Composite

Part:BBa_K4635006

Designed by: Ruohui Li   Group: iGEM23_NEU-CHINA   (2023-10-12)

pET28-mfp5-spycatcher

This part is used to express the fusion protein Mfp5-Spycatcher as one of the components of our project product in vivo functional biofilm.

Characterization

IPTG was used to induce its expression. After centrifugation, the bacteria solution was suspended with 40 mL PBS. After crushing, centrifugation was carried out (the rotation speed should be gradual: 2000 rpm, 20 min; 8000 rpm, 30 min). The supernatant was mixed with E.coli MG1655-ompR234 Δ csgA:: Kn-ccdB/pCA24N-csgA-spytag in orifice plate. After incubation at room temperature for 4 hours, the biofilm was scraped off with a cell scraper and smeared on the surface of X80 carbon steel. The adhesion of the biofilm was tested on an 80rpm shaking table.

verification-of-biofilm-adhesion.png

Fig.1 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.

Therefore, Mfp5-Spycatcher and CsgA-Spytag are connected, and Mfp5 enhances the adhesion ability of biofilm.

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:

electrochemical-ocp-data.png

Fig.2 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.

electrochemical-lpr-data.png

Fig.3 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.

electrochemical-polarization-data.png

Fig.4 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)

nyquist-plot-and-bode-plot1.png

Fig.5 EIS curve after coating on electrode for 1 day, from left to right, left: Nyquist plot, right: Bode plot

nyquist-plot-and-bode-plot2.png

Fig.6 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.

nyquist-plot.png

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

bode-plot.png

Fig.8 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.

Finally, we carried out the experiment of biomineralization. We used X80 carbon steel as the experimental material to observe the corrosion situation, and used simulated seawater as the environment of 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.

sem.png

Fig.9 SEM after 7 days of culture in simulated seawater (1).Blank control; (2).CsgA-Spytag; 
(3).CsgA-Spytag+E. coli BL21;(4).CsgA-Spytag+Mfp5-Spycatcher

After scanning by electron microscope, it can be clearly seen that there are obvious mineralized layers on the surface of the fourth group.

biomineralization-verification.png

Fig.10 Corrosion of materials under different coatings, (1).Blank control; (2).CsgA-Spytag; 
(3).CsgA-Spytag+E. coli BL21;(4).CsgA-Spytag+Mfp5-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)

Through the above experimental results, it can be seen that the biofilm material with mineralized layer still has anti-corrosion ability, and the action time is prolonged.

Reference:

[1].Kim E, Dai B, Qiao JB, Li W, Fortner JD, Zhang F. Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion. ACS Appl Mater Interfaces. 2018 Dec 12;10(49):43003-43012. doi: 10.1021/acsami.8b14890. Epub 2018 Nov 27. PMID: 30480422.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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