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Self-healing properties, originating from the natural healing process, are highly desirable for the fitness-enhancing functionality of biomimetic materials. Herein, we fabricated the biomimetic recombinant spider silk by genetic engineering, in which Escherichia coli (E. coli) was employed as a heterologous expression host. The self-assembled recombinant spider silk hydrogel was obtained through the dialysis process (purity > 85%). The recombinant spider silk hydrogel with a storage modulus of ~250 Pa demonstrated autonomous self-healing and high strain-sensitive properties (critical strain ~50%) at 25 °C. The in situ small-angle X-ray scattering (in situ SAXS) analyses revealed that the self-healing mechanism was associated with the stick-slip behavior of the β-sheet nanocrystals (each of ~2–4 nm) based on the slope variation (i.e., ~−0.4 at 100%/200% strains, and ~−0.9 at 1% strain) of SAXS curves in the high q-range. The self-healing phenomenon may occur through the rupture and reformation of the reversible hydrogen bonding within the β-sheet nanocrystals. Furthermore, the recombinant spider silk as a dry coating material demonstrated self-healing under humidity as well as cell affinity. The electrical conductivity of the dry silk coating was ~0.4 mS/m. Neural stem cells (NSCs) proliferated on the coated surface and showed a 2.3-fold number expansion after 3 days of culture. The biomimetic self-healing recombinant spider silk gel and thinly coated surface may have good potential in biomedical applications.

Developing materials with structural flexibility that permits self-repair in response to external disturbances remains challenging. Spider silk, which combines an exceptional blend of strength and pliability in nature, serves as an ideal dynamic model for adaptive performance design. In this work, a novel self-healing material is generated using spider silk. Dragline silk from spiderNephila pilipesis demonstrated with extraordinaryin situself-repair property through a constructed thin film format, surpassing that of two other silks from spiderCyrtophora moluccensisand silkwormBombyx mori. Subsequently, R2, a key spidroin associated with self-healing, is biosynthesized, with validated cohesiveness. R2 is further programmed with tunable healability (permanent and reversible) and conductivity (graphene doping; R2G) for electronics applications. In the first demonstration, film strips from R2 and R2G are woven manually into multidimensional (1D-3D) conductive fabrics for creating repairable logic gate circuits. In the second example, a reversibly-healable R2/R2G strip is fabricated as a re-configurable wearable ring probe to fit fingertips of varying widths while retaining its detecting capabilities. Such prototype displays a unique conformable wearable technology. Last, the remarkable finding of self-healing in spider silk could offer a new material paradigm for developing future adaptive biomaterials with tailored performance and environmental sustainability.

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.

Protein-level regulations have gained importance in building synthetic circuits, as they offer a potential advantage in the speed of operation compared to gene regulation circuits. In nature, localized protein degradation is prevalent in polarizing cellular signaling. We, therefore, set out to systematically investigate whether localized proteolysis can be employed to construct intracellular asymmetry in Escherichia coli. We demonstrate that, by inserting a cognate cleavage site between the reporter and C-terminal degron, the unstable reporter can be stabilized in the presence of the tobacco etch virus protease. Furthermore, the split protease can be functionally reconstituted by the PopZ-based polarity system to exert localized proteolysis. Selective stabilization of the unstable reporter at the PopZ pole can lead to intracellular asymmetry in E. coli. Our study provides complementary evidence to support that localized proteolysis may be a strategy for polarization in developmental cell biology. Circuits designed in this study may also help to expand the synthetic biology repository for the engineering of synthetic morphogenesis, particularly for processes that require rapid control of local protein abundance.

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.

Silk fibroin produced from silkworms has been intensively utilized as a scaffold material for a variety of biotechnological applications owing to its remarkable mechanical strength, extensibility, biocompatibility, and ease of biofunctionalization. In this research, we engineered silk as a novel trap platform capable of capturing microorganisms. Specifically, we first fabricated the silk material into a silk sponge by lyophilization, yielding a 3D scaffold with porous microstructures. The sponge stability in water was significantly improved by ethanol treatment with elevated β-sheet content and crystallinity of silk. Next, we biofunctionalized the silk sponge with a poly-specific microbial targeting molecule, ApoH (apolipoprotein H), to enable a novel silk-based microbial trap. The recombinant ApoH engineered with an additional penta-tyrosine was assembled onto the silk sponge through the horseradish peroxidase (HRP) mediated dityrosine cross-linking. Last, the ApoH-decorated silk sponge was demonstrated to be functional in capturing our model microorganism targets, E. coli and norovirus-like particles. We envision that this biofabricated silk platform, capable of trapping a variety of microbial entities, could serve as a versatile scaffold for rapid isolation and enrichment of microbial samples toward future diagnostics and therapeutics. This strategy, in turn, can expedite advancing future biodevices with functionality and sustainability.

