Design of temperature-responsive biofilms for the precise and reversible control of engineered living biomaterials
Cell Biology
Final Report Abstract
Bacterial biofilms hold great promise as innovative materials that could serve as biological replacements for chemical hydrogels and petrochemical plastics. However, to realize the full potential of these living materials, tuning and dynamic regulation of their physical properties will be required. The goal of this one-year project was to develop thermo-responsive biofilms based on the curli system in Escherichia coli for applications as living materials, thereby also advancing the field of thermally regulated proteins. Curli biofilms form upon extracellular oligomerization of a single protein: CsgA. By modulating its ability to oligomerize via mutagenesis or by fusing it to thermosensitive protein domains, we aimed to create CsgA variants capable of responding dynamically to temperature changes. After successfully establishing the required assays to study and characterize curli biofilms, we observed rather unexpected growth defects upon induction of the system, as well as the strong interdependence of two key components, which contradicted previous reports and precluded our initial screening strategy. Considering these findings, we decoupled the engineering of thermally responsive protein modules from the optimization of the biofilm expression system. Specifically, we screened various modifications of the curli operon to reduce the burden on cell growth and optimized the expression levels of individual protein components. These studies have provided valuable insights into the dynamics of curli production, while also successfully improving the performance of the overall system as intended. As a result, we are in the process of acquiring data for a manuscript on optimized refactored curli operons, which will be highly beneficial for any future endeavours on curli engineering by us and others. In parallel, toward the second aspect of the project, we screened and optimized temperature-responsive protein domains in E. coli. Our approach resulted in powerful protein modules based on the TlpA repressor and a LOV photoreceptor domain that exhibit a sharp response to temperature changes, including different transition temperatures. The careful characterization of these variants, as well as their use for conditional induction of curli expression will lead to a second publication on the general engineering of thermo-responsive proteins in bacteria, which we aim to publish early next year. Taken together, our work has led to exciting new insights into inducible curli systems and could greatly improve temperature-dependent regulation of proteins. Despite being only a one-year project, it will result in two publications on key aspects of the topic within the near future. Furthermore, our results on thermo-sensitive proteins will have implications far beyond the engineering of curli biofilms and will be highly beneficial for the design of thermoresponsive protein in general.
