In metal corrosion protection, biofilm can play a role in metal corrosion protection by removing corrosive cathode medium (such as oxygen), inhibiting the growth of corrosive microorganisms, and forming a protective layer on the metal surface. However, one of the shortcomings of biofilm for metal corrosion protection is its weak metal adhesion ability. In the iGEM2023 competition year, the NEU-CHINA team aimed to design a living functional biofilm that can be applied to marine metal corrosion protection. They named it BMCP (Biofilm for metal corrosion prevention). In this project, Escherichia coli amyloid protein CsgA and mussel foot silk protein Mfp were connected by in vitro assembly, aiming at improving the adhesion ability of the biofilm on the surface of metal materials, so as to play the role of corrosion protection better. In addition, in order to ensure that the engineered bacteria will not pose a threat to the environment, we integrated the toxic protein ccdB gene regulated by arabinose inducible promoter into the bacterial genome and then realized the controllable suicide death of bacteria. This strategy may provide a new biological control method for marine metal corrosion protection.

Corrosion of metals is a natural phenomenon. In a sense, "corrosion is inevitable". Every year, 30%-40% of the total steel output is scrapped due to corrosion in the world, and about one-third of it cannot be recycled. According to the statistics of the American International Association of Corrosion Engineers (NACE), the global economic loss caused by corrosion in 2013 was about 2.5 trillion US dollars, equivalent to 3.4% of the global GDP in that year. Moreover, serious corrosion will also cause catastrophic accidents such as damage to devices and facilities, leakage of toxic media, fire, explosion, etc., which will cause irreparable losses to human life safety, the environment, and society^[1].

Traditional corrosion protection methods include coating, surface treatment,corrosion resistant materials,corrosion inhibitors,electrochemical protection and so on.However,these technologies still face many inherent defects and deficiencies.

In recent years, more and more studies have found that some microorganisms form biofilms on metal surfaces, which play a role in metal corrosion protection by removing corrosive cathode media (such as oxygen)^[5], inhibiting the growth of corrosive microorganisms, and forming protective layers on metal surfaces. Compared with traditional corrosion protection methods, the microbial coating has low cost, wide application range, and strong operability, and using non-toxic and harmless microorganisms to prepare biofilms will not damage the environment and lead to pollution. However, it also has some defects, such as insufficient adhesion of the microbial coating on metal surfaces, discontinuous and uneven distribution, low coverage rate, and can not play a long-term role in corrosion protection^[6].

Inspired by the research on microbial coating and realizing that microbial coating materials are expected to be developed into new corrosion protection means, we decided to take it as the theme of our team's iGEM project this year. When we were thinking about how to make up for the defects of microbial coating materials and find a breakthrough way, news about the discovery of many barnacles on the hull of ships caught our attention, and we associated barnacles with mussels. The adhesion ability of mussels mainly depends on mussel foot silk protein Mfp. If biofilm protein and mussel foot silk protein can be connected by molecular biological means, it can have mussel-like adhesion ability, thus firmly adhering to the metal surface and giving better play to corrosion protection ability.

Create BMCP: A living functional microbial coating that can be applied to marine metal corrosion protection,with stronger and more stable adhesion ability.

We chose E.coli MG1655 as the chassis strain, because E.coli MG1655 is a common experimental strain, and there are a lot of related literatures and experimental data in genetic engineering, which is convenient for us to carry out genetic modification and experimental design based on the existing data. Moreover, it is a common and widely distributed strain, which will not cause common diseases and infections and is not corrosive^[7].

We integrated a toxic protein ccdB gene regulated by arabinose inducible promoter into the genome of chassis bacteria, so as to realize bacterial controllable suicide death after adding arabinose when necessary^[8].

Mfp, a macromolecular protein secreted from the foot silk glands of marine mussels, can ensure that mussels can adhere to the surfaces of various objects in the water environment. In iGEM2023, our team aims to connect Escherichia coli biofilm protein CsgA with mussel foot silk protein Mfp by self-assembly in vitro, so that the biofilm shows stronger and more stable adhesion ability than ordinary biofilm materials, thus firmly adhering to the surface of marine metal^[9].

Because the molecular weight of mussel foot silk protein is too large, if it is directly fused with the Curli amyloid protein encoded by E.coli, it cannot be secreted out of cells and its function cannot be effectively realized. Polypeptide fragments Spytag and Spycatcher have been proven to form hetero-peptide bonds spontaneously in vitro, realizing the binding of two polypeptide fragments^[10]. Therefore, we use gene editing technology to insert Spytag and Spycatcher domains into the mussel foot silk protein coding gene and Curli amyloid coding gene respectively, so as to realize the effective binding of these two proteins and enhance the adhesion ability of Escherichia coli biofilm.

In order to prolong the service life of biofilm for metal corrosion protection and further improve the biological safety of metal corrosion protection technology based on biofilm, this project will finally realize the long-term metal corrosion protection of biofilm by means of biomineralization^[11][12].

1. Idea innovation: It is a brand-new interdisciplinary attempt to optimize and transform biofilm by using synthetic biology technology, improve its metal adhesion ability, and apply it to the interdisciplinary field of metal corrosion protection.
2. Technological innovation: Synthetic biology technology and biomineralization technology are organically integrated to improve the adhesion ability of biofilm, prolong its service life of metal corrosion protection, and overcome the problem that living biofilm has potential biosafety hazards when applied to natural environment.

The application of BMCP is expected to provide a new biological control method for marine metal corrosion protection
    In our project, this product is mainly used for static anticorrosion, such as small metal parts of the hull, metal pipes, metal guardrails of the wharf, and steel pipe columns of the port.

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