Description

Overview

As one of the oldest forms of human art, ancient murals are widely present in various regions around the world. These ancient murals often contain important information about ancient cultures and history, and are of great value for studying ancient art, religion, architecture, and scientific achievements.

However, with changes in the environment and the impact of human activities, these cultural treasures with hundreds or even thousands of years of history are facing unprecedented challenges. The warming and humidification of the climate allows microorganisms to grow on the mural relics in regions that were originally relatively dry, causing the relics to lose their original value.

The physical and chemical reactions caused by the growth of microorganisms, especially filamentous bacteria, in the base layer will accelerate the separation of the matrix; the bacteria and fungi growing on the murals will consume the organic adhesives such as animal glue in the mural materials, reducing the cohesion of the pigment layer and causing it to flake off and be lost; the organic acids produced by the metabolism of fungi will dissolve and chelate the metal cations in the mineral pigments, thereby forming various secondary minerals, causing cracking, powdering, and flaking on the surface of the murals.

The vigorous metabolic reactions of microorganisms will affect the mineral pigments in the environment, causing changes in their colors, such as lead white forming black lead dioxide; to resist adverse conditions in the environment, microorganisms will produce various pigment compounds represented by melanin and carotenoids during their growth, which not only cause the murals to change color but also interact with the mural components, leading to the deterioration of the material of the artifact[1].

The current microbial control technologies for mural relics include physical methods such as scalpels and vacuum cleaners, and chemical disinfectants such as quaternary ammonium salts. However, the former are prone to direct damage to the murals, and the latter have ecological toxicity and are prone to generating new resistant populations[1]. Different from traditional methods, the biological method provides a safer, more economical, and adaptable method for the restoration of murals.

Inspiration

Considering the importance of microbial control for mural relics, we decided to explore new solutions in this year's iGEM project. During the discussion, an article on mural restoration using the overall utilization of microorganisms prompted us to consider using the entire microbial cells to achieve our expected goal. The article proposed using Serratia marcescens, a bacterium with multiple extracellular enzyme activities, to produce a new type of agar-sponge biological gel that can effectively remove various organic stains on the murals[2].

This article demonstrates a successful application of biological cleaning technology in the field of mural protection. Of course, there are still some areas that we can explore: The existence of microorganisms themselves and the compounds secreted by them is mutually reinforcing. In the microbial control process, efforts should be made to address both issues; the diverse enzyme activities may be equally effective against stains and the materials of the murals themselves, which may be detrimental to mural protection; unlike the wet murals used as experimental subjects in the article, dry murals have stricter requirements for water management, which poses new requirements for biological fixation carriers. In this year's project, we will take these points as breakthroughs.

Our Solution

We decided to engineer the chassis bacteria to achieve the killing and inhibition of pathogenic microorganisms through antimicrobial peptides in an environment where microorganisms are thriving. We also utilized enzyme-peptide complexes to specifically degrade microbial secretions, achieving more diverse and specific functions compared to previous solutions, as an effective application of synthetic biology.

1 Antibacterial Module

Antimicrobial peptides can disrupt the integrity of the cell membrane by insertion into it, thereby exerting an antibacterial effect[3]. This property determines that antimicrobial peptides have broad antibacterial activity and excellent sterilization effects, and have lower drug resistance compared to traditional antibiotics. We use this as a solution for inhibiting harmful microorganisms on the mural. The antimicrobial peptide GRP1 extracted from Limosilactobacillus fermentum GR-9 comes from our laboratory and can have a significant inhibitory effect on bacteria. Pn-AMP1 is an antimicrobial peptide obtained from Pharbitis nil, which can have a significant inhibitory effect on fungi including Saccharomyces cerevisiae and Candida albicans[4]. We introduced these two antimicrobial peptides into our engineered bacteria as a measure to deal with the main pathogenic microorganisms growing on the mural.

2 Removal of Microbial Secretions

Microbial secretions are of great significance for the survival of microorganisms and are also a difficulty in mural restoration and microbial prevention. In this project, we focus on melanin and carotenoids as the key targets for removal, as they play a crucial role in the stress resistance of microorganisms and are easily color-developed on the mural. The synergistic action of laccase CueO and dye decolorizing peroxidase DyP can effectively remove various pigments including melanin[5]. To avoid damage to the components of the mural matrix caused by the two broad-spectrum degrading enzymes, we modified CueO, DyP, and Scaffoldin from Clostridium, which can form a functional complex and bind melanin and carotenoids through the melanin-binding peptide and carotenoid-binding peptide fused on Scaffoldin[6][7], making it closer to the two enzymes and improving the efficiency and specificity of degradation.

