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Pluronic (Plu)-based hydrogels containing a fraction of Pluronic diacrylate (PluDA) chains form physical networks with additional covalent crosslinks. These hydrogels have been used to encapsulate bacteria or yeast in living materials and devices. Within Plu/PluDA hydrogels, the growth of the encapsulated organisms decreases with increasing density of covalent bonds. This has been attributed to the capability of the organisms to sense and respond to the mechanical properties of the embedding matrix. Here we investigate the rheological behavior of 30 wt% Plu/PluDA hydrogels with increasing covalent crosslinking adjusted by the PluDA fraction in the mixture. This hydrogel composition allowed hydrogel-regulated growth ofE. coliin living devices in previous reports. We aim to find experimental descriptors of the hydrogel’s rheological response that can be correlated to the reported growth behavior. We present stress relaxation and creep-recovery experiments and quantify the elastic, viscoelastic and plastic responses of the hydrogels as function of the covalent crosslinking degree, with a focus on critical yield strain/stress and relaxation parameters. An inverse correlation was observed between the storage modulus and the critical stress/strain for fluidization τF, of the hydrogel and the reported maximum volume ofE. colicolonies grown in the hydrogels. These results contribute to the biophysical understanding of bacteria behavior in confinement and are relevant for different application contexts, eg. to improve the performance and safety of living therapeutic implants and to maximize the yield of biotechnological production in immobilized cell reactors.Statement of significancePluronic-based hydrogels are increasingly used to encapsulate bacteria and yeast in engineered living materials (ELMs). The mechanical properties of these hydrogels impact the growth of the organisms in the confined state. This study presents a fundamental characterization of the viscoelastic response of Pluronic and Pluronic diacrylate hydrogels with different degrees of covalent crosslinking by rheology. It correlates rheological response with observed cell behavior in model encapsulated systems, thereby contributing to the understanding of bacteria-material interactions and their impact in ELMs.

Engineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in material’s composition and function. Understanding how the spatial confinement in 3D affects the behavior of the embedded cells is crucial to design and predict ELM’s function, regulate and minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and decreasing plasticity of the matrix, a decrease in colony volumes and an increase in their sphericity is observed. Protein production surprisingly follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that, bacterial mechanosensitivity can be used to control and regulate the composition and function of ELMs by thoughtful design of the encapsulating matrix, and by following design criteria with interesting similarities to those developed for 3D culture of mammalian cells.

Engineered living materials (ELMs) use encapsulated microorganisms within polymeric matrices for biosensing, drug delivery, capturing viruses, and bioremediation. It is often desirable to control their function remotely and in real time. Suitable, genetically engineered microorganisms respond to changes of their environment. Here, we combine this local sensitivity with a nanostructured encapsulation material to sensitize the ELM for infrared light. Previously, blue light has been used to stimulate microorganisms that contain optogenetic modules responsive to those wavelengths without the need for exogenous cofactors. Here, we use plasmonic gold nanorods (AuNR) that have a strong absorption maximum at 808 nm, a wavelength where human tissue is relatively transparent. Biocompatible composites of a Pluronic-based hydrogel and AuNR are prepared without agglomeration; they react to illumination by local heating. We measure a photothermal conversion efficiency of 47 % in transient temperature measurements. Steady-state temperature profiles from local photothermal heating are quantified using infrared photothermal imaging, correlated with measurements inside the gel, and applied to stimulate thermoresponsive bacteria. Using a bilayer ELM construct with the thermoresponsive bacteria and the thermoplasmonic composite gel in two separate but connected hydrogel layers, it is shown that the bacteria can be stimulated to produce a fluorescent protein using infrared light in a spatially controlled manner.

In engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.

