iGEM 2023 — Team neu-china

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[1. February](#1)

[2. March](#2)

[3. April and May](#3)

[4. June](#4)

[5. July](#5)

[5.1 The first group](#51)

[5.2 The second group](#52)

[6. August](#6)

[6.1 The third group](#61)

[6.2 The forth group](#62)

First of all, you should know our number of engineering strains, and then we will use this number to explain.

Number of engineering strains | || BMCP-B1 | E. coli K-12 MG1655 |
| BMCP-B2 | E. coli MG1655 ΔompR::kn-ccdB |
| BMCP-B3 | E. coli MG1655-ompR234 |
| BMCP-B4 | E. coli MG1655-ompR234 ΔcsgA::kn-ccdB |
| BMCP-B5 | E. coli BL21 |

## February

---

-NEU-China team established

    -Learn the basics of iGEM

## March

---

-NEU-China team experimental group was formally established

    -Establish the general direction and find relevant literature

## April and May

---

-Complete the project: BMCP (Biofilm for metal corrosion prevention)

    -The experimental group entered the laboratory in batches to participate in basic experimental training

## June

---

-The experimental group was divided into four groups, and the experimental part they were responsible for was trained pertinently

    -Refer to literature and optimize experimental design

## July

---

-Officially start the experiment

### The first group

---

#### 8th

---

-Electroconversion of pSIM6 vector into BMCP-B1

#### 9th

---

-Amplification of ompR-Kn-ccdB gene fragment from CR201 strain

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img1.jpg)

#### 12nd

---

-Construction of BMCP-B2 strain (first homologous replacement: Kn-pBAD-ccdB replacing ompR gene)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img2.jpg)

#### 13rd

---

-Amplification of ompR234 gene fragment from gene synthesis fragment

#### 16th

---

-Construction of BMCP-B3 strain (second homologous replacement: ompR234 replacing Kn-pBAD-ccdB gene)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img3.jpg)

#### 19th

---

-Construction of BMCP-B4 strain (Kn-pBAD-ccdB replacing csgA gene)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img4.jpg)

#### 20th

---

-Verification of ccdB suicide switch

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img5.jpg)

### The second group

---

#### 22nd

---

-PCR linearization vector pCA24N

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img6.jpg)

-Amplification of mfp5 gene fragment from vector pET28a-mfp5-spycatcher

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img7.jpg)

-Amplification of csgA fragment from BMCP-B1

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img8.jpg)

#### 23rd

---

-Construct the recombinant vector pCA24N-csgA-mfp5 by Gibson assembly

#### 25th

---

-Verification of pCA24N-csgA-mfp5 by double digestion(by Hind III，Xho I)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img9.jpg)

#### 26th

---

-Electroconversion of pCA24N-csgA-mfp5 to BMCP-B4

#### 28th-31th

---

-BMCP-B4 pCA24N-csgA-mfp5 biofilm forming ability test: Crystal violet test, Congo red test

    -PCR linearization vector pCA24N

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img10.jpg)

-The csgA-spyTag gene fragment was obtained by amplifying csgA from BMCP-B1 and introducing spyTag by primers

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img11.jpg)

-Construct the recombinant vector pCA24N-csgA-spyTag by Gibson assembly

    -Verification of pCA24N-csgA-spyTag by double digestion(by Hind III,  Xho I)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img12.jpg)

-Electroconversion of pCA24N-csgA-spyTag into BMCP-B4

    -Electroconversion of pET28a-mfp5-spycatcher into BMCP-B5

    -BMCP-B4 Biofilm Formation Tests: Crystal Violet Test, Congo Red Test

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img13.jpg)

## August

---

### The third group

---

#### 1st-15th

---

-Electrochemical test of corrosion of functional biofilm in vivo

    -OCP curve of corrosion electrochemical test

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img14.jpg)

-LPR curve of corrosion electrochemical test

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img15.jpg)

-Polarization curve of corrosion electrochemical test

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img16.jpg)

-EIS curve of corrosion electrochemical test

    -EIS curve after coating on electrode for 1 day, left: Nyquist plot, right: Bode plot

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img17.jpg)

-EIS curve after coating on electrode for 7 days, left: Nyquist plot, right: Bode plot

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img18.jpg)

-Nyquist plot of four groups of materials after one day, three days, five days and seven days

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img19.jpg)

-Bode plot of four groups of materials after one day, three days, five days and seven days

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img20.jpg)

#### 16th-18th

---

-Biofilm adhesion test in vivo (80rpm shaker)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img21.jpg)

Verification of adhesion ability of biofilm, from left to right:(a).E.coli MG1655-CsgA-spytag, (b).E.coli BL21, (c).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher.

