In the project, we proved that the direct connection of CsgA-Mfp5 is not ideal, and we used spytag-spycatcher to connect CsgA protein with Mfp5 protein, which will provide inspiration for subsequent teams.
    We use electrochemical technology to detect the degree of metal corrosion, which provides data and technical reference for subsequent teams who want to study corrosion.
    We use biomineralization technology to prolong the service life of biofilm, and provide ideas for the follow-up team dedicated to the research of biofilm materials.
    We compared the effects of five RBS sequences in Escherichia coli MG1655, which can help future igem teams choose RBS.
    We have codon optimized the mfp5 gene sequence, which will help the team who want to study Mfp5 protein to use this sequence in the future.
    We configure simulated seawater to study the biomineralization of Escherichia coli biofilm in marine environment, which provides research ideas for future teams who want to study microbial activities in marine environment.

This part is used to express the fusion protein CsgA-Mfp5, which is mainly used to verify the idea of enhancing the adhesion of biofilm.
    The csgA gene fragment was amplified from Escherichia coli genome by PCR, and the mfp5 gene fragment was amplified by PCR with mfp5-spycatcher (synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.) as template. The pCA24N linear particle was obtained by PCR method, and finally pCA24N-csgA-mfp5 was successfully constructed. We transferred it into the modified chassis bacteria E.coli MG1655-ompR234 Δ csgA:: Kn-ccdB for detection.

-Crystal violet and Congo red experiments
    For details, please see:http://parts.igem.org/Part:BBa_K4635005

This part is used to express the fusion protein Mfp5-Spycatcher as one of the components of our project product in vivo functional biofilm.
    We obtained the nucleotide sequence of mfp5 from the literature, optimized the codon for our chassis strain, designed the sequence of mfp5-spycatcher, synthesized it by Suzhou Jinweizhi Biotechnology Co., Ltd., obtained pET28 linear particle by PCR, and finally successfully constructed pET28-mfp5-spycatcher. We transferred it to E. coli BL21 for detection.

-Test the adhesion of biofilm (80rpm shaker)
-Corrosion electrochemical test
-Biomineralization and corrosion resistance test
    For details, please see:http://parts.igem.org/Part:BBa_K4635006

This section is used to express the fusion protein CsgA-Spytag as one of the components of our project product in vivo functional biofilm.
    We amplified csgA gene from Escherichia coli genome by PCR, obtained spytag fragment by primer with spytag sequence (synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), obtained pCA24N linear property particle by PCR, and finally successfully constructed pCA24N-csgA-spytag. We transferred it into the modified chassis bacteria E.coli MG1655-ompR234 ΔcsgA:: Kn-ccdB for detection.

-Crystal violet and Congo red experiments.
    For details, please see:http://parts.igem.org/Part:BBa_K4635007

Using bioinformatics, we screened five RBS sequences from E. coli MG1655 by software. We used yellow fluorescent protein YFP as reporter gene, and measured the fluorescence intensity in the bacterial solution by microplate reader to reflect the expression of YFP, thus verifying the corresponding RBS intensity. The following are the experimental results.

The results showed that the strain using RBS3 expressed the most yellow fluorescent protein, which indicated that the intensity of RBS3 was the highest among the five RBS.
We hope that the RBS information provided by our team will help future igem teams in choosing RBS.