Overview

Our project is built around three primary parts: BaCBM2, Barbie1, and Spidroin Nt2RepCt. These proteins play a crucial role in our Design-Build-Test-Learn (DBTL) cycles. As the project advanced and our goals shifted, we developed variants of these proteins to serve different functions in line with the evolving objectives and strategies. These variants were developed to facilitate the protein purification, characterization, and integration in our biosensor.

Key components across our protein engineering cycles: The first cycle employed the plastic-binding proteins BaCBM2 (yellow) and BARBIE1 (pink), combined with three tandem repeats of the magnetite-binding peptide Mad10 (silver) and red fluorescent protein (RFP, red). The second cycle introduced the chimeric spidroin protein Nt2RepCT (blue) alongside the SpyCatcher/SpyTag system for modular protein assembly. In the third cycle, we developed constructs fusing the spidroin Nt domain (dark blue) to a flexible linker and plastic-binding proteins (white), including BARBIE1, BaCBM2, or newly engineered variants.

BaCBM2 and Variations

BaCBM2 is a protein derived from Bacillus anthracis and is part of a larger family of Carbohydrate-Binding Modules, specifically from family 2 or CBM2. These modules are essential for efficient polysaccharide degradation by carbohydrate-active enzymes. The CBM2 proteins can enhance enzyme targeting and localization to specific substrates, increasing catalytic efficiency by bringing the catalytic domains closer to the polysaccharide surface and allowing microorganisms to convert the substrates into energy sources. Specifically, BaCBM2 is demonstrated to bind to specific types of plastics¹ that share, to some extent, structural similarities with polysaccharides.

Barbie1 and Variations

Barbie1 is a synthetic protein designed by our team. This protein has an increased affinity to bind to plastics compared to BaCBM2. To learn more about Barbie1, check our model page.

Spidroin Nt2RepCt and Variations

Spidroins are the main proteins from spider silk. These proteins have interesting mechanical properties, including strength, elasticity, and biodegradability. These proteins are synthesized in specialized glands within spiders and serve various functions, such as web construction, prey capture, and facilitating mobility. The Nt2RepCt spidroin is a synthetic mini-spidroin composed of an N-terminal domain (NT), a short repeat region, and a C-terminal domain (CT).

References

¹ Rennison, A. P., Westh, P. & Møller, M. S. Protein-plastic interactions: The driving forces behind the high affinity of a carbohydrate-binding module for polyethylene terephthalate. Science of the Total Environment 870, (2023).