### The fourth group

---

#### 23rd

---

-Characterization of biomineralization morphology of biofilm by SEM

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img22.jpg)

SEM after 7 days of culture in simulated seawater (1).Blank control; (2).E.coli MG1655-CsgA-spytag; (3).E.coli MG1655-CsgA-spytag+E.coli BL21；(4).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher.

#### 24th-30th

---

-Observation on anticorrosion effect of mineralized biofilm

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/notebook/img23.jpg)

Corrosion of materials under different coatings, (1).Blank control; (2).E.coli MG1655-CsgA-spytag; (3).E.coli MG1655-CsgA-spytag+E.coli BL21；(4).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher. From left to right:A.Just coated; B.After the coating is dried; C.After seven days in seawater; D.After seven days in seawater (sun exposure)

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[1. Part 1: Escherichia coli film formation](#1)

[1.1 First iteration](#1)

[2. Part 2: Biofilm adhesion](#2)

[2.1 Second iteration](#21)

[2.2 Third iteration](#22)

[3. Part 3: Biofilm anticorrosion](#3)

[3.1 The fourth iteration](#31)

[4. References](#4)

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.

## Part 1: Escherichia coli film formation

---

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

### First iteration

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).

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img1.png)

The following are the sequences of ompR and ompR234：

[Download ompR.pdf](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/ompr.pdf)

[Download ompR234.pdf](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/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.

## Part 2: Biofilm adhesion

---

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.

### Second iteration

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.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img2.jpg)

Please download the pdf file for details:

[Download pCA24N-csgA-mfp5.pdf](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/pca24n-csga-mfp5.pdf)

[Download pCA24N-csgA-spytag.pdf](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/pca24N-csga-spytag.pdf)

[Download pet28a-Mfp-spycatcher.pdf](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiment%27%29%20%7D%7D)

Results: The plasmid was successfully introduced.

### Third iteration

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiment%27%29%20%7D%7D)

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.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img3.jpg)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img4.jpg)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img5.jpg)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img6.jpg)

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.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img7.png)

Verification of adhesion ability of biofilm, from left to right:(a).E.coli MG1655-CsgA-spytag, (b).E.coli BL21, (c).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher.

## Part 3: Biofilm anticorrosion

---

### The fourth iteration

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.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img8.jpg)

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:

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img9.jpg)

SEM after 7 days of culture in simulated seawater (1).Blank control; (2).E.coli MG1655-CsgA-spytag; (3).E.coli MG1655-CsgA-spytag+E.coli BL21；(4).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher.

Obvious mineralized layers were observed on BMCP coating (4).

In addition, we further tested the anti-corrosion ability of BMCP after biomineralization in seawater.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/engineering/img10.jpg)

Corrosion of materials under different coatings, (1).Blank control; (2).E.coli MG1655-CsgA-spytag; (3).E.coli MG1655-CsgA-spytag+E.coli BL21；(4).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher. From left to right:A.Just coated; B.After the coating is dried; C.After seven days in seawater; D.After seven days in seawater (sun exposure)

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

## References

---

[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.

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#

[1. Overview](#1)

[2. Part 1:Biological transformation enhances
the film-forming ability of chassis bacteria](#2)

[2.1 Our finished work](#21)

[2.2 Our unfinished work](#22)

[3. Part 2: Enhance the adhesion of biofilm](#3)

[3.1 Plasmid construction](#31)

[3.2 Crystal violet and Congo red experiments](#32)

[3.3 Verification of adhesion ability of biofilm](#33)

[4. Part 3:Corrosion resistance test of
functional biofilm in vivo](#4)

[5. Part 4:Biomineralization and corrosion
resistance test](#5)

First of all, you should know our number of engineering strains, and then we will use this number to explain.

