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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 |
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.
CsgA expression is enhanced, aiming at forming more biofilms.
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:
See Engineering and Experiment for details
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 section.
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.
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.
The second part is to verify the feasibility of enhancing the adhesion of biofilm, experiments are carried out according to the two schemes.
Three plasmids were constructed in this experiment:
BBa_K4635005,BBa_K4635007,BBa_K4635006
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.
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.
The results of crystal violet experiment show that the value of (3) is low and the film forming effect is very poor.
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.
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.
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.
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.
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:
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.
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.
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)
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.
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.
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.
It can be seen from the above experimental results that the biofilm materials (C4, D4) with mineralized layers still have significant anti-corrosion ability.