Regulating Bacterial Behavior within Hydrogels of Tunable Viscoelasticity

Abstract

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.

Encapsulation of bacteria in bilayer Pluronic thin film hydrogels: a safe format for engineered living materials

“…Inducible protein production in our thin films was tested using an optogenetically engineered E. coli strain that expresses a red fluorescent protein (RFP) in response to blue light. [5][6] With pulsed light irradiation (470 nm, 500 ms ON, 10 min OFF), fluorescence was detectable in the inner film within 4 hours, although it was not homogeneous through the film. Higher fluorescence intensity was observed at the outer edge of the inner layer ( Figure 3a ).…”

“…The growth and the metabolic activity of bacteria embedded in hydrogels depends on the composition and physical properties of the hydrogel matrix. [3][5] In physically crosslinked hydrogels, like agarose or alginate, bacteria grow slower than in free suspension and eventually grow out of the hydrogel (i.e. into the surrounding medium) if there are enough nutrients available.…”

“…The variation of the diacrylate content in the mixture lead to hydrogels with different mechanical properties in terms of elastic moduli ( Table S1 ) and viscoelasticity as consequence of the introduction of covalent crosslinks into the physical network. [5] E. coli encapsulated in these hydrogels proliferated and kept their metabolic function to an extent which was dependent on the concentration of acrylate groups, i.e. the degree of covalent crosslinks in the hydrogel.…”

“…In DA X hydrogels with X<50, bacterial growth was found to be significantly less restricted and controlled. [5] Interestingly, highest rates of inducible protein production were observed in the gels with intermediate degrees of chemical cross-linking (DA X, 25< X <75), indicating that encapsulation influences both growth and metabolic activity of the bacteria in potentially different manner.…”

“…[7] Previous studies have demonstrated that Pluronic-based hydrogels are good matrices to encapsulate bacteria. [3][5][8][9] 30 wt% mixtures of Pluronic F127 and Pluronic F127 diacrylate at different concentrations (named DA X , where X represents the fraction of diacrylated Pluronic in the mixture, formed stable hydrogels after covalent crosslinking by photoinitiated radical polymerization of the acrylate groups. The variation of the diacrylate content in the mixture lead to hydrogels with different mechanical properties in terms of elastic moduli ( Table S1 ) and viscoelasticity as consequence of the introduction of covalent crosslinks into the physical network.…”