Number of engineering strains | || BMCP-B1 | E. coli K-12 MG1655 |
| BMCP-B2 | E. coli MG1655 ΔompR::kn-ccdB |
| BMCP-B3 | E. coli MG1655-ompR234 |
| BMCP-B4 | E. coli MG1655-ompR234 ΔcsgA::kn-ccdB |
| BMCP-B5 | E. coli BL21 |

## Overview

---

This year, our team hopes to create a biofilm material with stronger adhesion ability for metal corrosion protection. In order to prove the feasibility of this concept, we have carried out a series of molecular biology experiments, microbial experiments and electrochemical experiments. The following experimental results well prove the concept of our project. Welcome to visit our designs, experiments and results for more information.

## Part 1:Biological transformation enhances the film-forming ability of chassis bacteria

---

CsgA expression is enhanced, aiming at forming more biofilms.

### Our finished work:

The work we have done is to realize the mutation of ompR in E.coli MG1655 to ompR234.

    The following are the sequences of ompR and ompR234:

[Download ompR.pdf](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/ompr.pdf)

    [Download ompR234.pdf](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/ompr234.pdf)

See [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) and [Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiment%27%29%20%7D%7D) for details

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img11.jpg)

Figure1.1 Construction of BMCP-B2 strain (first homologous replacement: kn-pBAD-ccdB replacing ompR

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img12.jpg)

Figure1.2 Construction of BMCP-B3 strain (second homologous replacement: ompR234 replacing kn-pBAD-ccdB gene)

Through the mutation of ompR gene, the film formation rate of Escherichia coli was significantly increased

    We constructed a biofilm growth model of BMCP-B1 and mutant BMCP-B3. For details, please see the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) section.

### Our unfinished work:

1. RBS sequence: We modified six RBS sequences in Escherichia coli MG1655 to observe their effects on the expression of downstream YFP fluorescent protein gene, but due to time, we did not directly verify their effects on the translation process of CsgA-spytag and Mfp5-spycatcher genes in this project.

    2. Escherichia coli film formation conditions: Although we proved that the film formation effect was significantly improved after ompR mutation by measuring the film formation rate and establishing the biofilm growth model of E · coli MG1655, we did not explore the influence of various environmental factors on film formation (such as nutrients, pH, temperature, metal materials, etc.), which was planned to be done, but we did not have time to do it because of time.

    3. Optically controlled switch: We plan to achieve the controllability of biofilm growth through light control system, but this experiment has not been completed.

    In addition, we also integrated a toxic protein ccdB regulated by arabinose inducible promoter on the basis of BMCP-B3, in order to achieve controllable suicide death of engineering bacteria after adding arabinose. By knocking out the original csgA gene of BMCP-B3 and replacing it with kn-ccdB gene, we obtained an engineering strain (BMCP-B4) which successfully integrated suicide switch. Finally, we tested the effect of suicide switch by culturing BMCP-B2, BMCP-B3 and BMCP-B4 in culture medium and adding arabinose for comparison.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img14.jpg)

Figure1.3 Verification of suicide switch (1)BMCP-B2, (2)BMCP-B3, (3)BMCP-B4

As shown in the figure, after adding arabinose, no bacteria grew in groups 1 and 3 with kn-ccdB.This proves that our suicide switch is a success.

## Part 2: Enhance the adhesion of biofilm

---

The second part is to verify the feasibility of enhancing the adhesion of biofilm, experiments are carried out according to the two schemes.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img21.jpg)

Figure2.1 Our plan

Three plasmids were constructed in this experiment:

    [BBa\_K4635005](http://parts.igem.org/Part%3ABBa_K4635005),[BBa\_K4635007](http://parts.igem.org/Part%3ABBa_K4635007),[BBa\_K4635006](http://parts.igem.org/Part%3ABBa_K4635006)

