1.The original intention of the project
Many people's hair quality has become very poor due to perm and dyeing, so everyone wants to use a good conditioner to make their hair quality better, but in the experience of the people around us, there is no conditioner on the market can be a good solution to this problem. Therefore, in order to solve the above problem, we carried out a survey of conditioner brands and screening of ingredients, and finally our team decided to develop an amino acid conditioner that is suitable for the general public and has safe ingredients.
2.Experiment Part
Before the experiment formally started, we found two kinds of E. coli containing the target genes we need, they are DH5α/pET23b and DH5α/pSB1A3-insert, on the first day of the experiment we have to carry out the preparation of the experiment, we will first amplify the number of these two kinds of E. coli, so that we can extract the target genes later on, after the completion of the test tubes with the E. coli will be put into the shaking table at 37 degrees Celsius and wait for the bacterial amplification to be completed. After that, the test tube with E. coli will be put into a shaker at 37 degrees and wait for the bacterial amplification to be completed. The next day after the preparation, we started the next step, which is to extract the plasmid and use PCR to amplify our target gene fragments. Before using PCR we need to prepare an agarose gel, which is used to perform agarose gel electrophoresis. Once the gel is prepared we can start the PCR amplification. In this step we need our plasmid, two primers (IF and IR) and PCR product.After PCR, we start agarose gel electrophoresis, add the PCR product to the agarose gel, then add the DNA marker, and then start electrophoresis. At the end of electrophoresis, we can cut the gel, and put the cut gel into a centrifuge tube for storage. On the third day, we first need to do gel recovery of the gel cut on the previous day and determine the concentration of the recovered DNA using Nanodrop. The next step is to perform a double digest, we need to double digest the extracted plasmid pET23b and the amplified target DNA fragments using EcoRI and XhoI. All we need for this step is our DNA, EcoRI, XhoI, buffer and water. After preparation the digest was performed at 37 degrees C. After 4h the digest was finished and we added the digest to an agar gel and added our DNA marker again to start electrophoresis. At the end of electrophoresis, we looked at the image and cut the gel again and put it into a centrifuge tube. The next step is to recover the digested product from the gel, after recovery the DNA is stored at -20 degrees Celsius. The last step was to activate E. coli DH5α and E. coli Rosetta, and our E. coli Rosetta would be used to prepare the receptor cells that would be used later. On the fourth day we need to ligate the DNA from the previous day, in the ligation reaction we need T4 DNA ligase, DNA ligase buffer, linear plasmid, and insert DNA. we need to put it into a PCR machine at 16 degrees Celsius to get the pET23b-insert, and then ligate the product at 20 degrees Celsius at -20 degrees Fahrenheit. environment to ligate the product. After ligating we configured LB solid medium and poured it into a petri dish, then all we had to do was to transform the ligated product. The ligation product was added to the receptor cells, after which liquid medium was added for recovery and it was applied to the Amp antibiotic plate. By the fifth day, we need to have performed the PCR verification. After labeling the colonies we needed to pick some as templates and perform colony PCR using Taq enzyme. after PCR we reconfigured the agarose gel and electrophoresed it using a DNA marker. Next we picked the correct colonies and added them to the medium and then put in antibiotics for expansion. After expansion, the plasmid was reextracted from the correct colony and transformed pET23b-insert into E. coli Rosetta, and the recombinant Rosetta-pET23b-insert strain was inoculated into tubes containing Amp antibiotics to expand the culture. After the culture was completed, the E. coli Rosetta strain was inoculated into test tubes for activation to be used as a control. On the sixth day, we took samples and placed them on slides to look for our fluorescent proteins using a fluorescence microscope and collected images. After the acquisition we tested the expression of the silver light proteins with an enzyme marker and finally presented it in an excel spreadsheet.
3.How to do a good job of statistics
Our team's statistics include the brands of conditioners that people like in their life, the price range of conditioners that they often buy, the reviews of conditioners with high usage rate in the market, and the volume of conditioners that people often buy. These data will also be put on our wiki site for the next IGEM team to use. During the counting period, we used a questionnaire to collect the data, and checked the price, rating and ingredients of the brands in the questionnaire in the major shopping apps, and finally used an excel sheet to organize the data to make the results clearer.
