INTEGRATED HUMAN PRACTICES
Our human practices helped us thoroughly develop our project, from its initial conception to its design and execution. Our project was inspired by Dr. Che-Hong Chen, who came to our school to talk about ALDH2 deficiency. After we decided on our project, we interviewed Dr. Stephanie Hsieh, a medical doctor, and called hospitals to understand what is currently being done about ALDH2 deficiency. We learned that there is a much greater focus on other alcohol-induced diseases, and a relative lack of knowledge about ALDH2 deficiency and its increased cancer risks. This helped reaffirm the importance of both raising awareness and actually developing a treatment--producing recombinant ALDH2 to reduce acetaldehyde buildup in ALDH2 deficient people. As we tested the enzymatic activity of our recombinant ALDH2, we also consulted Dr. Chen, who helped troubleshoot some of the issues we were running into in our functional tests. At first, we ran tests using bacterial cell lysates, but Dr. Chen pointed out that there are too many other factors if we use lysate, and suggested that we add a HIS-tag and purify the protein to get clearer results. This helped tremendously, and we were able to obtain much better results with the purified proteins. Next, we wanted to target acetaldehyde buildup in the upper digestive and respiratory tracts, so we looked to the public for their preferred method of delivery. Our survey showed that people preferred delivery using probiotics, and only wanted to take this treatment if and when they drank. Thus, to learn more about common probiotic strains and the use of probiotics in manufactured food products, we visited Yakult, a yogurt manufacturer. Since Yakult uses Lactobacillus casei, a commonly used and widely known probiotic strain, we also tried to use it as the chassis. Our transformation attempts turned out to be unsuccessful, but fortunately, through the Asia Pacific iGEM Conference, we were introduced to Dr. Ying-Chieh Tsai, an expert in probiotic research, who suggested using two other strains: Escherichia coli Nissle 1917 and Lactococcus lactis. Every step of the way, our human practices work influenced the direction and outcomes of our project.
Read more about each activity below!
DR. CHE-HONG CHEN
Stanford ALDH2 Researcher & TAIES Cofounder
Dr. Che-Hong Chen is a molecular biologist and geneticist researching the ALDH family at Stanford University. In September 2017, Dr. Chen came to our school to give a talk as a part of our Research Speaker Series. In his talk, he discussed the misconceptions about Asian glow and its actual cause: ALDH2 deficiency. From this talk, we learned that there is a lack of awareness about this serious problem, and were inspired to develop a simple solution to mitigate the adverse health effects of ALDH2 deficiency.
DR. STEPHANIE HSIEH
We consulted Dr. Stephanie Hsieh to learn more about ALDH2 deficiency and about different types of treatment from a medical perspective. She believes that the health risks of drinking are overshadowed by those of other cultural habits, such as betel nut chewing and smoking--both have long been a part of Taiwanese culture, and their hazards are more publicized. In Taiwan hospitals, free screenings for oral diseases are provided for people over 30 who smoke and chew betel nut, but few provide screening for ALDH2 deficiency. (Interviewed by Catherine C, Tim H, Iris H, and Emily T)
What is currently being done about ALDH2 Deficiency?
To find out what is currently being done by healthcare systems, we called multiple hospitals in Taiwan. Like Dr. Hsieh said, all of the hospitals we called have a “Smoking Cessation” department, where patients can go through programs to quit smoking and get checkups for smoking-related health effects. When we asked about ALDH2 deficiency, however, we were surprised to find that most prominent hospitals in Taiwan do not have any form of testing for ALDH2 deficiency, and the few that did were part of extensive blood tests and expensive (2180 NTD). This helped reaffirm the need to both raise awareness of and actually develop a treatment for the adverse health effects of ALDH2 deficiency. (Calls made by Longan S, Catherine C, Colin H, Christy C, Chloe W, Nicole C, Iris H, Caroline C, Emily T)
DR. CHE-HONG CHEN
Stanford ALDH2 Researcher & TAIES Cofounder
In our experiments comparing the enzymatic activity of normal ALDH2*1 and mutant ALDH2*2, we first used E. coli cell lysates. Although our results showed that the produced ALDH2 enzymes were functional, Dr. Chen recommended that we purify our recombinant ALDH2. He pointed out that many other enzymes in the bacteria may also utilize NAD+ and convert it into NADH, which interferes with our test. We listened to his advice, and by purifying our ALDH2, we obtained much better results with the purified enzymes. (Interviewed by Longan S, Charlotte C, Justin L, Justin Y, Leona T, and Catherine C)
We created a questionnaire covering a broad spectrum of project topics. With close to 700 (n=697) responses, the results of our survey ultimately helped us shape our construct design and final delivery prototype.
