During the design process of this project, we made several key decisions regarding several issues. Here we will explain why we made such choices.
Physical methods of removing bacteria, such as brushes and scalpels, are difficult to completely eliminate microorganisms and are prone to damage the mural itself. Various chemical disinfectants, including quaternary ammonium salts, have been used to inhibit and remove microorganisms on mural surfaces. However, these chemical agents have certain ecological toxicity and are prone to failure due to the emergence of resistant populations. Moreover, chemical disinfectants are usually sprayed on mural surfaces in solution form, which is not suitable for the management of mural surface moisture.
Antimicrobial peptides can disrupt the cell membrane structure of microorganisms or cause cell death through non-cell membrane targeting. This mechanism makes antimicrobial peptides less likely to develop resistant populations after long-term application. Antimicrobial peptides have been widely used as antibacterial substances in fields such as food, which gives us confidence in applying them to our project.
We designed an enzyme system based on laccase and peroxidase to degrade melanin and carotenoids. Microorganisms produce various pigment compounds during their growth, which can help them tolerate adverse conditions such as radiation and dehydration. Moreover, these pigment compounds can interact with mural components and alter the material properties. Among these pigment compounds, melanin and carotenoids are the most common.
In our design, the chassis organism needs to produce multiple enzymes and peptides, which may impose an excessive metabolic burden. The normal environment on the mural surface is neutral or slightly alkaline, while the growth and metabolic processes of microorganisms sometimes make the microenvironment weakly acidic. Based on this, we designed a pH-inducible switch in our project, so that our engineered bacteria can automatically identify areas where microorganisms are actively growing and choose to prioritize the synthesis of antimicrobial peptides or enzymes to degrade organic pigments.
As a project that needs to be applied outside the laboratory, we need to design a method to prevent escape. Our chassis microorganisms will be carried on bacterial cellulose membrane carriers. We use arabinose as a reverse trigger condition, allowing the microorganisms to distinguish between the bacterial cellulose membrane (absorbed arabinose solution) and the mural surface. Once leaked onto the mural surface, they will start to synthesize the mazF protein, inhibiting cell survival.