Some people with autism spectrum disorders have been observed to experience difficulties with making correct inferences in conversations in social situations. However, the nature and origin of their problem is rarely investigated. This study used manipulations of video stimuli to investigate two questions. The first question was whether it is the number of people involved in social situations, that is, the source of problems in following conversations, or whether it is the increased mentalising demands required to comprehend interactions between several people. The second question asked was whether the nature and pattern of the errors that autism spectrum disorder participants show are the same as typically developing people make when they make an error. In total, 43 typically developed adults and 30 adults diagnosed with autism spectrum disorder were studied. We found that it was the amount of mentalising required, rather than the number of people involved, which caused problems for people with autism spectrum disorder in following conversations. Furthermore, the autism spectrum disorder participants showed a more heterogeneous pattern of errors, showing less agreement among themselves than the typically developed group as to which test items were hardest. So, fully understanding the observed behaviour consequent upon weakness in mentalising ability in people with autism spectrum disorders requires consideration of factors other than mentalising. People with autism spectrum disorders sometimes report difficulties with following observed conversations in social situations, especially those where several people are interacting with each other. But this has rarely been investigated directly. This study determines whether people with autism spectrum disorders do indeed have problems following observed conversations even when they perform well on IQ tests and investigates two possible reasons for any difficulty found: (1) some people may have a problem integrating stimuli from multiple speakers; (2) following a conversation between many people might make particularly high demands on mentalising abilities. We used a variety of video clips of people conversing together to investigate these two possibilities in 30 adults diagnosed with autism spectrum disorder and 43 age- and IQ-matched typical-developing adults. We found that it was the amount of mentalising required, rather than the number of people involved, which caused problems for people with autism spectrum disorder in following conversations. Furthermore, when the autism spectrum disorder participants made a mistake, the error they made was frequently not the same error that typically developed participants made, and the autism spectrum disorder population made a more varied set of errors than the typically developed participants. Together, these results suggest that people with autism spectrum disorders observe significant problems with following conversations between many people when they contain a lot of mentalising material, but where they do make a mistake, the conclusions they draw from the conversation they are observing may have a more complex cause than an impairment in mentalising alone.

Protein-level regulations have gained importance in building synthetic circuits, as they offer a potential advantage in the speed of operation compared to gene regulation circuits. In nature, localized protein degradation is prevalent in polarizing cellular signaling. We, therefore, set out to systematically investigate whether localized proteolysis can be employed to construct intracellular asymmetry in

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.

The superlative mechanical properties of spider silk and its conspicuous variations have instigated significant interest over the past few years. However, current attempts to synthetically spin spider silk fibers often yield an inferior physical performance, owing to the improper molecular interactions of silk proteins. Considering this, herein, a post-treatment process to reorganize molecular structures and improve the physical strength of spider silk is reported. The major ampullate dragline silk from Nephila pilipes with a high &beta, sheet content and an adequate tensile strength was utilized as the study material, while that from Cyrtophora moluccensis was regarded as a reference. Our results indicated that the hydrothermal post-treatment (50&ndash, 70 &deg, C) of natural spider silk could effectively induce the alternation of secondary structures (random coil to &beta, sheet) and increase the overall tensile strength of the silk. Such advantageous post-treatment strategy when applied to regenerated spider silk also leads to an increment in the strength by ~2.5&ndash, 3.0 folds, recapitulating ~90% of the strength of native spider silk. Overall, this study provides a facile and effective post-spinning means for enhancing the molecular structures and mechanical properties of as-spun silk threads, both natural and regenerated.

