Mr. Tony project is a large and systematic project of perming and dyeing, which also brings unprecedented challenges to our design and implementation plan. In the process of formulating specific plans, we always maintain close communication with scientists, entrepreneurs and practitioners, and listen to their stories, experiences and methods carefully. Finally, combined with their opinions, we conducted extensive discussions and formulated thorough project and specific implementation plans.Mr. Tony project is a large and systematic project of perming and dyeing, which also brings unprecedented challenges to our design and implementation plan. In the process of formulating specific plans, we always maintain close communication with scientists, entrepreneurs and practitioners, and listen to their stories, experiences and methods carefully. Finally, combined with their opinions, we conducted extensive discussions and formulated thorough project and specific implementation plans.

1 Professors and scientists

We have extensively visited many professors in various fields, and their opinions have played a vital role in our program formulation and problem improvement.

1.1 Xinhua Feng

Figure 1: Xinhua Feng

Professor Xinhua Feng is a member of the China Protein Professional Committee and he has a series of important research achievement in the fields of signal conduction and protein modification. During the experimental design phase, we consulted him how to assemble the PepACS peptide chain and achieve its extracellular expression and detection.

1.2 Dabiao Liu

Figure 2: Dabiao Liu

Associate Professor Dabiao Liu is a professor of micro and nanomechanics in Huazhong University of Science and Technology. When we first measured the mechanical properties of hair, we used a tensometer in the laboratory, and found that our experimental method might be problematic. Therefore, we visited Associate Professor Dabiao Liu who was engaged in micro and nanomechanics measurement. He suggested that we use Young ' s modulus, stress limit and tensile limit length as mechanical indexes to measure the degree of hair damage after perming and dyeing. At the same time, he suggested that we use stress relaxation as a mechanical index to measure the curling maintenance ability of hair after perming and dyeing. He allowed us to borrow the micro-tensile / micro-torsional mechanical testing instrument and three-dimensional depth of field microscope in his laboratory. Under the guidance of his graduate students, we tested the mechanical properties of hair and obtained quantitative experimental results.

1.3 Maoteng Li

Figure 3: Maoteng Li

Professor Maoteng Li is engaged in botany, genetics and other research. In vica-micronucleus test we found that the micronucleus rate of indigo group was less than the negative control group. Therefore, we consulted Professor Li Maoteng who studied botany. Professor Li believed that the high concentration of solution may inhibit the division of cells in the dividing zone, and the existing experimental results alone cannot explain the safety of indigo. Therefore, we took Li teacher ' s proposal to experiment the lower concentration of indigo dye, and got a more convincing conclusion.

1.4 Tie Ke

Figure 4: Tie Ke

Associate Professor Tie Ke is engaged in genetic research. After we finished the vica-micronucleus test, there were many questions about the analysis of experimental data and the reliability of the conclusion. Therefore, we consulted Associate Professor Tie Ke who studied genetics. Mr. Ke acknowledged our results, but also pointed out that abnormal data of indigo group, suggested that we should further experiment. In addition, Professor Ke was very interested in our developed algorithm for counting cell micronucleus, and expressed his willingness to use this tool in the subsequent teaching of this experiment.

1.5 Jiangyun Wang

Figure 5: Jiangyun Wang

Wang Jiangyun Research Group is engaged in new methods of protein and RNA labeling. We communicated with Wang Jiangyun researcher and Liu Xiaohong researcher in the same group, and discussed the selection of quantum dot labeling elements. Finally, 5-carboxyfluorescein was chosen.

1.6 Jun Hu

Figure 6: Jun Hu

Associate Professor Jun Hu is engaged in the research of nanomaterials. In the process of the communication with Associate Professor Jun Hu, we consulted the relevant information of quantum dots, which provided great help for the absorption verification experiment of short peptides. The team eventually decided to use common fluorescent molecules 5-carboxyfluorescein for experiments.

1.7 Xiao Yi

Figure 7: Xiao Yi

Dr. Xiao Yi is a researcher of Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, who has been engaged in the field of synthetic biology for a long time. Through the communication with Dr. Xiao Yi, the team members are more clear about the experimental ideas and development prospects of synthetic biology, and also obtained the relevant experience of rapid expression of target proteins in the laboratory. In addition, Dr Xiao Yi provides suggestions for the experimental design of our pathway and heterologous yeast chassis expression.

2 Enterprises and practitioners

The goal of our project is to produce a truly important product. We deeply realize that there is a huge gap between theoretical experiments and industrial applications. In order to shorten the difference between production, education and research as far as possible, we strengthen the close ties with enterprises from the beginning of the project implementation plan, and put their valuable opinions into the process of project progress.