In living therapeutic materials, organisms genetically programmed to produce and deliver drugs are encapsulated in porous matrices or hydrogels acting as physical barriers between the therapeutic organisms and the host cells. The therapeutic potential of such constructs has been highlighted in in vitro studies, but the translation to in vivo scenarios requires evaluation of the immune response to the presence of the encapsulated, living organisms. In this study, we investigate the responses of human peripheral blood mononuclear cells (PBMCs) exposed to a living therapeutic material consisting of engineered E. coli encapsulated in Pluronic F127-based hydrogels. The release of inflammation-related cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNFα and IFNγ) and cytotoxic proteins (granzyme A, granzyme B, perforin, granulysin, sFas, and sFasL) in response to the bacterial hydrogels, as well as the subsets of natural killer cells and T cells after exposure to the bacterial hydrogel for up to three days were examined. In direct contact with PBMCs, both E. coli and its endotoxin-free variant, ClearColi, induce apoptosis of the immune cells and trigger IL-6 release from the surviving cells. However, we found that encapsulation of the bacteria in Pluronic F127 diacrylate hydrogels considerably lowers their immunogenicity and practically abolishes apoptosis triggered by ClearColi. In comparison with E. coli, free and hydrogel-encapsulated ClearColi induced significantly lower levels of NK cell differentiation into the more cytolytic CD16dim subset. Our results demonstrate that ClearColi-encapsulated hydrogels generate low immunogenic response and are suitable candidates for the development of living therapeutic materials for in vivo testing to assess a potential clinical use. Nevertheless, we also observed a stronger immune response in pro-inflammatory PBMCs, possibly from donors with underlying infections. This suggests that including anti-inflammatory measures in living therapeutic material designs could be beneficial for such recipients.

Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due since their complex and thick cell walls limit our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as increase in plasmid size, different methylation patterns and limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on Gibson assembly and PCR to amplify the DNA to sufficient quantities for successful transformation in L. plantarum WCFS1. This advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.

Lactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules inLactiplantibacillus plantarumthat achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA, from a phylogenetically distant bacterium,Salmonella typhimurium, that drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tunable transient Genetically Modified Organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.

[EN] Biomaterials have evolved from inert materials that lack interaction with the body to biologically active, instructive materials that host and provide signals to surrounding cells and tissues. Engineered living materials contain living cells (responsive function) and polymeric matrices (scaffolding function) and, thus, can be designed as active and response biomaterials. In this Review, we discuss engineered living materials that incorporate microorganisms as the living, bioactive component. Microorganisms can provide complex responses to environmental stimuli, and they can be genetically engineered to allow user control over responses and integration of numerous inputs. The engineered microorganisms can either generate their own matrix, such as in biofilms, or they can be incorporated in matrices using various technologies, such as coating, 3D printing, spinning and microencapsulation. We highlight biomedical applications of such engineered living materials, including biosensing, wound healing, stem-cell-based tissue engineering and drug delivery, and provide an outlook to the challenges and future applications of engineered living materials., Support from EPSRC through a programme grant (EP/P001114/1) is acknowledged. M.S.-S. and M.J.D acknowledge support from a grant from the UK Regenerative Medicine Platform "Acellular/Smart Materials - 3D Architecture" (MR/R015651/1). S.S. and A.d.C. acknowledge support from the Deutsche Forschungsgemeinschaft's Collaborative Research Centre, SFB 1027 and the Leibniz Science Campus on Living Therapeutic Materials, LifeMat.

Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form a heteroternary complex with cucurbit[8]uril (CB[8]) and surface-tethered methylviologen (MV2+). The binding affinity of the knottins with CB[8] and MV2+ surfaces was evaluated using surface plasmon resonance spectroscopy. Specific binding occurred, and the affinity increased with the valency of tryptophans on the knottin. Additionally, increased multilayer formation was observed, attributed to homoternary complex formation between tryptophan residues of different knottins and CB[8]. Thus, we were able to control the surface coverage of the knottins by valency and concentration. Cell experiments with mouse myoblast (C2C12) cells on the self-assembled knottin surfaces showed specific integrin recognition by the RGD-displaying knottins. Moreover, cells were observed to elongate more on the supramolecular knottin surfaces with a higher valency, and in addition, more pronounced focal adhesion formation was observed on the higher-valency knottin surfaces. We attribute this effect to the enhanced coverage and the enhanced affinity of the knottins in their interaction with the CB[8] surface. Collectively, these results are promising for the development of biomaterials including knottins via CB[8] ternary complexes for tunable interactions with cells.

