We added new information learned from literature and the experimental characterization to the previous parts (BBa_K3040118, and BBa_K861050). Furthermore, we have documented the way to culture Propionibacterium acnes with the help of China Center for Type Culture Collection. In addition, we manufactured a high-throughput microfluidic chip for directed evolution and documented the 3D printing process of the part of the hardware assisting the construction of microfluidics.
All of these may be helpful to other teams, and we hope it will make some contribution to the iGEM community.
FadE, the acyl-CoA dehydrogenases, catalyze the first reaction of the β-oxidation cycle. Acyl-CoA dehydrogenase carry noncovalently (but tightly) bounds FAD, which is reduced during the oxidation of the fatty acid. As shown in Figure1, FADH2 transfers its electrons to an electron transfer flavoprotein (ETF). Reduced ETF is reoxidized by a specific oxidoreductase (an iron–sulfur protein), which in turn sends the electrons on to the electron-transport chain at the level of coenzyme Q. The mitochondrial oxidation of one FAD in this way eventually results in the net formation of about 1.5 molecules of ATPs. The mechanism of the acyl-CoA dehydrogenase involves deprotonation of the fatty acid chain at the α-carbon, followed by hydride transfer from the β-carbon to FAD.
In our experiment, we hoped to improve the β-oxidation capacity of our engineered bacteria by overexpressing FadE protein. As is shown in Figure 2, we constructed a recombinant plasmid containing FadE gene and introduced it into our engineered bacteria.
After confirming that we correctly constructed and transferred the recombinant plasmid into the engineered strain E. coli DH5α, we used IPTG to induce the expression of FadD protein and tested its improvement on the fatty acid decomposition ability of engineered bacteria. Our experimental results showed that inducing the overexpression of FadE did not significantly improve the fatty acid decomposition ability of engineered bacteria, and we did not reproduce the experimental results in references.
In order to explore the reasons for the failure of the experiment, we detected the protein expression more carefully. After inducing the expression of FadD and FadE proteins, they were purified with Ni2+ affinity chromatography column. After purification, SDS-PAGE results showed that the molecular weight of our target band was about 20 kDa, lower than the predicted value. The endogenous protease system of strain E. coli DH5α we used has not been artificially knocked out, so the overexpressed protein is easy to be degraded. Considering this possibility, we replaced our engineered strain with E. coli BL21 strain. Then we purified the protein by Ni2+ affinity chromatography column and detected it with SDS-PAGE. The results showed that the bands of FadD and FadE protein were in line with our prediction. However, due to time constraints, we have not been able to test the improvement of fatty acid decomposition ability of engineered bacteria after overexpressing the FadD protein with correct molecular weight in E. coli BL21 strain.
Since we designed a directed evolution pipeline to further optimize the function of the repressor FadR, we have searched a lot of relevant literature and added content for BBa_K861050. We found that the N-terminus binds DNA, and C-terminal domain binds acyl-CoA. In addition, we added the information of a promoter that can respond to FadR well.
3. Document troubleshooting that would be helpful to future teams——Culture of Propionibacterium acnes
We have documented the way and notes to culture Propionibacterium acnes with the help of China Center for Type Culture Collection.
We wanted to build a high-throughput microfluidic platform for directed evolution, and have completed the design, iteration, and fabrication of microfluidic chips. We provided a detailed manufacturing process and corresponding data of microfluidic chip and proved the feasibility of our idea through pre-experiment. For teams that intend to design microfluidic chips, it may be of great help.
We have also documented the 3D printing process of a part of the hardware assisting the construction of microfluidics:
The droplet flow in the chip is observed in real time under inverted fluorescence microscope Experimental results:
1. The chip cannot be fixed and observed by negative pressure at the same time when viewed from the front up beyond the observation range of inverted fluorescence microscope (about 7000μm)
2.The back of the chip can be observed normally and clearly by looking up. However, there are gaps between the chip and the bottom support glass, which may cause the beads and cells to pour out and not in the same glue plane.
The scaffold is designed by 3D printing to match the inverted fluorescence microscope and meet the demand of frontal and upward observation.
The 3D-printed stand has grooves that fits the pipe, allowing the chip and channel to fit perfectly, making it easy to observe the capture performance of the chip.
Through the use of 3D printing model, we successfully solve the problem that it is difficult to observe the chip under the microscope. The chips and tubes will be embedded in the grooves of the 3D-printed model, and the glass slides used to seal it will fit perfectly. Our 3D printed model helps us to realize real-time fluid observation under the microscope, which will better detect the chip performance and carry out subsequent experiments.
Please see our Hardware for more detailed information.
We are delighted to share our modeling methods and experience with future iGEMers. Our differential equation model provides a method for future teams to predict the changes in the number of engineered bacteria and pathogens. The second model of our program offers a method to determine which enzymes should be overexpressed. Future iGEM teams can refer to our method to reduce the number of their wet-lab experiments.
Please see our Model for more detailed information.
In order to give us more knowledge about synbio and make suitable advice to our project, we have done much in Human Practice. First, by interviewing professors, enterprises stakeholders (for example, the shop assistants), etc., we aimed our project to solve the problems that people are troubled and concerned about contrapuntally.
Moreover, in education part, we not only expected others to know acnes scientifically, but to be familiar to synthetic biology as well. It’s our pleasure to lead Skin Alliance this year to produce a meaningful educational brochure. At the same time, we carried out science popularization for all ages and the response is great.
Last but not least, to build a large family of synthetic biology, we have conducted rich communication and cooperation with 5 alliances and 12 partners, deepening our understanding of synthetic biology and our own project.
Briefly, our team this year fulfilled the goals of inclusivity and education in a reciprocal way.
It’s our pleasure to exchange our project with Ascentage Pharama, Humanwell Healthcare and Junweian Live Technology Company, and obtain affirmation and support from them. They provided data to help us complete the market research and analysis of acne drug in Chinese market, and make a complete business plan for our project, so as to comprehensively consider and design the commercial promotion and application prospect of our products in the future.