2.1 Yantai Forestry Textile Printing and Dyeing Company Ltd

Figure 8: Yantai Forestry Textile Printing and Dyeing Company Ltd

Yantai Forestry Textile Printing and Dyeing Company Ltd is a private enterprise which independently develops plant dyes to print and dye textiles. Its plant dye products also win many awards. In order to understand the coloring of plant dyes and to explore better mordant conditions, we visited Li Wei, the technical director of the company. The team members learned that the company had made many important breakthrough scientific and technological achievements in the field of plant dyes, understood the importance of the development of science and technology to the emerging environmental protection industry, and also clarified the major advantages of plant dyes in environmental protection. Director Li also provided advice on the selection of our mordant. Finally, combined with their professional knowledge and the technical experience of the company, the team members discussed about dye development prospects. The company ' s manager also expressed confidence and expectations for our project prospects and downstream applications.

2.2 Lianchuan Biology

Figure 9: Lianchuan Biology

Team leaders Changxin Fan and Jiacheng Shi interviewed Feiran Han, the technical director of Lianchuan Biology, communicated with them about the understanding of synthetic biology and our project design. Director Han pointed out that synthetic biology is now a rapidly developing field. Synthetic biology can be used for research in the upper, middle and lower reaches of biological fields such as basic research, technological development and industrial application. Its core idea is the idea of design and redesign and engineering systematization. With these thoughts, Director Han put forward his own opinions on our project : our project conformed to the standardization of synthetic biology in the construction process and it was also the embodiment of systematic thinking. But there was a lack of some engineering ideas in it. He pointed out that on the one hand, we could integrate multiple functions that we want to achieve; on the other hand, we could add logic gates in it so that more functions could be achieved through regulation in a smaller system, and it is easier to achieve quantitative regulation of each function. Based on the proposal of Director Han, we integrate the two devices of dyeing and fading, and use two different promoters to regulate them, so that the other part is closed when one part works.

2.3 Nine-source gene

Figure 10: Nine-source gene

Team leader Changxin Fan and Jiacheng Shi had a discussion with Feihu Xu, the head of Jiuyuan Gene Development Department. Both of them discussed the characteristics of various chassis for synthetic biology applications, especially for Pichia pastoris GS115. Director Xu has a wealth of experience in heterologous expression using yeast as a chassis organism. He pointed out that signal peptides played a very important role in heterologous extracellular expression using the eukaryotic system, while signal peptides from different sources and different structures are different. Some signal peptides may not secrete a certain protein, but another signal peptide or protein can be synthesized and secreted in large quantities. Eukaryotic system expression is a very complex process, the transformation, screening, replication, transcription, translation, folding, transportation and other processes all need to be considered and optimized. Based on the suggestions of Director Xu, we chose the α-factor operon with commonly used signal peptide for extracellular expression, and the operon without signal peptide for intracellular expression. All components were expressed in both intracellular and extracellular ways and we should verify which expression is more appropriate.

2.4 Barbershop

Figure 11: Barbershop

In the preliminary experiment of the project, we found that with temperature and other conditions the coloring effect of chemical dyes and plant dyes fluctuated greatly. In order to find a solution, iGEM HUST-China team members visited a private barbershop in Wuhan, Hubei Province. During the conversation with the manager, we learned that in the practical application process, hair dye does not act solely on hair, and the coloring effect itself is closely related to temperature and oxidant content. The manager also recommended some tentative plans for perming and dyeing hair. In addition, we have learned that the low prevalence of plant hair dyes is mainly due to their relatively high prices and poor color fixation effect. The barber 's manager also revealed expectations that technology could help improve products.

3 Our design

After a series of visits and consultations, we finally confirmed Mr. Tony ' s overall project plan combined with the suggestions of scientists and professors.

Mr. Tony project combines promoter, signal peptide, gene encoding protein, terminator and other parts to form a device that can express a certain kind of protein, and then combines functional related devices to form a system. We use molecular biology and microbiotechnology to construct the system, which can express some enzymes to synthesize a kind of pigment or short peptide to perm, and express other enzymes to degrade a kind of pigment or straighten the hair. We then transfer the constructed three systems into three engineering yeasts using Pichia pastoris GS115 as the chassis strain. Through this way, when glycerol is used as carbon source, engineering yeast could express short peptides that can make hair curls and enzymes that can synthesize pigments. These enzymes could synthesize three kinds of pigments respectively with the added components in the system as substrates. When xylose is used as carbon source, engineering yeast could express short peptides used to straighten hair and enzymes that degrade pigments, using three pigments as substrates, respectively. After the fermentation of engineering yeast, the pigments, decolorizing enzymes and short peptides are separated and extracted. We carry out a series of experiments with the extracts to determine the optimal working environment of the project and compare the advantages and disadvantages of traditional chemical perm and dye and biological perm and dye in this project.

I'm so impressed!!! Thank you for reminding me that the process of developing a plan also needs to be accompanied by communication and practice!! I'm even more curious about your third part: Project development and assessment!