Here we report on the covalent attachment of photoresponsive azobenzene moieties to cowpea chlorotic mottle virus (CCMV). The modified virus capsids can be reversibly immobilized on cucurbit[8]uril (CB[8]) bearing surfaces via supramolecular complexation.

Background Collagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders this stability is lost, either due to mutations in collagens or in the chaperones involved in collagen assembly. This leads to chronic wounds, skin fragility, and blistering. Existing approaches to treat such conditions rely on administration of small molecules to simulate collagen production, like 4-phenylbutyrate (4-PBA) or growth factors like TGF-β. However, these molecules are not specific for collagen synthesis, and result in unsolicited side effects. Hsp47 is a collagen-specific chaperone with a major role in collagen biosynthesis. Expression levels of Hsp47 correlate with collagen deposition. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) to skin cells, including specific collagen subtypes quantification. Results Here we quantify the collagen deposition level and the types of deposited collagens after Hsp47 stimulation in different in vitro cultures of cells from human skin tissue (fibroblasts NHDF, keratinocytes HaCat and endothelial cells HDMEC) and mouse fibroblasts (L929 and MEF). We find upregulated deposition of fibrillar collagen subtypes I, III and V after Hsp47 delivery. Network collagen IV deposition was enhanced in HaCat and HDMECs, while fibril-associated collagen XII was not affected by the increased intracellular Hsp47 levels. The deposition levels of fibrillar collagen were cell-dependent i.e. Hsp47-stimulated fibroblasts deposited significantly higher amount of fibrillar collagen than Hsp47-stimulated HaCat and HDMECs. Conclusions A 3-fold enhancement of collagen deposition was observed in fibroblasts upon repeated dosage of Hsp47 within the first 6 days of culture. Our results provide fundamental understanding towards the idea of using Hsp47 as therapeutic protein to treat collagen disorders.

OA hybrid A novel class of aggregation-induced emissive bis(phenylthio)phthalonitrile dyes were synthesized. These dyes assembled into nanoparticles that were equipped with mannose units. The nanoparticles underwent selective interactions with lectins and bacteria. The bright fluorescent aggregates aid in the visualization of the agglutination of bacteria.

Unambiguous, rapid, cost-effective and simple methodologies for the detection of microbial pathogens in environmental and medical samples are vital for effectively controlling infectious diseases. Recently, the daunting task of early identification of pathogens has been addressed by using gold nanoparticles coated with antibodies that have shown to increase the sensitivity of detection several fold. However this usually affects the specificity of detection since background responses due to non-specific interactions also get amplified. In this study we have exploited multivalent supramolecular interactions between antibody-coated gold nanoparticles to achieve super selective detection of pathogenic bacteria. We term these as supramolecular immuno-nanoparticles (SINs) and used them to develop a simple, rapid and cost-effective immuno dot-blot detection method to selectively detect pathogenic bacteria even at extremely low densities (LOD: 50 cells per spot). Furthermore, we performed analytical dot-blot and ELISA assays using the antibodies, both in their free form and as SINs, to gain fundamental insights into the reasons behind the remarkable detection characteristics. We found that the combined effects of steric hindrance, signal amplification and surface density of the SINs resulted in the best detection characteristics in an immuno dot-blot format reported so far. These fundamental insights can be widely applied to improve various multivalent nanoparticle based detection methods.

