SporoPrinting – 3D matrices for immobilizing and controlling functionalized SporoBeads of B. subtilis as a structured protein displaying ELM platform with adaptive functions

• Applicants: Professor Dr. Michael Gelinsky; Professor Dr. Thorsten Mascher
• Subject Area: Microbial Ecology and Applied Microbiology ; Biomaterials ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 541298315

Genetically programmable engineered living materials (ELM) is a field of high potential that gained momentum with the advent of the Synthetic Biology era. Endospores of Bacillus subtilis hold great potential for ELM, since they provide a second layer to implement engineered functionalities that can be genetically programmed. Upon starvation, B. subtilis forms highly resistant endospores that protect the DNA by the cell wall cortex and three different protein layers. By translationally fusing a gene-of-interest to a gene encoding a suitable anchor protein, the target proteins can be immobilized. The resulting SporoBeads remain stable for long time, but can germinate within hours upon induction.In the context of this PP, we will combine the SporoBead concept with bioprinting to integrate functionalized endospores in matrices containing inducing cues for then performing adaptive functions. We will develop protocols and polymeric and composite materials that allow controlling both spore formation and germination. We will then combine the activity displayed on the spore surface with a second functionality, which is expressed after germination, thereby generating two adaptive functions that can be switched, by either inducing the SporoBead state or activating the engineered vegetative cells. Different bioprinting technologies (extrusion, core-shell and drop-on-demand) will enable us to position these spores in multiple arrangements to allow meaningful readouts.Both partners will develop their technologies to establish a versatile SporoPrinting platform, to develop three different demonstrators: (i) an ELM that switches fluorescence from green to red shall serve as the feasibility study. It will be based on a dually fluorescently labelled strain that displays one fluorophore on the spore surface, while producing a second, compatible fluorophore in vegetative cells. This strain thereby allows monitoring the switch from spore to vegetative cell by the change in fluorescence. Bioinks of suitable composition and different bioprinting strategies and patterns based on material design and construct morphology will be explored to control the transition between the different developmental stages. Once established, we will then (ii) expand the SporoPrinting platform by introducing the streptomycetes, to develop protocols that allow growing two different groups of microorganisms in a highly defined spatiotemporal arrangement. We will develop an antibiotic-responsive biosensor set-up that allows screening potential streptomycete producers for particular types of antibiotics, based on the specific induction of B. subtilis biosensors. Ultimately (iii) an ELM with a programmable dark-and-light pattern based on a melanin-producing activity displayed on SporoBeads, in combination with a melanin-decolorizing activity expressed and secreted from vegetative cells will be developed.

• DFG Programme: Priority Programmes
• Subproject of: SPP 2451: Engineered Living Materials with Adaptive Functions
• Co-Investigator: Dr. Anja Lode