### Plasmid construction:

We successfully constructed BBa\_K4635005, BBa\_K4635007, BBa\_K4635006, BBa\_K4635005, BBa\_K4635007 plasmids, were introduced into BMCP-B4 and BBa\_K4635006 plasmids were introduced into BMCP-B5.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img22.jpg)

Figure2.2 Verification of pCA24N-csgA-mfp5 by double digestion(by Hind III，Xho I)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img23.jpg)

Figure2.3 Verification of pCA24N-csgA-spyTag by double digestion(by Hind III，Xho I)

### Crystal violet and Congo red experiments:

Through crystal violet and Congo red experiments, it is proved that the film formation effect of CsgA and Mfp5 is not ideal, so we choose the second scheme, that is, spytag-spycatcher.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img24.jpg)

Figure2.4 The results of crystal violet experiment,from left to right:(1) pCA24N, (2)pCA24N-csgA,(3) pCA24N-csgA-mfp5

The results of crystal violet experiment show that the value of (3) is low and the film forming effect is very poor.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img25.jpg)

Figure2.5 The results of Congo red experiment,from left to right:(4) Congo Red Blank Control, (5)MG1655, (6)MG1655-ompR234,(7) pCA24N,(8) pCA24N-csgA, (9) pCA24N-csgA-spytag, (10) pCA24N-csgA-mfp5

The results of Congo red experiment showed that the value of (7) group was the lowest, which proved that E · coli MG1655 with pCA24N-csgA-spytag had the best film-forming ability.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img26.jpg)

Figure2.6 Experimental results of crystal violet with pCA24N-csgA-spytag,from left to right: (11)pCA24N, (12)pCA24N-csgA,(13) pCA24N-csgA-spytag, (14)pCA24N-csgA-mfp5

After adding (13) to the crystal violet experiment, it can be found that the film-forming ability of (13) group is far greater than that of (14) group, which further proves the feasibility of the second scheme. Comparing (12) with (13), it was found that the addition of Spytag had no obvious effect on the film-forming ability.

For more information, please see the detailed data of crystal violet and Congo red experiment.

### Verification of adhesion ability of biofilm

At the end of this part, we tested the adhesion of biofilm on a 80rpm shaking table after film formation on the surface of X80 carbon steel.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img27.jpg)

Figure2.7 Verification of adhesion ability of biofilm, from left to right:(a).E.coli MG1655-CsgA-spytag, (b).E.coli BL21, (c).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher.

The experimental results show that the corrosion degree of X80 carbon steel in group (c) is the lowest, which proves that the adhesion performance of biofilm (named BMCP) produced by E.coli MG1655-CsgA-spytag + E.coli BL21-Mfp-spycatcher is improved.

## Part 3:Corrosion resistance test of functional biofilm in vivo

---

This part of the experiment is used to evaluate the protective effect of biofilm materials on metal corrosion in the project. After testing, the mixed solution of BMCP has the best anti-corrosion effect.

    In this experiment, simulated seawater immersion electrodes were used in three-electrode electrolytic cells, and four groups of controls were set up

    Group I: Control group, blank electrode (Sterile)

    Group 2:E.coli-MG1655-CsgA-spytag strain was evenly coated on the test surface of the working electrode

    Group 3:E.coli-MG1655-CsgA-spytag + E.coli-BL21 crushing solution was uniformly coated on the test surface of the working electrode

    Group 4: BMCP was uniformly coated on the test surface of the working electrode

    In this experiment, the Gamry Framework program was used to test the corrosion electrochemistry, and each group of experiments lasted for 7 days

    The following are the experimental results and analysis:

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img31.jpg)

Figure3.1 OCP curve of corrosion electrochemical test

From the analysis of OCP curve: Eocp is an open circuit potential, and the larger its value, the smaller its corrosion tendency. In the experiment, the values of 2, 3 and 4 groups were higher than those of the blank control group, which proved that compared with the blank control group, the other three groups had less corrosion tendency, and after the third day, the 3 and 4 groups showed better anti-corrosion ability.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img32.jpg)

Figure3.2 LPR curve of corrosion electrochemical test

Analyze from LPR curve: The ordinate Rp of LPR curve represents the resistance value, and the larger the Rp value, the more corrosion-resistant the group is. From the image, the fourth group shows a higher Rp value as a whole.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img33.jpg)