Biomolecular scaffolds were engineered by genetically fusing robust miniproteins in a sequence, like a chain. By fusing these miniprotein chains to a teal fluorescent protein (TFP), an efficient strategy was devised for their production in E. coli. Miniproteins that bind β-trypsin, VEGF and HIV-1 Nef proteins were used to make 4 individual chains, each with 3 miniproteins arranged in different orders. Their stability and binding properties were analyzed with native ESI-mass spectrometry, microscale thermophoresis, surface plasmon resonance imaging and a trypsin activity assay. Miniproteins within the chains were found to be functional and significantly robust. The trypsin activity assay showed that miniprotein chains containing two β-trypsin inhibitors clearly inhibit β-trypsin more than two fold stronger compared to chains containing only one β-trypsin inhibitor indicating that multivalent interactions occurs. SPR imaging results indicated that these constructs had the potential to simultaneously bind multiple target proteins. However, each miniprotein chain exhibited different binding affinities towards the target proteins, especially VEGF and HIV-1 Nef possibly due to issues dealing with folding, miniprotein orientations, non-specific adhesion and slow proteolytic degradation of the miniproteins. Analysis of these issues provided insights into parameters that need to be taken into consideration while designing such multi-specific protein constructs. By careful optimization, such constructs have the potential to be used in a very versatile manner for various molecular engineering applications.

Developing materials to encapsulate and deliver functional proteins inside the body is a challenging yet rewarding task for therapeutic purposes. High production costs, mostly associated with the purification process, short-term stability in vivo, and controlled and prolonged release are major hurdles for the clinical application of protein-based biopharmaceuticals. In an attempt to overcome these hurdles, herein, the possibility of incorporating bacteria as protein factories into a material and externally controlling protein release using optogenetics is demonstrated. By engineering bacteria to express and secrete a red fluorescent protein in response to low doses of blue light irradiation and embedding them in agarose hydrogels, living materials are fabricated capable of releasing proteins into the surrounding medium when exposed to light. These bacterial hydrogels allow spatially confined protein expression and dosed protein release over several weeks, regulated by the area and extent of light exposure. The possibility of incorporating such complex functions in a material using relatively simple material and genetic engineering strategies highlights the immense potential and versatility offered by living materials for protein-based biopharmaceutical delivery.

Hsp47 is a chaperone protein with a fundamental role in the folding, stability and intracellular transport of procollagen triple helices. A light-responsive Hsp47 recombinant protein, engineered to control in situ the production and assembly of cellular collagen is here demonstrated. This novel light-driven tool enables unprecedented fundamental studies of collagen biosynthesis and associated diseases.

A living hydrogel has been developed with metabolically and optogenetically engineered E. coli encapsulated within an agarose-based hydrogel matrix to produce and release deoxyviolacein in response to blue light irradiation. Localized, tunable and prolonged drug release have been demonstrated.

Hsp47 is a chaperone protein with a fundamental role in the folding, stability and intracellular transport of procollagen triple helices. A light-responsive Hsp47 recombinant protein, engineered to control in situ the production and assembly of cellular collagen is here demonstrated. This novel light-driven tool enables unprecedented fundamental studies of collagen biosynthesis and associated diseases.

Hsp47 is a chaperone protein with a fundamental role in the folding, stability and intracellular transport of procollagen triple helices. A light-responsive Hsp47 recombinant protein, engineered to control in situ the production and assembly of cellular collagen is here demonstrated. This novel light-driven tool enables unprecedented fundamental studies of collagen biosynthesis and associated diseases.

Living materials are a rapidly emerging material class, infused with the productive, adaptive and regenerative properties of living organisms. Property regulation in living materials requires external control of the activity of the living components, in order to achieve desired functions and performance. As a first step, a light-activatable E. coli-based system that can be externally triggered to interact with mammalian cells has been genetically engineered as an active component for developing optoregulated living-biomaterials. This has been achieved by combining optogenetic activation of gene expression using a photo-activatable inducer molecule and bacterial surface display technology to present an integrin-specific miniprotein on the outer membrane of an endotoxin-free E. coli strain. The bacteria are immobilized on surfaces and in situ light-activation of the E. coli results in mammalian cells specifically responding to them. Possible delivery of a fluorescent protein from the bacteria to the mammalian cells when they are interacting is also observed, indicating the potential of such a bacterial material to deliver complex cargo to cells in a targeted manner.

Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly synthesize a β-trypsin inhibitory knottin with supramolecular guest tags that enable it to adhere to self-assembled monolayers of the supramolecular host cucurbit[8]uril (CB[8]). We have described a strategy to easily express knottins in E. coli by conjugating them to a fluorescent protein after which they are cleaved and purified. Knottin constructs that varied in the number and position of the supramolecular tag at either the N- or C-termini or at both ends have been verified for their trypsin inhibitory function and CB[8]-binding properties in solution and on surfaces. All of the knottin constructs showed strong inhibition of trypsin with inhibition constants between 10 and 30 nM. Using microscale thermophoresis, we determined that the supramolecular guest tags on the knottins bind CB[8] with a Kd of ∼6 μM in solution. At the surface, strong divalent binding has been determined with a Kd of 0.75 μM in the case of the knottin with two supramolecular guest tags, whereas only weak monovalent binding occurred when only one guest tag was present. We also show successful supramolecular surface immobilization of the knottin using CB[8] and prove that they can be used to immobilize β-trypsin at the surface.

Biomimetic and stimuli-responsive cell-material interfaces are actively being developed to study and control various cell-dynamics phenomena. Since cells naturally reside in the highly dynamic and complex environment of the extracellular matrix, attempts are being made to replicate these conditions in synthetic biomaterials. Supramolecular chemistry, dealing with noncovalent interactions, has recently provided possibilities to incorporate such dynamicity and responsiveness in various types of architectures. Using a cucurbit[8]uril-based host guest system, we have successfully established a dynamic and electrochemically responsive interface for the display of the integrin-specific ligand, Arg-Gly-Asp (RGD), to promote cell adhesion. Due to the weak nature of the noncovalent forces by which the components at the interface are held together, we expected that cell adhesion would also be weaker in comparison to traditional interfaces where ligands are usually immobilized by covalent linkages. To assess the stability and limitations of our noncovalent interfaces, we performed single-cell force spectroscopy studies using fluid force microscopy. This technique enabled us to measure rupture forces of multiple cells that were allowed to adhere for several hours on individual substrates. We found that the rupture forces of cells adhered to both the noncovalent and covalent interfaces were nearly identical for up to several hours. We have analyzed and elucidated the reasons behind this result as a combination of factors including the weak rupture force between linear Arg-Gly-Asp and integrin, high surface density of the ligand, and increase in effective concentration of the supramolecular components under spread cells. These characteristics enable the construction of highly dynamic biointerfaces without compromising cell-adhesive properties., ACS Nano, 11 (4), ISSN:1936-0851, ISSN:1936-086X

On-demand and long-term delivery of drugs are common requirements in many therapeutic applications, not easy to be solved with available smart polymers for drug encapsulation. This work presents a fundamentally different concept to address such scenarios using a self-replenishing and optogenetically controlled living material. It consists of a hydrogel containing an active endotoxin-free Escherichia coli strain. The bacteria are metabolically and optogenetically engineered to secrete the antimicrobial and antitumoral drug deoxyviolacein in a light-regulated manner. The permeable hydrogel matrix sustains a viable and functional bacterial population and permits diffusion and delivery of the synthesized drug to the surrounding medium at quantities regulated by light dose. Using a focused light beam, the site for synthesis and delivery of the drug can be freely defined. The living material is shown to maintain considerable levels of drug production and release for at least 42 days. These results prove the potential and flexibility that living materials containing engineered bacteria can offer for advanced therapeutic applications.