Mineral trioxide aggregate (MTA) is widely used in various dental endodontic applications such as root-end filling, furcal perforation repair, and vital pulp therapy. In spite of many attempts to improve handling properties and reduce the discoloration of MTA, the ideal root canal filling material has yet to be fully developed. The objective of this study was to investigate the setting time, mechanical properties, and biocompatibility of MTA set by a silk fibroin solution. A 5 wt% silk fibroin (SF) solution (a novel hydration accelerant) was used to set SavDen&reg, MTA and ProRoot&reg, white MTA (WMTA). Changes in setting time, diametral tensile strength (DTS), material crystallization, in vitro cell viability, and cell morphology were assessed by Vicat needle measurement, a universal testing machine, scanning electron microscopy (SEM), and WST-1 assay, respectively. The initial setting time of ProRoot&reg, MTA and SavDen&reg, MTA experienced a drastic decrease of 83.9% and 42.1% when deionized water was replaced by 5 wt% SF solution as the liquid phase. The DTS of SavDen&reg, MTA showed a significant increase after set by the SF solution in 24 h. A human osteoblast-like cell (MG-63)-based WST-1 assay revealed that both ProRoot&reg, MTA hydrated using SF solution did not significantly differ (p &gt, 0.05) in cell viability. MG-63 cells with pseudopodia attachments and nuclear protrusions represent a healthier and more adherent status on the surface of MTA when set with SF solution. The results suggest that the 5 wt% SF solution may be used as an alternative hydration accelerant for MTA in endodontic applications.

In addition to engineering new pathways for synthesis, synthetic biologists rewire cells to carry out "programmable" functions, an example being the creation of wound-healing probiotics. Engineering regulatory circuits and synthetic machinery, however, can be deleterious to cell function, particularly if the "metabolic burden" is significant. Here, a synthetic regulatory circuit previously constructed to direct Escherichia coli to swim toward hydrogen peroxide, a signal of wound generation, was shown to work even with coexpression of antibiotic resistance genes and genes associated with lactose utilization. We found, however, that cotransformation with a second vector constitutively expressing GFP (as a marker) and additionally conferring resistance to kanamycin and tetracycline resulted in slower velocity (Δ~6 μm/s) and dramatically reduced growth rate (Δ > 50%). The additional vector did not, however, alter the run-and-tumble ratio or directional characteristics of H2 O2 -dependent motility. The main impact of this additional burden was limited to slowing cell velocity and growth, suggesting that reprogrammed cell motility by minimally altering native regulatory circuits can be maintained even when extraneous burden is placed on the host cell. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2778, 2019.

Electronic textiles (E-textiles) have been an area of intense industrial and academic research for years due to their advanced applications. Thus, the goal of this study was to develop highly conductive silk fibroin electrochromic nanofibers for use in E-textiles. The silk nanofibers were prepared by an electrospinning technique, and the conductive polyaniline (PANI) was added to impart the electrical conductivity and electroactive property to the resultant electrospun silk composite nanofibers. The experimental results showed that tuning the electrospinning procedure could control the morphology of the composite nanofibers, thus altering their mechanical properties and surface wettability. Furthermore, the developed PANI/silk composite fibers possess electroactive and electrochromic properties, such as adjusting the applied voltage. The developed strategy demonstrated the feasibility of incorporating not only electrical functionality but also electroactivity into sustainable silk nanofibers using electrospinning technique.

In spite of all the efforts towards deciphering the silk spinning process of spiders, the underlying mechanism is yet to be fully revealed. In this research, we designed a novel approach that allowed us to quantitatively evaluate the concentration change of silk dope during the liquid-to-solid spinning process of the orb-weaver Nephila pilipes. As a prior characterization of the optimal silking conditions, we first gauged the influence of silking-rate, ranging from 1.5 to 8.0 m/min, on dragline silk diameters and silk tensile strengths obtained from the spiders. Next, to evaluate the liquid content of the silk dope, the major ampullate gland was dissected and the concentration of the sac portion was measured by thermogravimetric analysis (TGA). The solid content of the dragline fibers leaving the spinneret was investigated by calculating the ratio of collected dried silk to the weight loss of the spider recorded in situ upon spinning. As the results indicate, the tensile strength and diameter of the spun dragline fibers were 800–1100 MPa and 8–11 μm, respectively. The liquid content of silk stored in the major ampullate sac (50.0 wt%) was significantly lower than that of silk leaving the spinnerets (80.9–96.1 wt%), indicating that a liquid supplying mechanism might be involved during the spinning process. This reveals, for the first time, quantitative evidence in support of the lubricative hypothesis proposed formerly, namely that a liquid coating layer is supplemented to compensate for silking resistance during the spinning process of a spider. The spigot, at the exit of the spinneret, is speculated to serve as a valve-like controller that regulates the lubrication process along with fiber formation. Taken together, these findings provide understanding of the physiological functions in the spider spinning process and could further shed some light on the future biomimetic development of silk material fabrication.

