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- ItemToward light‐regulated living biomaterials(Hoboken, NJ : Wiley, 2018) Sankaran, Shrikrishnan; Zhao, Shifang; Muth, Christina; Paez, Julieta; Del Campo, AránzazuLiving materials are an emergent material class, infused with the productive,adaptive, and regenerative properties of living organisms. Property regulation in living materials requires encoding responsive units in the living components to allow external manipulation of their function. Here, an optoregulated Escherichia coli (E. coli)-based living biomaterial that can be externally addressed using light to interact with mammalian cells is demonstrated. This is achieved by using a photoactivatable inducer of gene expression and bacterial surface display technology to present an integrin-specific miniprotein on the outer membrane of an endotoxin-free E. coli strain. Hydrogel surfaces functionalized with the bacteria can expose cell adhesive molecules upon in situ light-activation, and trigger cell adhesion. Surface immobilized bacteria are able to deliver a fluorescent protein to the mammalian cells with which they are interacting, indicating the potential of such a bacterial material to deliver molecules to cells in a targeted manner.
- ItemEncapsulation of bacteria in bilayer Pluronic thin film hydrogels: A safe format for engineered living materials(Amsterdam : Elsevier, 2023) Bhusari, Shardul; Kim, Juhyun; Polizzi, Karen; Sankaran, Shrikrishnan; del Campo, AránzazuIn 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.
- ItemDiscovery of a high-performance phage-derived promoter/repressor system for probiotic lactobacillus engineering(Cold Spring Harbor : Cold Spring Harbor Laboratory, NY, 2023) Blanch-Asensio, Marc; Tadimarri, Varun Sai; Wilk, Alina; Sankaran, ShrikrishnanBackground: The Lactobacillus family comprises many species of great importance for the food and healthcare industries, with numerous strains identified as beneficial for humans and used as probiotics. Hence, there is a growing interest in engineering these probiotic bacteria as live biotherapeutics for animals and humans. However, the genetic parts needed to regulate gene expression in these bacteria remain limited compared to model bacteria like E. coli or B. subtilis. To address this deficit, in this study, we selected and tested several bacteriophage-derived genetic parts with the potential to regulate transcription in lactobacilli. Results: We screened genetic parts from 6 different lactobacilli-infecting phages and identified one promoter/repressor system with unprecedented functionality in L. plantarum WCFS1. The phage-derived promoter was found to achieve expression levels nearly 9-fold higher than the previously reported strongest promoter in this strain and the repressor was able to almost completely repress this expression by reducing it nearly 500-fold. Conclusions: The new parts and insights gained from their engineering will enhance the genetic programmability of lactobacilli for healthcare and industrial applications. Competing Interest Statement: A patent application has been filed based on the results of this work (Application no. is DE 102022 119024.2).
- ItemRegulating Bacterial Behavior within Hydrogels of Tunable Viscoelasticity(Weinheim : Wiley-VCH, 2022) Bhusari, Shardul; Sankaran, Shrikrishnan; del Campo, AránzazuEngineered 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 can regulate the behavior of the embedded cells is crucial to design and predict ELM's function, 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 elastic response to deformation of the matrix, a decrease in colony volumes and an increase in their sphericity are observed. Protein production follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that matrix design can be used to control and regulate the composition and function of ELMs containing microorganisms. Interestingly, design parameters for matrices to regulate bacteria behavior show similarities to those elucidated for 3D culture of mammalian cells.
- ItemPhotoactivatable Hsp47: A tool to control and regulate collagen secretion & assembly(Hoboken, NJ : Wiley, 2018) Khan, Essak; Sankaran, Shrikrishnan; Paez, Julieta; Muth, Christina; Han, Mitchell; Del Campo, AránzazuCollagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen-specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non-natural light-responsive tyrosine (o-nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor-mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.
- ItemNovel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli(Oxford : Wiley-Blackwell, 2023) Dey, Sourik; Blanch‐Asensio, Marc; Balaji Kuttae, Sanjana; Sankaran, ShrikrishnanLactobacilli 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 in Lactiplantibacillus plantarum that 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, which 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 tuneable 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.
- ItemExpanding the genetic programmability of Lactiplantibacillus plantarum(Oxford : Wiley-Blackwell, 2024) Blanch‐Asensio, Marc; Dey, Sourik; Tadimarri, Varun Sai; Sankaran, ShrikrishnanLactobacilli are ubiquitous in nature and symbiotically provide health benefits for countless organisms including humans, animals and plants. They are vital for the fermented food industry and are being extensively explored for healthcare applications. For all these reasons, there is considerable interest in enhancing and controlling their capabilities through the engineering of genetic modules and circuits. One of the most robust and reliable microbial chassis for these synthetic biology applications is the widely used Lactiplantibacillus plantarum species. However, the genetic toolkit needed to advance its applicability remains poorly equipped. This mini-review highlights the genetic parts that have been discovered to achieve food-grade recombinant protein production and speculates on lessons learned from these studies for L. plantarum engineering. Furthermore, strategies to identify, create and optimize genetic parts for real-time regulation of gene expression and enhancement of biosafety are also suggested.
- ItemCell adhesion on RGD-displaying knottins with varying numbers of tryptophan amino acids to tune the affinity for assembly on cucurbit[8]uril surfaces(Washington D.C. : American Chemical Society, 2017) Sankaran, Shrikrishnan; Cavatorta, Emanuela; Huskens, Jurriaan; Jonkheijm, PascalCell 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.
- ItemIn vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1(San Francisco, California, US : PLOS, 2023) Blanch-Asensio, Marc; Dey, Sourik; Sankaran, ShrikrishnanLactobacilli 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 to their complex and thick cell walls limiting 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 an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The 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.
- ItemCell Adhesion on Dynamic Supramolecular Surfaces Probed by Fluid Force Microscopy-Based Single-Cell Force Spectroscopy(Washington D.C. : American Chemical Society, 2017) Sankaran, Shrikrishnan; Jaatinen, Leena; Brinkmann, Jenny; Zambelli, Tomaso; Vörös, Janos; Jonkheijm, PascalBiomimetic 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.