For Integrated Human Practices, what we should do should have directly “impacted [our] project purpose, design and execution.” With the criteria as our guidance, we derived five purposes for our HP works. There are: studying the market so that the product will be desirable once it comes out and more people will be free from salmonella infection (this has to do with “impacting the design”), learning how the current food testing industry works to make sure that our product will be applicable once it comes out (“execution”), learning relevant experimental techniques through interviews and workshops so that we can actually produce a product that is workable and beneficial (“project purpose”), collaborating with multiple iGEM teams to build connections and learn collectively, and finally educating the younger generation and passing synthetic biology down and to the public. As you can see, everything we did was directly impacting on the project purpose, design, and execution, while also adding spreading awareness and building connections.
As mentioned, since there were five purposes for human practices, what we’ve done can also be divided into five categories. First, for our project design, we conducted market investigations to understand the market; they include conducting a public survey on the purchase habits of eggs, and understanding the egg market through multiple visits to chicken ranches. Secondly, after understanding the market, we needed more background knowledge on how the inspection industry works and the execution of the product (test kit), so we investigated the current food testing methods. We interviewed a third-party food inspection agency (SuperLab), had a workshop with Creative Microbiologicals, and visited the national lab of the Food and Drug Administration of Taichung to understand their methods of food testing. Thirdly, to ensure that our experiment reaches this goal, we interviewed Dr. Steven Hagens from Micreos and Dr. Chih-Hsin from I-Shou University. They gave us sufficient knowledge on bacteriophage modification, so that we can perfectly execute our experiments. The fourth category is education, in which we came up with a material (board game) that we then brought on education trips to schools and even to the Taiwanese National Museum of Natural Science. Lastly, we met up with multiple iGEM teams and even had a long-term partnership with iGEM CSMU Taiwan. The works in all human practices are multidirectional, broad, and holistic.
Understanding the market has always been crucial when developing a product and it wouldn’t be an exception for our project. First or all, our team conducted a public survey with the local community through having walks into the neighborhood, holding stands in the park, sending surveys electronically, and us simply asking people around us. With a total of 1,025 survey results being collected, we had a very clear image of what our product should look like for it to be applicable to the market.
In order to investigate the market, we asked eight questions on their purchase habits. Starting with the question regarding their age. Since the target market is mostly for people who shop for eggs, homemakers most likely, we mainly surveyed people at the age from 25 to 65 (they took up about 50.7%), while people from 19-25, who also have little market power, still accounted for another 5%. Overall, the surveyed audience fit very well to our target market, meaning that it is an effective survey. We then asked them if they had heard of salmonella. It turned out that 84.9% of the interviewees have in fact heard of it. This surprisingly large percentage indicates that many people do recognize that there’s such a crisis that needs to be resolved. However, do they really understand how serious this problem is? This is the most crucial question.
When we asked them if they know how many people are infected with salmonella each year, only 13.3% of the respondents answered correctly. With an incorrection rate of over 86.7%, who all guessed “below” the correct answer, a hundred thousand to one million. This shows that most of our interviewees are unaware of the seriousness of the problem. Therefore, we then taught them more thoroughly what salmonella is and how it infects people. For electronic surveys, we directly inserted a text box explaining what it is. By asking this question, we were more certain that we were doing the right thing because we were able to save more innocent people from infection.
The question followed after making sure that the interviewees have a complete understanding of what salmonella is, we asked them about their preference of a salmonella detector-- which kind of product they prefer to buy. The first question asked if they would prefer having the upstream companies/labs detect salmonella for them or having them do the detection process themselves. Within our expectations, 82% of the respondents preferred having others test for them even if the price would be higher. Some interviewees even mentioned that “what is the purpose of us testing it after we’ve already purchased the eggs, if there is really salmonella present, we can’t refund it!” or some said that “why should the consumers be testing themselves, they are the ones being served!,” (translated). These are all solid claims which made us believe that a product for the labs and companies will be more applicable than a product for the general public.
We then asked what kind of product they will want to buy or have the companies/labs use, the options are a spray, a test paper, and a test-kit.The options were the three solutions that we were considering to produce at the early stage, so their preference would help us decide what we would then focus on. 25.4% of the surveyed population prefered a spray, 34% chose a test paper, while 54.2% preferred a test kit. The reasons given were very straightforward, the most common one being “a test kit sounds more professional and is more hygienic” (translated).
