Advancing New Living Materials by Understanding Assembly Principles

Like natural materials such as wood and bone, engineered living materials (ELMs) require specific conditions to facilitate proper assembly. By elucidating these assembly parameters in relation to the mechanism of material formation, we become able to better understand a material’s potential application space. Our research group has recently developed the first example of a <i>macroscopic</i> <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>) in the bacterium <i>Caulobacter crescentus</i>¹.<i> </i>To understand and optimize the conditions that promote proper material assembly, we grew BUD-ELM cultures under various shaking conditions. We have found that this material not only requires shaking to assemble from planktonic culture, but also that the assembly process is sensitive to specific shaking conditions. To describe these conditions under a single parameter, we developed the modified volumetric power input (P_v,a), which takes into account both the sheer forces experienced by the culture and the size of the air-water interface onto which the material forms. By analyzing the resulting size of material samples grown under different P_v,a values, we were able to determine an optimal P_v,a range for maximum material formation. Additionally, we used this model to predict the optimal shaking conditions for larger flasks sizes, demonstrating the potential to scale up cultures for increased material production. By interpreting this data in conjunction with Western blot analysis and atomic force microscopy, we propose that during BUD-ELM assembly, <i>Caulobacter </i>cells adhere to a secreted BUD-protein matrix that forms at the air-water interface. Based on this mechanism, we hypothesized that this sequential assembly process would allow for integration of non-engineered bacterial species into the material’s structure. To explore this concept, we developed a co-culturing approach for BUD-ELM-forming <i>C. crescentus</i> and the model cyanobacterium <i>Synechocystis </i>sp. PCC 6803. We observed the stochastic incorporation of live <i>Synechocystis </i>cells within the material by fluorescence microscopy. This result not only provides further support to our proposed assembly mechanism, but also enables a novel platform for photosynthetic, co-cultured ELMs. By further engineering <i>Syenchocystis </i>cells, we hope to expand this platform to provide a novel class of ELMs with reduced nutrient requirements, increased lifespan, and user-defined functionality.