Contribution of BioBricks

As soon as our team was formed, we started working on different project ideas. Ultimately, we decided on “ReFiBa – Enzyme based Recycling of Textile Fibers using Bacillus subtilis”. Besides literature research, we looked at the work of previous iGEM teams which addressed the textile waste issue. For example, Chalmers-Gothenburg 2020 aimed to produce an enzyme cocktail by Escherichia coli for elastane degradation, Edinburgh 2021 immobilized cellulases on silica beads and Greatbay SCIE 2022 tested cell surface display systems for textile degradation using PETases and cellulases.

Inspired by their work as well as various expert talks (see Human Practices), we divided our project into two main strategies: 1) Induced expression as a reference approach, in which we aimed to find active cellulases and PETases for working with B. subtilis as expression host, and 2) Spore surface display as final strategy, in which chosen enzymes are immobilized on the surface of B. subtilis spores.
The latter approach is not new to iGEM, as we got inspired by the iGEM Team LMU Munich 2012, who introduced protein display on Bacillus spores to the iGEM competition. Based on their work, the project “Sporobeads” was continued in the research group General Microbiology of Prof. Thorsten Mascher in Dresden. Having benefited from the local expertise of his team as well as one of our team members, who already worked on this topic before, we planned the immobilization strategy, i.e. fusing enzyme candidates to an anchor protein of the spore crust (CotY).

By testing different enzyme candidates, we aim to make a useful contribution to future iGEM teams. First, we started with induced expression as a reference approach and constructed translational units containing the ribosome binding site for B. subtilis (BsRBS) and the gene of each enzyme candidate (e.g. BsEglS), listed in the following table of composite parts (Tab. 1). As these composite parts were ordered by gene synthesis (IDT), associated basic parts were designed, but not constructed in the laboratory (Tab. 2). Only the composite parts were tested and characterized (see respective Parts Registry pages).
After performing activity assays, promising enzymes were chosen for spore surface display. This approach included design and amplification of basic parts like the promoter P_cotYZ, the anchor gene cotY, the terminator B0014 as well as each gene of interest (Tab. 2). Three different linkers (L1-L3) were tested for fusing the protein of interest with the anchor protein. Ultimately, these basic parts were assembled to transcriptional units shown in the table of composite parts (Tab. 1) and characterized by activity assays (see respective Parts Registry pages).