Figure3.3 Polarization curve of corrosion electrochemical test

From the polarization curve, it is analyzed that the tangent line extends from the corrosion potential (horizontal line) and the cathode linear area respectively (in the lower right corner, the tangent point can generally be located at about 120mV below the corrosion potential). The abscissa corresponding to the intersection point is the self-corrosion current density, and the ordinate is the self-corrosion potential. The self-corrosion current density can indirectly express the corrosion rate. The smaller the self-corrosion current density, the lower the corrosion rate, which proves that the corrosion resistance of this group is stronger.

Group | Corrosion current | Standard deviation | Corrosion potential | Standard deviation || 1 | -0.90795 | 0.04331 | 63.56 | 7.73613 |
| 2 | -0.90727 | 0.04014 | 51.86833 | 6.88913 |
| 3 | -0.90178 | 0.03873 | 54.97824 | 5.28404 |
| 4 | -0.89722 | 0.03308 | 25.9975 | 7.66928 |

    From the data in the above table, it can be seen that the corrosion current and corrosion voltage of the fourth group are significantly lower than those of the other three groups, which proves that its corrosion resistance is strong.·

    In addition, we also compared 2, 3 and 4 groups of one-day and seven-day EIS curves, which were divided into Nyquist plot (left) and Bode plot (right)

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img34.jpg)

Figure3.4 EIS curve after coating on electrode for 1 day, from left to right, left: Nyquist plot, right: Bode plot

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img35.jpg)

Figure3.5 EIS curve after coating on electrode for 7 days, from left to right, left: Nyquist plot, right: Bode plot

Analysis from Nyquist plot: mainly observe the radius of arc. The larger the radius, the stronger the corrosion resistance.

    Analyze from Bode diagram: Bode diagram is divided into a mountain diagram and a slope diagram. To judge the corrosion resistance of materials, the slope diagram is mainly observed. The value of slope diagram is also called modulus value, and the modulus value in low frequency region is the equivalent impedance of circuit. Therefore, the larger the value of intersection point between slope diagram and ordinate, the more corrosion-resistant this group of materials is.

    Therefore, it can be explained that the fourth group of coatings did not show superior anticorrosion ability after 1 day, but after 7 days, both figures proved that this coating had better anticorrosion ability. The results not only prove that the coating is better after 7 days on the electrode, but also fully prove the anticorrosion effect of the coating.

    EIS graphs for four groups of materials for one day, three days, five days and seven days are shown below.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img36.jpg)

Figure3.6 Nyquist plot of four groups of materials after one day, three days, five days and seven days

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img37.jpg)

Figure3.7 Bode plot of four groups of materials after one day, three days, five days and seven days

Generally speaking, our BMCP coating has ideal anticorrosion effect.

## Part 4: Biomineralization and corrosion resistance test

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We use X80 carbon steel as the experimental material for observing corrosion and simulated seawater as the environment for biomineralization. Like electrochemical experiments, four groups of experiments were set up, which were cultured in simulated seawater for 7 days, and mineralization was photographed by electron microscope. The following are the experimental results.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img41.jpg)

Figure4.1 SEM after 7 days of culture in simulated seawater (1).Blank control; (2).E.coli MG1655-CsgA-spytag; (3).E.coli MG1655-CsgA-spytag+E.coli BL21；(4).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher.

SEM can clearly see that there are obvious mineralized layers on the surface of the fourth group.

    In addition, we further tested the anti-corrosion ability of BMCP after biomineralization in seawater.

![...](https://static.igem.wiki/teams/4635/wiki/pages/page-img/results/img42.jpg)

Figure4.2 Corrosion of materials under different coatings, (1).Blank control; (2).E.coli MG1655-CsgA-spytag; (3).E.coli MG1655-CsgA-spytag+E.coli BL21；(4).E.coli MG1655-CsgA-spytag+E.coli BL21-Mfp-spycatcher. From left to right:A.Just coated; B.After the coating is dried; C.After seven days in seawater; D.After seven days in seawater (sun exposure)

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

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