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.

Liposomes are nanoscale containers that are typically synthesized from lipids using a high-shear process such as extrusion or sonication. While liposomes are extensively used in drug delivery, they do suffer from certain problems including limited colloidal stability and short circulation times in the body. As an alternative to liposomes, we explore a class of container structures derived from erythrocytes (red blood cells). The procedure involves emptying the inner contents of these cells (specifically hemoglobin) and resuspending the empty structures in buffer, followed by sonication. The resulting structures are termed nanoerythrosomes (NERs), i.e., they are membrane-covered nanoscale containers, much like liposomes. Cryo-transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS) are employed for the first time to study these NERs. The results reveal that the NERs are discrete spheres (∼110 nm diameter) with a unilamellar membrane of thickness ∼4.5 nm. Remarkably, the biconcave disc-like shape of erythrocytes is also exhibited by the NERs under hypertonic conditions. Moreover, unlike typical liposomes, NERs show excellent colloidal stability in both buffer as well as in serum at room temperature, and are also able to withstand freeze-thaw cycling. We have explored the potential for using NERs as colloidal vehicles for targeted delivery. Much like conventional liposomes, NER membranes can be decorated with fluorescent or other markers, solutes can be encapsulated in the cores of the NERs, and NERs can be targeted to specifically bind to mammalian cells. Our study shows that NERs are a promising and versatile class of nanostructures. NERs that are harvested from a patient's own blood and reconfigured for nanomedicine can potentially offer several benefits including biocompatibility, minimization of immune response, and extended circulation time in the body.

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.

Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as "tunable" factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user-specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (as seen with nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as "pseudotaxis." Escherichia coli, absent the motility regulator cheZ, swim sporadically, missing the traditional "run" in the run:tumble swimming paradigm. Upon introduction of cheZ in trans and its signal-generated upregulation, engineered bacteria can be "programmed" to swim toward the source of the chemical cue. Here, engineered cells that encounter sufficient levels of the small signal molecule pyocyanin, produce an engineered CheZ and swim with programmed directionality. By incorporating a degradation tag at the C-terminus of CheZ, the cells stop running when they exit spaces containing pyocyanin. That is, the engineered CheZ modified with a C-terminal extension derived from the putative DNA-binding transcriptional regulator YbaQ (RREERAAKKVA) is consumed by the ClpXP protease machine at a rate sufficient to "brake" the cells when pyocyanin levels are too low. Through this process, we demonstrate that over time, these engineered E. coli accumulate in pyocyanin-rich locales. We suggest that such approaches may find utility in engineering probiotics so that their beneficial functions can be focused in areas of principal benefit.

Individually, advances in microelectronics and biology transformed the way we live our lives. However, there remain few examples in which biology and electronics have been interfaced to create synergistic capabilities. We believe there are two major challenges to the integration of biological components into microelectronic systems: (i) assembly of the biological components at an electrode address, and (ii) communication between the assembled biological components and the underlying electrode. Chitosan possesses a unique combination of properties to meet these challenges and serve as an effective bio-device interface material. For assembly, chitosan’s pH-responsive film-forming properties allow it to “recognize” electrode-imposed signals and respond by self-assembling as a stable hydrogel film through a cathodic electrodeposition mechanism. A separate anodic electrodeposition mechanism was recently reported and this also allows chitosan hydrogel films to be assembled at an electrode address. Protein-based biofunctionality can be conferred to electrodeposited films through a variety of physical, chemical and biological methods. For communication, we are investigating redox-active catechol-modified chitosan films as an interface to bridge redox-based communication between biology and an electrode. Despite significant progress over the last decade, many questions still remain which warrants even deeper study of chitosan’s structure, properties, and functions.

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.