All in all, through this survey on a valid population, with the majority having actual market power, we were able to narrow down on our product. The first thing we learned was that although many people might have heard Salmonella before, they weren’t fully understanding the real impact of it, which made us more certain to produce a product that tackles this problem. Secondly, people prefer having the eggs to be tested by upstream companies and labs, meaning that we should design a product that is designated for them. Finally, a test kit is most likely to be accepted by the general public as it is considered to be more “professional” and “hygienic.”
After knowing from the survey that we should better be designing a product for the upstream companies, we decided to understand how the companies detect salmonella on their own products since they have changed into our main market now.
We reached out to Ho Fong Chicken Ranch, a chicken ranch in Nantou, Taiwan, famous for their perfect chicken raising environment. Located in the mountains of Nantou, with an altitude of 500m, they have an ideal water source, perfect temperature and geography. One of the reasons we decided to reach out to them was that they do not rely on another company that sells their products, they are not only manufacturers. They usually sell their products directly to their customers, meaning that they have their own inspection problem, making it worth the visit.
We would like to appreciate Ho Fong Chicken Ranch for giving us the opportunity to visit their chicken farm and understand the production process of eggs and most importantly, the testing process of eggs. They introduced us to the concept of washed and unwashed eggs. Eggs are divided into washed and unwashed. Washed eggs, as its name suggests, are eggs that have original pathogenic bacteria on the chicken washed away, but the original mucosa will also be washed away, meaning the protection will disappear. Non-washed eggs, on the other hand, have more pathogenic bacteria on them, leading to a great possibility of them appearing on the food. Smaller chicken ranches mainly produce non-washed eggs due to the lack of machineries, meaning that the eggs that ranches like Ho Fong sell have a greater possibility of having salmonella. After hearing about this problem, we introduced our project to the founder of Ho Fong chicken ranch because a test kit might come in handy for companies like them that have no laboratories for further inspections. We introduced our idea to them and they gave us an honest opinion, the founder of the chicken ranch said that “it seems like a good solution for smaller chicken ranches like us because we don’t have a lab of our own like other bigger franchises.” This made us certain that our project does have its own market and can actually help many people and companies.
After understanding the general public and our potential customers, upstream companies, we moved on to getting more information and knowledge regarding the process of salmonella testing. We have to learn how similar companies are tackling this problem.
SuperLab is the third party food inspection agency that collaborates with the government quite often, many upstream manufacturers also rely on them for their products’ safety inspection. They accepted our request for a real life workshop at our school when we were still uncertain what to do; they came and lectured us on how they inspect Salmonella on food, the equipment they used, the processes undertaken… etc. For example, they listed their common food contamination detection methods (especially for Salmonella).
1. The amount of pathogenic bacteria tested is at least 25 g (mL)
2. Dilute 10 times for trophic enrichment of bacteria, cultivate for 24 hours
3. Take a small amount for selective enrichment and culture for 24 hours
4. Inoculate selective medium and cultivate for 24~48 hours
5. Transplant suspected colonies to nutrient medium and cultivate for 24 hours
6. Conduct biochemical and serological identification
From the above list that they’ve given to us, it can be said that the process takes a long time and isn’t efficient. After explaining everything, they mentioned the difficulty of the process, which is, as mentioned, that the process usually takes up to a week and requires many kinds of laboratory materials and culture media (for nourishment, selection, identification). This inspired us to target the inefficiency of the process, shortening the process to within a day.
Founded in 1975, Creative Microbiologicals is currently Taiwan’s largest supplier of microbial products. It is a manufacturing company of multiple test kits, not only for food inspection, but also for common virus infections including COVID. We would like to thank Creative Microbiologicals for joining our collaborative workshop. During that experience, experts from the company introduced us to their current ways of Salmonella detection and provided some feedback on our method of detecting Salmonella. We understood that they are using a different approach to detect salmonella on eggs. They also use bacteriophage to infect the bacteria, but they are eliminating all the bacteria other than salmonella. Once all other bacteria are killed, they will have a testing paper to see if there are any salmonella on the egg. This is the complete opposite of how we approached the project. The down side to their method, as they recalled, is that the time required for other bacteria to die off takes a long time and there are too many variables to that method as you don’t exactly know what other bacteria are on the eggs. Therefore, engineering a bacteriophage that only focuses on salmonella, which is our method, “might have been better,” said Mrs. Hsieh, an executive experimenter from Creative Microbiologicals. Moreover, they also taught us about the current regulation and laws regarding food security in our country so that we could have a better understanding of our market.
