Our goal is to provide corrosion protection for metals in marine environments. Traditional anticorrosive coatings have hidden dangers of environmental pollution. In recent years, there has been increasing research showing that certain microorganisms can form biofilms on metal surfaces. These biofilms help protect against metal corrosion by removing corrosive cathodic media (such as oxygen)^[1], inhibiting the growth of corrosive microorganisms, and forming protective layers on the metal surfaces without causing environmental pollution^[2].

    Inspired by the research on microbial coating, we realize that microbial coating materials are expected to be developed into new corrosion protection technologies. However, if biofilm wants to show its talents in the field of metal corrosion, the problems such as coating adhesion and coating coverage need to be solved urgently.
    Therefore, we plan to design an engineering strain by using the method of synthetic biology, so that it can form a living functional biofilm BMCP (Biofilm for metal corrosion prevention) with high metal coverage and strong adhesion on the metal surface.

After further literature search, we found that many biofilms formed by microorganisms attached to metal surfaces, which may lead to increased metal corrosion. After many attempts, we finally chose E.coli MG1655 as the chassis strain^[3].

    As a common experimental strain, E.coli MG1655 has many advantages:
      (1) There have been a large number of related literatures and experimental data in genetic engineering, which makes it convenient for us to carry out genetic modification and experimental design on the basis of the existing data.
      (2) As a common and widely distributed strain, it does not cause common diseases and infections.
      (3) It is non-corrosive and does not aggravate metal corrosion.

Our team mainly wants to design and produce BMCP through the following processes: Genetic modification enhances the film-forming efficiency of chassis bacteria, design induced suicide switch to eliminate potential biosafety hazards, synthesize biological design BMCP, test the function of BMCP by corrosion electrochemistry, and enhance the long-term effect of BMCP by biomineralization of biofilm. Finally, we expect that BMCP produced by the engineering strain constructed by the above steps can be helpful for marine metal corrosion protection.

By consulting the literature, we found a study on the increase of csgA expression by ompR mutation. The first step of bacterial biofilm formation is the contact between cells and solid surface, in which fimbriae are the key to promote irreversible adsorption of cells and are regulated by EnvZ-OmpR two-component system. This study proves that mutation of ompR can significantly increase the expression of csgA gene^[4]. Inspired by this study, we decided to mutate ompR in E.coli MG1655 into ompR234 by gene knockout technology, so as to increase the film-forming efficiency of chassis bacteria^[5].

We integrated an arabinose inducible promoter-regulated virulent protein ccdB gene into the genome of E.coli MG1655, so that arabinose can be added when necessary to achieve bacterial controllable suicide death^[6].
    We plan to verify the effect of suicide switch by culturing ccdB-integrated and unintegrated E.coli MG1655 in culture medium and adding arabinose for comparison.

Due to the lack of adhesion of Escherichia coli biofilm itself, we got inspiration from a photo of barnacles covered with hull, hoping to use mussel foot silk protein Mfp5 to increase the adhesion of biofilm. Our team tried to connect CsgA with Mfp5 and designed two schemes:
      ① Connect CsgA with Mfp5 directly
      ② Use polypeptide fragments spytag and spycatcher to spontaneously form heteropeptide bonds in vitro to realize the combination of two polypeptide fragments, that is, to connect through spytag-spycatcher^[7].
    We plan to verify the film-forming effects of the two methods through crystal violet and Congo red experiments: compare the OD values of the biofilm produced on the metal surface by comparing the advantages and disadvantages of the two schemes.

After BMCP is produced, we plan to evaluate the protection of living functional biofilm materials against metal corrosion by corrosion electrochemical test^[8].

We hope to further improve the biological safety of metal corrosion protection technology based on biofilm through biomineralization, and prolong the service life of biofilm for metal corrosion protection^[9][10]. We finally decided to realize long-term metal corrosion protection of biofilm through biomineralization.

    We plan to culture the biofilm with E.coli-MG 1655-CsgA-spytag in a 24-well plate, and then add the crushed solution of E.coli-BL 21-Mfp-spycatcher into it for co-incubation. After CsgA-spytag and Mfp-spycatcher are successfully connected, scrape off the biofilm and smear it on the surface of carbon steel X80 metal block, and cultured in simulated seawater for 7 days to induce its natural mineralization. Finally, the biomineralized layer is photographed by SEM.

We hope that the application of BMCP can provide a new and environmentally friendly biological control method for marine metal anticorrosion. In our vision, our products will eventually be used for static metal anticorrosion in marine environment, such as small metal parts of hull, metal pipes, metal guardrails of wharf and steel pipe columns of port, etc.
    Facing the users who have the above requirements, we hope to deliver BMCP products to users in the form of an anticorrosive coating. We make BMCP anticorrosive coating has the following advantages: familiar and simple use greatly simplifies the user's operation steps, has better production standards and is more suitable for mass production, and the preservation conditions are not harsh. In addition, we can also provide users with matching coating spray bottles or coating brushes.
    To learn more about our product implementation plan, please visit {Implementation}
    We are committed to marine metal anticorrosion and are very concerned about biosafety issues, and we are very worried that our products will cause biosafety problems. So we added suicide switches so that our engineered strains would not escape into the environment and harm nature.

[1]Earthman J C, Jayaraman A, Wood T K. Corrosion inhibition by aerobic biofilms on SAE 1018 steel [J]. Appl. Microbiol. Biot., 1997, 47(1): 62

[2]Pedersen A, Kjelleberg S, Hermansson M. A screening method for bacterial corrosion of metals [J]. Microbiol. Methods., 1988, 8(4): 191

[3]Xinran,Wang,Qian,et al.Identification of riboflavin: revealing different metabolic characteristics between Escherichia coli BL21(DE3) and MG1655[J].FEMS Microbiology Letters, 2015.DOI:10.1093/femsle/fnv071.

[4]Vidal O, Longin R, Prigent-Combaret C, Dorel C, Hooreman M, Lejeune P. Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression. J Bacteriol. 1998 May;180(9):2442-9. doi: 10.1128/JB.180.9.2442-2449.1998. PMID: 9573197; PMCID: PMC107187.

[5]WANG Liliang, GAO Chunhui, WU Yichao, HUANG Qiaoyun, CAI Peng. Research progress on mechanism of surface sensing in Escherichia coli. Journal of Zhejiang University(Agriculture & Life Sciences), 2017, 43(6): 685-690. DOI: 10.3785/j.issn.1008-9209.2017.07.261.

[6]Bernard P, Couturier M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA- topoisomerase Ⅱ complexes [J]. J Mol Biol, 1992，226(3):735-745.

[7]Wang XW, Zhang WB. SpyTag-SpyCatcher Chemistry for Protein Bioconjugation In Vitro and Protein Topology Engineering In Vivo. Methods Mol Biol. 2019;2033:287-300. doi: 10.1007/978-1-4939-9654-4_19. PMID: 31332761.

[8]Xia, J., Xu, D., Nan, L., Liu, H., Li, Q., & Yang, K. (2016). Study on Mechanisms of Microbiologically Influenced Corrision of Metal from the Perspective of Bio-electrochemistry and Bio-energetics. Chinese Journal of Materials Research, 30, 161-170.

[9]Voigt, O., et al., Carbonic Anhydrases: An Ancient Tool in Calcareous Sponge Biomineralization. Frontiers in Genetics, 2021. 12: 624533.

[10]Kim, I.G., et al., Biomineralization-based conversion of carbon dioxide to calcium carbonate using recombinant carbonic anhydrase. Chemosphere, 2012. 87(10): 1091-1096.