The ability to interconvert information between electronic and ionic modalities has transformed our ability to record and actuate biological function. Synthetic biology offers the potential to expand communication ‘bandwidth' by using biomolecules and providing electrochemical access to redox-based cell signals and behaviours. While engineered cells have transmitted molecular information to electronic devices, the potential for bidirectional communication stands largely untapped. Here we present a simple electrogenetic device that uses redox biomolecules to carry electronic information to engineered bacterial cells in order to control transcription from a simple synthetic gene circuit. Electronic actuation of the native transcriptional regulator SoxR and transcription from the PsoxS promoter allows cell response that is quick, reversible and dependent on the amplitude and frequency of the imposed electronic signals. Further, induction of bacterial motility and population based cell-to-cell communication demonstrates the versatility of our approach and potential to drive intricate biological behaviours., Synthetic biology offers the ability to explore new ways of manipulating gene expression and function. Here the authors demonstrate an electrogenetic device that allows control of transcription by an exogenous electrical signal.

By use of a strategy incorporating the green fluorescent protein (GFP), facile and rapid monitoring and visualization of baculovirus infection in insect cells is possible in vivo. This chapter describes two techniques for simple determination of virus titer in the baculovirus expression system using GFP co-expression and rapid monitoring of Sf-9 insect cell infection using a combination of GFP and the early-to-late (ETL) promoter of the virus vector. Because of its early appearance, GFP, when placed under the control of ETL promoter, will facilitate vector construction, virus isolation, and titer determination.

A technique is described for synthesizing and transfecting double stranded RNA (dsRNA) for RNA interference (RNAi) in Sf-21 cell culture. Transfection with dsRNA only requires an hour and the cells usually recover within 12 h. Suggestions for designing dsRNA are included in the methods. Furthermore, websites are provided for rapid and effective dsRNA design. Three kits are essential for using the described methods: RNAqueous®-4PCR, Megascript™ T7 kit, and the Superscript™ III kit from Life Technologies, Inc.

Probing the baculovirus infection process is essential in optimizing recombinant protein production. Typically, researchers monitor the infection process in stirred reactors that contain cells that have been infected at different times after virus inoculation, particularly if cells pass the primary infection and become infected by progeny virus. This chapter describes several alternative bioreactor systems for baculovirus infection. We provide an example alternative system that holds promise to avoid asynchronous distributions in infection time. Namely, we describe a two-stage reactor system consisting of an upstream continuous stirred tank reactor and a downstream tubular reactor with segmented plug flow for probing baculovirus infection and production.

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.

By use of a strategy incorporating the green fluorescent protein (GFP), facile and rapid monitoring and visualization of baculovirus infection in insect cells is possible in vivo. This chapter describes two techniques for simple determination of virus titer in the baculovirus expression system using GFP co-expression and rapid monitoring of Sf-9 insect cell infection using a combination of GFP and the early-to-late (ETL) promoter of the virus vector. Because of its early appearance, GFP, when placed under the control of ETL promoter, will facilitate vector construction, virus isolation, and titer determination.

Probing the baculovirus infection process is essential in optimizing recombinant protein production. Typically, researchers monitor the infection process in stirred reactors that contain cells that have been infected at different times after virus inoculation, particularly if cells pass the primary infection and become infected by progeny virus. This chapter describes several alternative bioreactor systems for baculovirus infection. We provide an example alternative system that holds promise to avoid asynchronous distributions in infection time. Namely, we describe a two-stage reactor system consisting of an upstream continuous stirred tank reactor and a downstream tubular reactor with segmented plug flow for probing baculovirus infection and production.

A technique is described for synthesizing and transfecting double stranded RNA (dsRNA) for RNA interference (RNAi) in Sf-21 cell culture. Transfection with dsRNA only requires an hour and the cells usually recover within 12 h. Suggestions for designing dsRNA are included in the methods. Furthermore, websites are provided for rapid and effective dsRNA design. Three kits are essential for using the described methods: RNAqueous®-4PCR, Megascript™ T7 kit, and the Superscript™ III kit from Life Technologies, Inc.

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.

We present a successful demonstration of a reusable Surface Acoustic Wave (SAW) sensor for bacterial biofilm growth monitoring in an animal serum and bacterial growth media. Bacterial biofilms produce harmful metabolic by-products and are a characteristic of severe infections. Thus, continuous monitoring of bacterial biofilm growth is critical. Here, we report a highly sensitive SAW sensor for biofilm growth monitoring fabricated by depositing zinc oxide (ZnO) piezoelectric thin film by pulsed laser deposition (PLD). To prevent ZnO damage from long term exposure to bacterial growth media or to an animal serum, the ZnO layer of the sensor was effectively protected by aluminum oxide (Al 2 O 3 ) using atomic layer deposition (ALD). As a result, the sensor was reusable for consecutive biofilm formation experiments. The detection limit of the SAW sensor was approximately 5.3 pg. The SAW sensor was tested with Escherichia coli W3110 in Lysogeny Broth (LB) media, and in 10% diluted Fetal Bovine Serum (FBS) as an approximation to an in vivo environment. The resonant frequency shift measured at the output of the SAW sensor in both LB media and 10% FBS corresponded to natural biofilm growth. These repeatable results support the novel application of a SAW sensor for real time biofilm sensing.