Since we seek to combat acetaldehyde buildup in the head and neck regions, we considered treatment options targeting these areas, such as oral medication, nasal sprays, and probiotics. When asked the preferred method of treatment, we found that 53.4% of the people preferred using probiotics as the method of delivery. Since probiotics are commonly used, beneficial, and most importantly, safe to consume, it is no surprise that this is preferred method of treatment. In addition, most people preferred treatment only when they drink (as opposed to taking a product daily, weekly, etc.). This would require the product to remain in the mouth, so we envision delivering our ALDH2 enzyme as a throat lozenge to be consumed before or during drinking. (Survey questions by Emily T, Iris H, Justin L, Chloe W, Catherine C, Christy C, Nicole C, Colin H, and Longan S)
YAKULT FACTORY VISIT
Yakult yogurts (Image from here)
Based on our survey results, we found that the general public preferred to have treatment delivered through probiotics. To learn more about common probiotic strains used in foods, the culturing process of lactic acid bacteria, how the final probiotic products are manufactured, and how safety is ensured, we visited a major yogurt manufacturer, Yakult.
Yakult uses Lactobacillus casei (L. casei). Not only are there more than a hundred peer-reviewed publications involving human studies with L. casei, but Yakult was also awarded Food for Specified Health Use (FOSHU) status in Japan in 1998 (Yakult, n.d.). “FOSHU is the only type of food product (not ingredients) that can carry health claims and is composed of functional ingredients that affect the structure/function (physiological functions) of the body” (Saito, 2007).
After our visit and learning more about Yakult’s culturing and use of L. casei, we hoped to also use L. casei as a potential chassis for delivery. We obtained a pure strain of L. casei from the Bioresource Collection and Research Center (BCRC) and we were able to cultured it in lab, but our attempts to transform L. casei by electroporation were unsuccessful. (Visit by Catherine C, Leona T, Tim H, Justin W, and Justin L)
ASIA PACIFIC IGEM CONFERENCE
& DR. YING-CHIEH TSAI
Lactic Acid Bacteria Researcher
At the Asia-Pacific iGEM conference, we presented our project to a group of professors and over 20 other iGEM teams.
There, we received helpful feedback about our project, and more importantly, we were able to get into contact with an expert in the field of probiotics, Dr. Ying Chieh Tsai.
Dr. Tsai is a molecular biology professor at National Yang Ming University, founder of Taiwan Association of Lactic Acid Bacteria, and former president of Asian Federation of Societies for Lactic Acid Bacteria. Prior to meeting Dr. Tsai, we were having trouble transforming L. casei. He helped us troubleshoot our transformation process, but also told us that electroporation of L. casei is uncommon in Taiwan. Instead, Dr. Tsai suggested using two other probiotic strains, Escherichia coli (E. coli) Nissle 1917 and Lactococcus lactis (L. lactis): E. coli Nissle 1917 for its ability to be chemically transformed, and L. lactis for its ability to efficiently synthesize proteins. (Conference attended by Tim H, Catherine C, Anna C, Justin W, Longan S, and Yasmin L; Correspondence with Dr. Tsai by Leona T, Tim H)
PUBLIC ENGAGEMENT AND EDUCATION
Through public engagement and education both within and outside of our school, we sought to ensure that our project is ethical, responsible, and beneficial to our community. In a bioethics roundtable discussion, we examined our project through different perspectives; this helped us think about some of the possible unintentional consequences (e.g. increased alcoholism) and determine how to carry out our project responsibly. Through our marketing plan, we considered the health food regulations as well as safety and labeling requirements needed to ensure that our product would be created, tested, and marketed responsibly. Our examination of current alcohol labeling regulations and policy brief also sought to encourage the Taiwanese government to develop better health warning labeling practices. This hopefully will promote more responsible drinking habits in a country where almost half the population is ALDH2 deficient and susceptible to increased cancer risks. Finally, through our talks in health classes and class meetings at our high school, we encouraged students to be more conscientious of the health risks associated with ALDH2 deficiency as they approach Taiwan’s legal drinking age of eighteen.
Read more about each activity below!
BIOETHICS ROUNDTABLE DISCUSSION
In February, we hosted a Bioethics Roundtable, where we invited students and teachers to discuss the social, ethical, and medical aspects of our project. Hoping to generate discussion and new perspectives, we developed a set of questions, assigned different roles to participants, and asked for opinions from the respective views of each assigned role.
“Should ALDH2 testing be a free test service for people? In what form would you want ALDH2 testing to be available? Kit? Hospital testings? Who should pay for the test?”
The designated roles for this question included Government Official, College Student, Hospital Administrator, Employer, and Alcohol Manufacturer.
There was no apparent preference for home-kits versus hospital tests, but a strong preference for tests to be covered by the government through the National Health Insurance. Some hospital administrators even suggested that the government should raise taxes on alcohol in order to fund these tests. On the other hand, government officials argued that the government should not be held responsible for providing testing services because drinking is a personal choice.