The Food and Drug Administration of Taichung city granted us an opportunity to visit the national lab used for food safety tests. It gave our team new insights on how the industry operates. They also introduced us to all of the regulations on dairy products (since our targeted problem appears on multiple dairy foods), the microbiological hygiene standards, and the current micro, which helped us make sure that our product is applicable to the actual market.
Specifically, what we learned was that if the bacterial strain on a dairy product is identified as suspected Salmonella-infected by the commercial biochemical kit, then an ontology group antiserum test and a flagellar serum test are required. If both are positive, it is judged to be Salmonella positive. That’s why it is needed for our product to be conducted in the lab for further testing for confirmation. Moreover, they’ve also demonstrated their experimental flow chart of methods of testing Salmonella in detail, giving us an opportunity to compare with how it differs from our approach. Not to our surprise, their method requires at least five days. The first four days are for bacteria enrichment, adding RV broth or TT broth for water-bath, adding XLD, BS and HE agar for culturing, and then for TSI and LIA agar slant culture-medium. This reassures the competitiveness of our product as our method seems to be faster and more effective. Having a chance to get a sneak peak in a national laboratory is once-in-a-lifetime, we really appreciate the Food and Drug Administration of Taichung for this opportunity.
After understanding the market and also how other people approach the problem, how others inspect salmonella, we moved on to the execution part of the project, we had to gain more knowledge to perfectly execute our plan. Therefore, we turned to professionals and professors who had experience dealing with salmonella and bacteriophage.
Prof. Hung is a professor at the Department of Chemical Engineering of I-Shou University. He was kind enough to provide us with an opportunity for an interview and a visit. To begin with, we made a visit to their P2 laboratory, where the professor shared some skills about his past experiments with us. Later on, during the interview, we received lots of important information regarding phage, including where to extract phage and how to isolate phages against a specific bacterial strain in nature, which both of these helped a lot when we were getting the “main character” of our experiment. He even taught us the different kinds of phage and suggested that we use Salmonella Phage Lambda1105. He helped us build up a phage engineering system in our lab and guided us setting up standard protocols to deal with phages. Everything that he has given to us has been very essential to our project. We greatly thank him for his enthusiasm to educate high school students and his invaluable experience in phage research.
Understanding how biotechnology companies deal with bacteriophage is also crucial and will be a crucial guide for us conducting the experiment. Therefore, at the very beginning of the project, we reached out to Micreos, one of the biggest bacteriophage engineering companies in the world, and arranged an online conference. In the conference we were met by the chief security officer from Micreos, Dr. Steven Hagens, and had an hour-long meeting with him. He first guided us through the usual process of phage modification and gave us some tips to better infect a phage with DNA. We also introduced our idea to him, and were lucky enough to receive remarkable feedback from such an experienced professional.
Firstly, he gave us a crucial comment that changed our experimental direction completely. Since it was the start of our project, we hadn’t really finalized our plan yet and the plan wasn’t good enough. Dr. Hagens helped us point out a major flaw in the first plan-- GFP’s lack of ability to transmit and magnify signals (their effect would be unnoticeable on the eggs). He pointed out that “after [the] cell gets infected, it only produces protein for a short period before its death.” It was not only because of the short amount of time given, he said, it was also because “the bacteria are quite widely spread on the egg and even if the amount of bacteria is enough, they might not be concentrated enough to show visible effect.” This was proven by our first trial when we found out that the fluorescent effect couldn’t even be seen because there weren’t enough reactants for it to react. Eventually, we moved on to using phi29 DNA polymerase to initiate rolling circle amplification and do what GFP couldn’t do, magnify the signal.
After that crucial comment, he then talked about how phages will interfere with normal cell life, so no other proteins will be produced except the one from the phage promoter, indicating that a promoter is essential. With this kept in mind, our later experiments reflected on this statement by conducting a T7 promoter (more in Project). Another general comment that he gave while listening to our presentation was that washing the egg and detecting the bacteria in the water used for washing may also be a solution. This is another great idea that we later on considered doing, but after conducting the market research, we found out that this might not be applicable as it will become a product that is for the customers not for what the surveyed audience wanted-- the upstream companies. However, this will definitely be our extension project for us to conduct in the near future.
