Engineering of bacterial synthetic multicellular systems and materials hold promise for many health- relevant applications such as modular drug biosynthesis, living diagnostic devices, and synthetic biofilm research models. To date, bacterial synthetic biology has largely focused on the scales of molecules and single cells. Equivalent work on bacterial synthetic consortia is much less advanced, in significant part due to the previous lack of suitable synthetic and genetically encoded cell-cell adhesion tools to control the assembly, development, and functionality of multicellular systems. We recently developed the first such synthetic cell-cell adhesion toolbox, as well as tools for optogenetically controlling cell-surface deposition and patterning. The specific objectives of this research are to significantly advance these synthetic cell-adhesion tools, and to develop design principles and predictive modeling tools that enable consortia engineering and patterning that integrate all relevant length scales (i.e., molecular, cellular, and multicellular), and ultimately pave the way for medially relevant applications. Our main hypothesis is that we can significantly advance our control over the strength, specificity, and subcellular localization of synthetic adhesion proteins in Escherichia coli, which will allow rational tuning of consortium-level biophysical properties such as porosity and viscoelasticity, and which will ultimately enable versatile multicellular consortium engineering and patterning. This work will constitute a foundation for various biomedical applications such as biocompatible materials, multicellular plug-and-play pathway engineering, targeted in-vivo drug delivery, and living diagnostic devices. Our interdisciplinary methodology combines synthetic biology, biophysics, instrumentation and modeling. All experiments will be done in a quantitative manner. The proposed investigations include three independent yet synergistic Specific Aims motivated by our hypothesis: (Aim 1) Advance the functionality of the synthetic adhesin toolkit at the subcellular level; (Aim 2) Achieve engineering control over synthetic consortium properties such as viscoelasticity and porosity at the scale of 10-100 µm; and (Aim 3) Achieve higher-level consortium patterning on the scale of centimeters and demonstrate potential for medical applications. The PI (Prof. Riedel-Kruse) and his team are well-suited for this project as we have significant expertise in synthetic biology, biophysics, instrumentation (e.g., microfluidics, imaging), and modeling genetic circuits and biophysical systems across scales. We developed the first synthetic cell-cell and optogenetic cell-surface adhesion toolboxes in bacteria. Multiple collaborators provide additional domain expertise in key areas. Overall, this project's innovation lies in establishing synthetic adhesins as an essential and integral component of the synthetic circuit-engineering toolbox and in establishing a novel para...