The NEU-China team did a lot of thinking while exploring this research direction of biofilm anticorrosion and tried to optimize the project. This page will show the iterative process of the NEU-China team experimental project.

In this project, biofilm is used to achieve antiseptic effect, so it is necessary to improve the film-forming ability of Escherichia coli.

Objective: 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^[1]. 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^[2].We plan to significantly improve the film-forming ability of chassis bacteria by mutating ompR gene into ompR234.
    Design: Firstly, pSIM6 plasmid expressing λ-Red recombinase (with kanamycin resistance) was transferred into wild-type E.coli MG1655 (we named BMCP-B1). After pSIM6 expressed λ-Red recombinase sufficiently, kn-ccdB (kn for kanamycin resistance gene, ccdB for arabinose-induced toxicity protein gene) was extracted and amplified using CR201 colony as template. kn-ccdB was transferred into BMCP-B1 by electric shock transformation, and was coated on a plate with kanamycin and glucose to obtain positive colony. The target strain (we named BMCP-B2) with ompR gene replaced by kn-ccdB gene was verified by PCR); The plasmid containing ompR234 was synthesized in Suzhou Jinweizhi Biotechnology Co., Ltd., and then transferred into BMCP-B2 by electric shock transformation. Finally, the target strain with kn-ccdB gene replaced by ompR234 gene was obtained by colony screening and PCR verification (we named it BMCP-B3).

The following are the sequences of ompR and ompR234：
Download ompR.pdf
Download ompR234.pdf
    Experiment: After the replacement of ompR234, BMCP-B1 and BMCP-B3 were cultured on the surface of 24-well plate and carbon steel X80 metal block respectively, and the biofilm on the surface of orifice plate and metal block was scraped off at a given time point (2h/4h/6h/8h/10h/12h/24h/36h/48h).
    Results: We tested the film-forming ability after mutation.
The film-forming ability of BMCP-B3 is much higher than that of BMCP-B2.

When applied in marine environment, the adhesion of biofilm determines whether the anticorrosive coating is firm or not, and directly affects its anticorrosive ability. In order to enhance the adhesion of Escherichia coli biofilm on metal surface and meet the requirements of metal corrosion protection in marine environment, we decided to use mussel foot silk protein Mfp to increase the adhesion of biofilm after multi-literature investigation. We designed two schemes: 1. CsgA and Mfp are directly connected in vivo; 2. spytag-spycatcher was used for in vitro ligation.

Objective: To use mussel foot silk protein to increase the adhesion ability of biofilm.

Design:
    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.

Please download the pdf file for details:
Download pCA24N-csgA-mfp5.pdf
Download pCA24N-csgA-spytag.pdf
Download pet28a-Mfp-spycatcher.pdf
Experiment: The constructed plasmids were introduced into the target strains respectively. Please refer to the Experiment section for the specific process.     Access to Experiment
Results: The plasmid was successfully introduced.

Objective: To leave the best scheme among the two schemes.
Design: The film-forming ability of E.coli MG1655 modified in two schemes was compared by crystal violet Congo red test.
Experiment: Carry out crystal violet Congo red test, please refer to Experiment section for specific process.     Access to Experiment
Results: The results showed that CsgA-Mfp direct bonding had a poor film-forming effect, while CsgA-spytag bonding had a good film-forming effect, so the first scheme was abandoned and the spytag-spycatcher bonding mode was selected.

After two iterations, we decided to use the second scheme, and then we verified the biofilm adhesion ability after spytag-spycatcher bonding on a shaking table. According to the following figure, the adhesion ability of group c is the best.

Objective: After verifying the anti-corrosion ability of BMCP, we try to optimize the anti-corrosion ability of biofilm by other means.
    Design: We consider biomineralization not only to prolong the service life of biofilm, but also to improve its biological safety.

Experiment: The biofilm was cultured in a 24-well plate with E.coli-MG 1655-CsgA-spytag, and then the crushed solution of E.coli-BL 21-Mfp-spycatcher was added into it to incubate together. After CsgA-spytag and Mfp-spycatcher were successfully connected, the biofilm was scraped off and smeared on the surface of metal block X80, and cultured in simulated seawater for 7 days. Finally, the biomineralized layer was photographed by SEM.
Results:

Obvious mineralized layers were observed on BMCP coating (4).
In addition, we further tested the anti-corrosion ability of BMCP after biomineralization in seawater.

It can be seen from the above experimental results that the biofilm materials (C4, D4) with mineralized layers still have significant anti-corrosion ability.

[1]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.

[2]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.