An understanding of how microorganisms react and respond to chemical and physical cues within a 3D microenvironment is severely lacking. Conversely, in mammalian systems (e.g., stems cells), the impact of the spatial chemical and physical environment on cell phenotype is a widely studied field. Establishing these key parameters that control the behaviours of microbes in 3D would enable engineered living material (ELM) designers to effectively modulate the emergent responsive, adaptative, and self-healing properties of ELMs. The viability of microorganisms is often characterised within 3D hydrogels, and confinement has begun to be explored as a method for control of microcolony growth. In this project, we will explore both chemical cues through the addition of various bioactive molecules (branched polysaccharides, quorum sensing molecules), as well as mechanical cues through alteration of the pore structure and viscoelastic properties of a hydrogel matrix, to ascertain design principles for the control of microorganism growth in 3D.

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Microorganism growth control in 3D. a) Microscope image of P. pastoris within a 3D printed alginate bioink immediately after crosslinking. b) Image of a P. pastoris laden hydrogel after one day of culture showing microcolony formation. c, d) Fluorescence microscopy of a live (green)/dead (red) stained 3D printed microbial construct, immediately after crosslinking (c), and after three days of culture (d). scale bars 500 µm.