3 Regulation of Inhibition and Removal

We hope that the engineered bacteria can inhibit microbial activity when microorganisms are present and then remove microbial secretions later. The environment on the mural varies depending on the technique; it can be neutral to alkaline. Fungi, as the main pathogens on the mural, will secrete organic acids during their growth, making the microenvironment acidic. We constructed a switch using the low pH-induced Pasr promoter[8] and tetracycline control system to release antimicrobial peptides in acidic environments and enzymes and Scaffoldin in neutral to alkaline environments.

4 Control of Bacterial Escape

For an iGEM project on microbial prevention, avoiding the escape of microorganisms into the environment is crucial. We designed an Arabinoxylan-mediated suicide element. In a system with Arabinoxylan, the engineered bacteria can survive normally; but once it leaves the environment we want it to be in (such as the mural surface), the enzyme will start to synthesize due to the absence of Arabinoxylan, inducing the suicide of the engineered bacteria.

Conclusion

In conclusion, SynBio-MuralShield combines the inhibition of microorganisms, the degradation of organic pigments, environmental sensing, and escape control. Together, they ensure the inhibition of microbial growth on the mural and the removal of organic pigments, while reducing the impact on the mural surface. For more details about our project design, please visit our design page.

References

The following references provide key theoretical and experimental support for the research on mural biological damage and the development of synthetic biology-based protection solutions.

• [1] 武发思, 贺东鹏, 苏敏. 中国古代壁画生物病害研究现状与展望. 中国文物保护技术协会第十次学术年会, 中国文物保护技术协会, 2018.
• [2] Ranalli, G., Zanardini, E., Rampazzi, L., Corti, C., Andreotti, A., Colombini, M.P., Bosch‐Roig, P., Lustrato, G., Giantomassi, C., Zari, D., Virilli, P. (2019). Onsite advanced biocleaning system for historical wall paintings using new agar‐gauze bacteria gel. Journal of Applied Microbiology, 126(6), 1785–1796. https://doi.org/10.1111/jam.14275
• [3] Huan, Y., Kong, Q., Mou, H., Yi, H. (2020). Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Frontiers in Microbiology, 11, 582779. https://doi.org/10.3389/fmicb.2020.582779 (PMID: 33178164; PMCID: PMC7596191)
• [4] Koo, J.C., Lee, B., Young, M.E., Koo, S.C., Cooper, J.A., Baek, D., Lim, C.O., Lee, S.Y., Yun, D.J., Cho, M.J. (2004). Pn-AMP1, a plant defense protein, induces actin depolarization in yeasts. Plant and Cell Physiology, 45(11), 1669–1680. https://doi.org/10.1093/pcp/pch189 (PMID: 15574843; PMCID: PMC2672105)
• [5] Shin, S.K., et al. (2019). Effective melanin degradation by a synergistic laccase-peroxidase enzyme complex for skin whitening and other practical applications. International Journal of Biological Macromolecules, 129, 181–186.
• [6] Ballard, B., et al. (2019). In vitro and in vivo evaluation of melanin-binding decapeptide 4b4 radiolabeled with 177Lu, 166Ho, and 153Sm radiolanthanides for the purpose of targeted radionuclide therapy of melanoma.
• [7] Janssen, G.G., Baldwin, T.M., Winetzky, D.S., Tierney, L.M., Wang, H., Murray, C.J. (2004). Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid-binding peptide. Journal of Peptide Research, 64(1), 10–24. https://doi.org/10.1111/j.1399-3011.2004.00150.x (PMID: 15200474; Erratum in: J Pept Res. 2005 Apr;65(4):472)
• [8] Seputiene, V., Motiejūnas, D., Suziedelis, K., Tomenius, H., Normark, S., Melefors, O., Suziedeliene, E. (2003). Molecular characterization of the acid-inducible asr gene of Escherichia coli and its role in acid stress response. Journal of Bacteriology, 185(8), 2475–2484. https://doi.org/10.1128/JB.185.8.2475-2484.2003 (PMID: 12670971; PMCID: PMC152617)