In the second part of that meeting, we asked them questions regarding the safety issues of bacteriophage modification. Listed below are some of the major questions that were raised during the conference (the answers have been summarized).
Q1: In order to remain safe, what should we be cautious of when it comes to modifying a phage? What if it leaks?
A1: As long as the experiment is conducted in the lab, then there will be no issues. However, if it happens to escape into the environment, because the gene used is not really dangerous, the environmental effect should be low. These modified phages may have evolutionary disadvantages when compared to natural phages so it might not be an issue as well. Lastly, just don’t give [inject] it to humans.
Q2: Can products like this (with a modified phage) be published? What will be some concerns (safety-wise) for this product to be published?
A2: If it works, it would be profitable with little safety concerns as long as it was conducted in the lab. May need to be careful with gene and technology patents.
Q3: What should we keep in mind when modifying a phage in order to keep it alive? What are some potential threats to phage? What will affect the activeness of phage? (Temperature, humidity… etc.)
A3: If putting a gene in random position may stop normal phage mechanism, then a clear promoter and position will be needed (look for a terminator of a portion of the gene and insert it there). Some phages can not accept extra genome material, but this is dependent on the phage.
Overall, for safety issues, there weren’t many concerns as long as the experiment was conducted in labs and with all safety precautions being made. For modifying a phage and keeping it alive, a promoter is much needed, which we did add in the end. Much thanks to him for first introducing their ways of modifying bacteriophage and beyond that, for pointing out the major flaw as well as giving aforementioned comments that were extremely crucial. We can’t thank him enough.
Education and communication have been done as usual (more in Education and also Communication). In short, for education we can divide the work into two parts, one is us educating others, another is us getting educated. For the first type of education, we came up with a material (board game) on the topic of bacteriophage modification. It is designed to educate the public on how bacteriophage helps solve problems around the world (in short, each player is playing as a bacteriophage, trying to find a matching DNA to solve the crisis given). It is an interesting way for people to learn how bacteriophage works and, most importantly, as they slowly realize how powerful a phage can be when facing all the global issues, it turns into a game that evokes their passion for synthetic biology, changing people’s mind on synthetic biology. We even brought this game to educate elementary and junior high students, and also to the STEAM fair to teach high school students about synthetic biology, and we also took this material with us to present at the National Museum of Natural Science, influencing hundreds of audience on the importance of synthetic biology.
For the second part of education, we were first taught by multiple college professors on bacteriophage engineering, including the aforementioned Chih Hsin Hung. We mainly discussed engineering phage with synthetic biology and its application, and safety concerns. We then reached out to Genmont Biotechnology Inc. to discuss and learn from their biotechnology product development experience. We also learned from the Food and Drug Administration of Taichung city, in which we discussed the bottleneck between traditional law and new technology. For details and more please visit both Education and Communication.
Collaboration has always been crucial for human practices and it is also what we value a lot as we believe that iGEM is also a place to know more people from around the world and build connections (for more details of each please visit Collaborations and Partnership). We mainly partnered up (long term) with team CSMU_Taiwan on a series of year-long projects. While also maintaining an almost one-year series of projects with TAS_Taipei as well (more of these two in Partnership). Other outreach projects include the booklet on synthetic biology that we came up with together with iGEM Warwick, the Emoji competition that helps spread synthetic biology to over 1,000 people online with team Aix-Marrseille, the “expARTiment” project initiated by team NOUS, the guidance we gave for team KCIS_NewTaipei at the beginning, and the traditional meeting we have with team NCKU_Tainan. From experimental collaboration to spreading awareness, we were able to collaborate with multiple teams under this hard time; collaboration is something that should never be let go of when it comes to iGEM. For details and more please visit both Collaboration and Partnership.
In conclusion, everything that we did directly “impacted [our] project purpose, design and execution.” From understanding the market, understanding how the industry works, understanding the skills and techniques required to run this industry, to education and collaboration, the HP team has carried out multiple works that complement the entire project. Without the HP team, the project couldn’t have been so successful. Big thanks to the Food and Drug Administration, MOHW, SuperLab, Creative Microbiologicals, Ltd, Genmont Technology Inc., Ho Fong Chicken Ranch, Dr. Steven Hagens, Prof. Chih Hsin Hung, and others who helped us with our HP group.