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 spite of all the efforts towards deciphering the silk spinning process of spiders, the underlying mechanism is yet to be fully revealed. In this research, we designed a novel approach that allowed us to quantitatively evaluate the concentration change of silk dope during the liquid-to-solid spinning process of the orb-weaver Nephila pilipes. As a prior characterization of the optimal silking conditions, we first gauged the influence of silking-rate, ranging from 1.5 to 8.0 m/min, on dragline silk diameters and silk tensile strengths obtained from the spiders. Next, to evaluate the liquid content of the silk dope, the major ampullate gland was dissected and the concentration of the sac portion was measured by thermogravimetric analysis (TGA). The solid content of the dragline fibers leaving the spinneret was investigated by calculating the ratio of collected dried silk to the weight loss of the spider recorded in situ upon spinning. As the results indicate, the tensile strength and diameter of the spun dragline fibers were 800&ndash, 1100 MPa and 8&ndash, 11 &mu, m, respectively. The liquid content of silk stored in the major ampullate sac (50.0 wt%) was significantly lower than that of silk leaving the spinnerets (80.9&ndash, 96.1 wt%), indicating that a liquid supplying mechanism might be involved during the spinning process. This reveals, for the first time, quantitative evidence in support of the lubricative hypothesis proposed formerly, namely that a liquid coating layer is supplemented to compensate for silking resistance during the spinning process of a spider. The spigot, at the exit of the spinneret, is speculated to serve as a valve-like controller that regulates the lubrication process along with fiber formation. Taken together, these findings provide understanding of the physiological functions in the spider spinning process and could further shed some light on the future biomimetic development of silk material fabrication.

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.

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.

Funding for Open Access provided by the UMD Libraries Open Access Publishing Fund., BACKGROUND/OBJECTIVES: The use of electric fields in combination with small doses of antibiotics for enhanced treatment of biofilms is termed the ‘bioelectric effect’ (BE). Different mechanisms of action for the AC and DC fields have been reported in the literature over the last two decades. In this work, we conduct the first study on the correlation between the electrical energy and the treatment efficacy of the bioelectric effect on Escherichia coli K-12 W3110 biofilms. METHODS: A thorough study was performed through the application of alternating (AC), direct (DC) and superimposed (SP) potentials of different amplitudes on mature E. coli biofilms. The electric fields were applied in combination with the antibiotic gentamicin (10 μg/ml) over a course of 24 h, after the biofilms had matured for 24 h. The biofilms were analysed using the crystal violet assay, the colony-forming unit method and fluorescence microscopy. RESULTS: Results show that there is no statistical difference in treatment efficacy between the DC-, AC- and SP-based BE treatment of equivalent energies (analysis of variance (ANOVA) P > 0.05) for voltages < 1 V. We also demonstrate that the efficacy of the BE treatment as measured by the crystal violet staining method and colony-forming unit assay is proportional to the electrical energy applied (ANOVA P < 0.05). We further verify that the treatment efficacy varies linearly with the energy of the BE treatment (r2 = 0.984). Our results thus suggest that the energy of the electrical signal is the primary factor in determining the efficacy of the BE treatment, at potentials less than the media electrolysis voltage. CONCLUSIONS: Our results demonstrate that the energy of the electrical signal, and not the type of electrical signal (AC or DC or SP), is the key to determine the efficacy of the BE treatment. We anticipate that this observation will pave the way for further understanding of the mechanism of action of the BE treatment method and may open new doors to the use of electric fields in the treatment of bacterial biofilms.

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.

