RESUMEN
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.
Asunto(s)
Hidrogeles , Poloxámero , Hidrogeles/farmacología , Poloxámero/química , Polímeros , BacteriasRESUMEN
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 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.
Asunto(s)
Bacterias , Hidrogeles , Animales , MamíferosRESUMEN
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.
Asunto(s)
Bioingeniería/métodos , Optogenética/métodos , Proteínas Recombinantes/metabolismo , Células Inmovilizadas , Escherichia coli , Hidrogeles/química , Proteínas Recombinantes/genética , Sefarosa/químicaRESUMEN
A facile modular approach to rapidly prepare pH-responsive hydrogels by crosslinking polysaccharides with polyamines is demonstrated. Hydrogels are prepared by first reacting the less reactive polysaccharides with the cross-linker epichlorohydrin and completed by the addition of polyamines. The crosslinking of polysaccharides with polyamines provides a facile method for incorporating functionality into polysaccharide based hydrogels. This process is demonstrated with the polysaccharides dextran, pullulan and carboxymethyl cellulose and with the polyamines polyallylamine and polyethylene imine. The hydrogels were characterized by FTIR and swelling studies, which showed pH-dependent swelling due to the presence of the polyamine. The hydrogels can also be tailored by varying the mass ratio between the polysaccharide and polyamine. Absorption studies of organic analytes showed the polyamine content affecting the uptake of a charged substrate (methylene blue) and no effect on a neutral substrate (6-methyl coumarin). This synthetic method was also used to prepare hydrogels with antibacterial activity against E. coli and S. aureus by utilizing an amphiphilic polyallylamine.