Controlling the Properties of Macroscopic Living Materials by Genetically Altering Their Matrix

Living cells assemble into hierarchical structures, forming complex materials with remarkable properties. Inspired by nature, the emerging field of Engineered Living Materials (ELMs) aims to predictively design cells to grow into materials with tailored properties. Contributing to this goal, our lab has recently reported the first <b>B</b>ottom-<b>U</b>p <b><i>D</i></b><i>e novo</i> <b>E</b>ngineered <b>L</b>iving <b>M</b>aterials (<b>BUD-ELMs</b>) using the bacterium <i>Caulobacter crescentus. </i>BUD-ELMs are comprised of a secreted protein matrix consisting of surface-layer protein and an elastin-like polypeptide (<b>ELP</b>) domain that assembles cells into centimeter-scale materials and defines their mechanical and physical characteristics. However, a fundamental knowledge gap we have yet to address is understanding how genetic modifications affect bulk material characteristics. Thus, our goal is to elucidate sequence-structure-property relationships of BUD-ELMs to achieve predictive design for targeted mechanical properties. We hypothesized that the mechanical properties of the purified ELPs, which are known to be tunable as a factor of sequence length, would be mimicked in BUD-ELMs. Therefore, to test this hypothesis, we engineered strains expressing ELPs of different lengths, grew the strains to form BUD-ELMs, and characterized each material’s viscoelastic behavior by rheology measurements. Our frequency sweep results show that shortening the ELP increases the storage and loss moduli, showing stiffer resulting material, while lengthening the ELP does not affect the moduli. Interestingly, flow sweeps show strong shear-thinning under low shear rates, implying a fragile microstructure network among all the resulting materials from each strain. Additionally, to elucidate the relationship between each material’s mechanical properties and microstructure, we employed confocal microscopy. The results show clear differences in each variant’s matrix structure. The shorter ELP variant produces spiderweb-like strands over the cells, while the longer ELP sequence forms thinner fiber-like strands between cells. Based on this data, we attribute the changes in this material’s mechanical properties to the interconnected network formed by the matrix of the shorter ELP. In comparison, the thin fiber-like strand formation formed by the longer ELP does not impact its rheological characteristics. Furthermore, the different structures produced by each material result in distinct yield stresses. Thus, this exploration into the genetic tunability of BUD-ELMs leads us toward establishing sequence-structure-property relationships for ELMs with programmable mechanical and structural properties for applications such as 3D printing and tissue engineering.