SUMMARY Recent studies have demonstrated that orb-weaving spiders may alter web structures, foraging localities or silk output in response to prey variations. In this study we conducted field surveys and food manipulations to examine whether orb-weaving spiders may also adjust the protein of silk to prey variations. A comparison of dragline silks collected from nine giant wood spider Nephila pilipes populations in Taiwan showed a spatial variation. The percentage of all amino acids (except alanine and glycine)exhibited significant differences among populations. A survey of prey composition also revealed a significant spatial variation among N. pilipes populations. To determine whether prey variation was responsible for silk protein variation, we fed N. pilipes with different types of prey (dipteran vs orthopteran) then compared the percentage of five major dragline amino acids and secondary structures. The results showed that dragline of N. pilipes fed with orthopteran prey contained significantly higher proline and glutamine but lower alanine. Congruent with this result were those from FTIR spectroscopy, which showed that dragline of N. pilipes fed with crickets exhibited significantly higher percentage of proline- and glutamine-containing β turns, and lower percentage of alanine-containing β sheet structures. Since the results of feeding manipulations showed that diet significantly affected the compositions of dragline silks, the observed spatial variation seemed to reflect the different types of prey these spiders had consumed. Results of this study thus indicated that orb-weaving spiders can alter dragline protein in response to prey variations.

The X-ray diffraction of dragline silks, produced by Nephila and Cyrtophora spiders, were measured by synchrotron radiation in their original states or in situ during stretching and heating. Nephila pilipesspiders construct a two-dimensional orb web that must be rebuilt in one or 2 days, but Cyrtophora spiders form a three-dimensional tent web that can exist for several weeks in a tropical forest. Diffraction patterns of N. pilipes and Cyrtophora draglines resemble each other. Crystals of two kinds are identified in these draglines; one is aligned parallel to the silk direction and another is less oriented. The less oriented crystal in Cyrtophoradragline is aligned better than that in N. pilipes dragline, which generates about three times stronger diffract intensity. Crystals in N. pilipes and C. moluccensisdragline silks have remarkable thermal stability. Equatorial reflections remain undiminished until 350 and 450 ◦ C for N. pilipes and C. moluccensis, respectively. In contrast, the meridional reflections S and (0 0 2), which are parallel to the silk thread, disappear at a temperature less than 100 ◦ C for C. moluccensis but remain for Nephila up to 100 ◦ C. Meridional reflections S and (0 0 2) shift to a smaller angle during stretching, whereas equatorial reflections remain constant in a range 1.0–1.3 times the original length. The position of the S reflection shifts rapidly in the first 10% of elongation from the original length but remains constant during subsequent stretching, whereas the (0 0 2) reflection shifts rapidly during the first 5% elongation from the original length and continues to shift subsequently. In contrast, the features of N. pilipes dragline alter insignificantly during stretching. Examination of the composition of amino acids of the draglines of N. pilipes and C. moluccensis indicates that a dragline of N. pilipes contains more glycine, but much less alanine, than that of C. moluccensis. © 2004 Elsevier B.V. All rights reserved.

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.g., hours), sufficient to evaluate cell phenotype. The device exploits a unique simple bilayer configuration that enables rapid setup and quick reproducible introduction of cells. Its reusability represents an additional advantage in that it need not be limited to settings with microfluidics expertise. We have successfully demonstrated the applicability of this tool in studying the chemotactic response of Escherichia coli to glucose. When coupled with our recent Python program, quantified metrics such as speed, ratio of tumble to run, and effective diffusivity can be obtained from slow frame rate videos. Moreover, we introduce a chemotaxis partition coefficient that conveniently scores swimming behavior on the single-cell level.

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 sensing (QS) is a highly conserved mechanism of bacterial cell-cell communication and QS-based synthetic signal transduction pathways represent a new generation of biotechnology toolbox members. Specifically, the E. coli QS master regulator, LsrR, is uniquely positioned to actuate gene expression in response to a QS signal. In order to expand the use of LsrR in synthetic biology, two novel LsrR switches were generated through directed evolution: an "enhanced" repression and derepression eLsrR and a reversed repression/derepression function "activator" aLsrR. Protein modeling and docking studies are presented to gain insight into the QS signal binding to these two evolved proteins and their newly acquired functionality. We demonstrated the use of the aLsrR switch using a coculture system in which a QS signal, produced by one bacterial strain, is used to inhibit gene expression via aLsrR in a different strain. These first ever AI-2 controlled synthetic switches allow gene expression from the lsr promoter to be tuned simultaneously in two distinct cell populations. This work expands the tools available to create engineered microbial populations capable of carrying out complex functions necessary for the development of advanced synthetic products.