M.Sc. Miguel Baños Maestro

Engineered living materials (ELMs) promise genetically programmable functions by coupling biological regulation to synthetic material responses. Here, we introduce a strategy for genetically driven bidirectional shape-morphing in a peptide-crosslinked polyethylene glycol (PEG) hydrogel whose network density is modulated by opposing genetically encoded enzyme pairs that induce crosslinking or hydrolysis. These molecular transformations switch the hydrogel between deswelling, swelling, or partial disintegration, producing two- to five-fold changes in mechanical properties. By fabricating a bilayer hydrogel composed of a responsive layer and a passive counterlayer, these network-level modulations are translated into directional actuation with bending angles exceeding 80° and shape recovery. We further show that genetically engineered bacteria and mammalian cells can function as programmable enzyme sources, thereby coupling genetic programs to hydrogel network remodeling and material deformation. Using opposing out-of-equilibrium biochemical reactions with dynamically changing relative reaction rates, we demonstrate hybrid-enzymatic genetic control over bending and autonomous shape recovery in bilayer hydrogels. This work establishes a customizable framework for genetically directed mechanical actuation in ELMs, in which living cells regulate macroscopic shapes through programmed network remodeling. More broadly, it provides proof-of-concept for genetically programmed shape-morphing of ELMs and opens opportunities for future biohybrid actuators, adaptive material systems, and dynamic biomedical interfaces.

Molecular optogenetics allows the control of molecular signaling pathways in response to light. This enables the analysis of the kinetics of signal activation and propagation in a spatially and temporally resolved manner. A key strategy for such control is the light-inducible clustering of signaling molecules, which leads to their activation and subsequent downstream signaling. In this work, an optogenetic approach is developed for inducing graded clustering of different proteins that are fused to eGFP, a widely used protein tag. To this aim, an eGFP-specific nanobody is fused to Cryptochrome 2 variants engineered for different orders of cluster formation. This is exemplified by clustering eGFP-IKKα and eGFP-IKKβ, thereby achieving potent and reversible activation of NF-κB signaling. It is demonstrated that this approach can activate downstream signaling via the endogenous NF-κB pathway and is thereby capable of activating both an NF-κB-responsive reporter construct as well as endogenous NF-κB-responsive target genes as analyzed by RNA sequencing. The generic design of this system is likely transferable to other signaling pathways to analyze the kinetics of signal activation and propagation.