4.How to make more people understand and learn about synthetic biology
We have learned that many people can't learn synthetic biology completely, so we have a general plan to let more people get in touch with and learn about this discipline. Firstly, we hope that there can be a section in our wiki page for people to learn, and secondly, we will also prepare some professional books and electronic resources for people to use. We hope that in the future, other iGEM teams can build on our foundation and improve the content of our resources, so as to jointly promote the learning of synthetic biology.
5. How to publicize the product and find suitable investors
Here we have a total of two options, option one is to go to the mall to find the brand counter attendant or store manager, show our product efficacy and use, ask them if they are willing to cooperate or invest with our team in the future, option two is to find the official customer service of the relevant brand or email, online to understand the situation, but also to get in touch with the PR of the major brands, introduce our products and try to get with them Business cooperation. We will also organize and summarize the channels we use into a table for your reference.
6.How do we find inspiration in wiki design?
For wiki design, the most important thing is the style and background of the wiki, we looked up the wiki interfaces of many 2022 and 2021 teams that won individual awards in wiki design from the official website of the event to learn and organize, and we also looked up the designs of the bigwigs on other websites that we thought were of reference significance, and in the course of continuous discussion, our wiki finally took shape.
7. How to find experimental ideas
With the basic idea in mind, we have a lot to learn about synthetic biology, which includes the principles behind the experiments and manipulations. Since most of the people suffer from hair loss, dandruff and hair damage, we will go back to the root of the problem and start from 0 to find the root cause of the problem. After pre-investigating the causes of the aforementioned problems, we started looking for countermeasures. In order to make our conditioner safer, we decided to use biology as a starting point to find the genes to improve the problem in bacteria, and when we found them, we started our genetic engineering, and finally completed the experiment by multiplying the successfully converted bacteria and taking out the parts we needed. Our team agreed that our research tools can shine the light for future IGEM teams to follow our team's research ideas to complete their own team's experimental projects, which can greatly shorten the time and improve efficiency.
8.data
Hair Dyeing Systems
Standard curve for melanin determination
LB medium was added with different concentrations of melanin and we measured and recorded the absorbance at 400 nm.
Measurement of melanin content
Dopa (L-DDOPA) and tyrosine (L-Tyrosine) were added as substrates in LB medium and the bacteria were cultured in a shaker. Every 4 h, ml of bacterial solution was transferred to EP tubes, the bacteria were broken and centrifuged, the absorbance at 400 nm was measured and recorded, and the production curves were measured over a 24-h period.
Effect of temperature on melanogenesis
The bacteria were incubated in a shaker at 37 and 25 degrees. OD400 was measured at 24h and the melanin concentration was converted from the standard curve.
Effect of pH on melanogenesis
The pH of the LB medium was adjusted to 7, 6, 5 using hydrochloric acid. the bacteria were incubated in a shaker at 37 degrees. OD400 was measured at 24h and the melanin concentration was converted from the standard curve.
GshF enhanced GSH production (2h) （picture A）
The engineered strain was constructed by coupling GshF downstream of the constitutive promoter plac.To test the production of GSH, the strain was resuspended in LB medium to an OD600 of 0.1 and incubated at 37 degrees Celsius for 2 h. The content of GSH was determined retrogradely by a reduced glutathione (GSH) content assay kit.
Effect of GshF on bacterial growth （picture B）
The engineered strains were constructed by coupling GshF downstream to the constitutive promoter plac. Initially, the strain was suspended in LB medium until the OD600 was 0.1 and incubated at 37 degrees C. Samples were taken at 4, 6, 8, and 12 to measure the OD600 and to determine the effect of GshF on the bacterial growth.
Effect of glutathione on bacterial growth （picture C）
Wild type E. coli, resuspended in LB medium to an OD600 of 0.1, incubated at 37 degrees Celsius, different amounts of glutathione were added, and samples were taken at 4, 6, 8, and 12 to determine the OD600, and the effect of glutathione on bacterial growth.
Effect of precursors on GSH production (2h) （picture D）
Whether to add amino acids
Enzyme Viability Test （picture E）
Effect of oxygen on GSH production (2h) （picture F）
Constructed engineered bacteria. The tested engineered bacteria were statically incubated in a CO2 incubator (adjusted O2 concentration of 0%, 20% and 30%). after incubation at 37 degrees for 2h, the GSH content was determined by a reduced glutathione (GSH) content assay kit.
picture A to F
