexed output. The resultant nano-guided cell network assesses QS activity and conveys molecular information as a ‘bio-litmus' in a manner read by simple optical means., Living cells have to interpret and react to changes in local environmental conditions. Here the authors exploit that by combining magnetic nanoparticles and bacterial quorum sensing to investigate and convey alterations in the molecular landscape.

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. Indeed, significant advances in the study of bacterial cell–cell signalling have been reported, including at length and time scales of the cells and their responses. Few reports exist, however, where signalling events that span physiologically relevant time scales are monitored and coordinated. Here, we employ principles of biofabrication to assemble, in situ, cell communities that are (i) spatially adjacent within partitioned microchannels for studying near communication and (ii) distally connected within longitudinal microfluidic networks so as to mimic long distance signalling among intestinal flora. We observed native signalling processes of the bacterial quorum sensing autoinducer-2 (AI-2) system among and between these communities. Cells in an upstream device successfully self-reported their activities and also secreted autoinducers that were carried downstream to the assembled networks of bacteria that reported on their presence. Furthermore, active signal modulation of among distal populations was demonstrated in a “programmed” manner where “enhancer” and “reducer” communities were assembled adjacent to the test population or “reporter” cells. The modulator cells either amplified or attenuated the cell–cell signalling between the distal, already communicating cell populations. Modulation was quantified with a bioassay, and the reaction rates of signal production and consumption were further characterized using a first principles mathematical model. Simulated distribution profiles of signalling molecules in the cell–gel composites agreed well with the observed cellular responses. We believe this simple platform and the ease by which it is assembled can be applied to other cell–cell interaction studies among various species or kingdoms of cells within well-regulated microenvironments.

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 in Escherichia coli are subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of the lsr (for “ luxS regulated”) operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of the lsr operon and its regulators, LsrR and LsrK, previously reported in Salmonella enterica serovar Typhimurium, are confirmed here for E. coli. The elucidation of cAMP-CRP involvement in E. coli autoinduction impacts many areas, including the growth of E. coli in fermentation processes.