For nearly over a decade, a wide variety of dynamic and responsive supramolecular architectures have been investigated and developed to address biological systems. Since the non-covalent interactions between individual molecular components in such architectures are similar to the interactions found in living systems, it was possible to integrate chemically-synthesized and naturally-occurring components to create platforms with interesting bioactive properties. Bacterial cells and recombinant proteins derived from them have been occasionally addressed and incorporated in such supramolecular biological systems, however a broader investigation into the various prospects of combining these two worlds has not yet been reported. This thesis represents an attempt to explore the possibilities of developing novel platforms by combining supramolecular chemistry with bacterial systems. The work described in this thesis has been organized in three sections – 1) using bacteria to produce recombinant proteins that can interact with supramolecular hosts and target proteins in a multivalent and multi-specific manner, 2) developing supramolecular platforms that display bioactive ligands in a photo-responsive manner or as gradients to address bacteria as pathogens and 3) genetically engineering bacterial cells with a supramolecular binding motif in an attempt to incorporate them as living entities in supramolecular architectures. These studies provide a glimpse into some of the versatile ways in which supramolecular chemistry and bacterial systems can be combined. The components and concepts investigated in these chapters can be used interchangeably and also extended towards other fields to develop innumerable other systems. Some of these possibilities have been addressed in the epilogue where preliminary results from three projects involving mammalian cells, viral protein cages and aggregation-induced emission compounds have been briefly described. The general outlook gathered from these explorations indicates that a ripe future lies ahead for such supramolecular bacterial systems where the possibilities are limited only by imagination.

Supramolecular assemblies, formed through noncovalent interactions, has become particularly attractive to develop dynamic and responsive architectures to address living systems at the nanoscale. Cucurbit[8]uril (CB[8]), a pumpkin shaped macrocylic host molecule, has been successfully used to construct various self-assembled architectures for biomedical applications since it can simultaneously bind two aromatic guest molecules within its cavity. Such architectures can also be designed to respond to external stimuli. Integrating living organisms as an active component into such supramolecular architectures would add a new dimension to the capabilities of such systems. To achieve this, we have incorporated supramolecular functionality at the bacterial surface by genetically modifying a transmembrane protein to display a CB[8]-binding motif as part of a cystine-stabilized miniprotein. We were able to confirm that this supramolecular motif on the bacterial surface specifically binds CB[8] and forms multiple intercellular ternary complexes leading to aggregation of the bacterial solution. We performed various aggregation experiments to understand how CB[8] interacts with this bacterial strain and also demonstrate that it can be chemically reversed using a competitor. To confirm that this strain can be incorporated with a CB[8] based architecture, we show that the bacterial cells were able to adhere to CB[8] self-assembled monolayers (SAMs) on gold and still retain considerable motility for several hours, indicating that the system can potentially be used to develop supramolecular bacterial biomotors. The bacterial strain also has the potential to be combined with other CB[8] based architectures like nanoparticles, vesicles and hydrogels.

Studying binding interactions involving living cells requires a platform that carefully mimics the physiological parameters that govern these phenomena. Very often the amount of ligands that receptors can bind affect overall binding strength as is the case in cell adhesion. In addition, the physical environment can strongly influence these processes. This is exemplified by the effect of shear stress in catch-bond-mediated binding of bacteria. Traditional analysis techniques do not allow to probe these factors simultaneously. To this end, continuous ligand gradients in locked-in supported lipid bilayers (SLBs) are prepared in a microfluidic device to control fluid flow. This platform allows for one-pot characterization of cell surface binding events and 1) the effect of ligand density and 2) shear stress, simultaneously. The model interaction between the FimH receptor found on Escherichia coli and mannose found on the mammalian cell membrane is used to evaluate the platform. Using a single chip, specific E. coli ORN 178 adhesion (K d of 0.9 × 10-21 m), detachment and displacement are shown to depend on the mannose-density and shear stress. For the first time, these effects are studied in a single chip device with high quality. This chip provides entry to further our understanding of other cell-cell interactions.