We also asked participants their opinions on ALDH2 deficiency and suggestions for ways to distribute our product in the future. (Whole team activity)
Will people drink more?
One of the main ethical concerns we had about our project was that reducing the health effects of ALDH2 deficiency may encourage people to drink more or lead to alcoholism. We brought the issue up in both the survey and our Bioethics Roundtable. The majority (close to 82%) of survey participants stated that they would not drink more even if ALDH2 deficiency could be treated. This was reassuring, as our focus was on eliminating the cancer risks associated with ALDH2 deficiency rather than promoting the consumption of alcohol.
Governmental Change Needed
NATIONAL HEALTH INSURANCE (NHI)
Taiwan has the largest ALDH2*1 deficient population in the world. Despite this, nothing is being done at the governmental level to combat this issue. We tried to find out more about this lack of action. From our calls to local hospitals, we learned that ALDH2 deficiency tests are not readily available, yet, a variety of smoking-related tests are commonly provided for free by Taiwan’s Ministry of Health and Welfare under “Smoking Cessation” programs. We were also surprised to find that citizens (30 and above) who chew betel nut or smoke have access to free oral cancer screenings every 2 years.
We reached out to Dr. Cheng-hua Lee, the Deputy Director General of the National Health Insurance Administration (NHI) to ask some questions relating to ALDH2 health issues. We first asked why Taiwan’s health insurance covers smoking-related issues but not alcohol related issues. Dr. Lee believes there is not a clear relationship between alcohol and increased cancer risks. He says that unlike smoking and betel nut chewing, which have direct cause-and-effect relationships with certain cancers, the combination of drinking and ALDH2 deficiency have yet to demonstrate a direct relationship with cancers (head and neck/oral). Dr. Lee also said that current statistics show that drunk driving is the largest alcohol-induced problem, and not the deficiency. He went on to say that the deficiency might even be a form of protection as people are likely to drink less because of their flushed symptoms.
We asked about the possibility of ALDH2 tests being covered by health insurance, but Dr. Lee believes that since the flushing symptoms are so apparent, there is no need to advocate for the test. He says that since the deficiency is not a “sickness,”, confirmatory diagnostics are not required.
When presented with results from our survey, Dr. Lee does realize that current health education programs are in need for reforms, and that maybe “greater implications” exist regarding alcohol ingestion. We also wanted to see if our product has the potential to be covered by insurance, unfortunately, he said only prescription drugs can be covered.
ALCOHOL WARNING LABELS
Current Practices and Policy Brief
Concerned about the large ALDH2 deficient population in Taiwan, we tried to look for policies aimed to educate the public about alcohol consumption and their ramifications on health. We found that little attention has been paid to this matter in terms of policy and regulation. For example, unlike cigarette health warning labels, alcohol products lack similar rigid health-oriented labels, despite having similar detrimental impacts on health. We also surveyed several alcohol manufacturers on their labeling practices and looked at alcohol warning labels at supermarkets and convenience stores. Only about half of the labels directly linked excessive drinking to negative health effects.
We looked at alcohol warning labels (boxed in yellow) in a convenience store, and found that less than half of the products (in this picture, only 2 out of 9 labels) mentioned health risks associated with alcohol consumption. Furthermore, these labels blend in with the product labels and are very small in size--current regulations only require warning text to be larger than 2.65mm in size.)
Using tobacco regulations as a model, we composed a policy brief with suggestions for modifying current alcohol labeling practices. We seek to inform more people about the health effects of excessive alcohol consumption. (Policy Brief by Iris H.)
We reached out to Dr. Vera Wu, former Global Asset Lead at Pfizer, to learn more about drug development and marketing. Dr. Wu walked us through the three stages of drug development: discovery & research, development, and manufacturing. Mrs. Michelle Bruce, our business and marketing teacher, then guided us through the final process of developing a marketing plan for our potential product. In our marketing plan, we formulated a description for our envisioned product, conducted a market analysis with the help of BioHealthWays CEO Brain Pan, and examined the regulations required for our product to be approved for sale.
To ensure our product is safe and marketed responsibly, we researched current food and drug regulations. For example, there are regulations in place for introducing new genetically engineered bacteria strains into the market. The approval process is laid out by the Taiwan Food and Drug Administration’s Guideline for Food Safety Assessment of Genetically Modified Foods Derived from Recombinant-DNA Organisms, which requires characterization of the organism, of any genetic modifications, and most importantly, a safety assessment. We also decided to market our product as a health food, defined by Taiwan’s Health Food Control Act as a “food with health care effects,” but not a “medical treatment aimed at treating or remedying human diseases.” To receive a health food permit, a product must be shown to 1) be harmless, and 2) carry definite health effects. Before any product can be launched, extensive tests must be performed so we can be sure our product is safe and useful for reducing the accumulation of acetaldehyde. (Interviewed by Nicole C, Emily T, Austin H, Tim H, and Iris H. Marketing Plan by Nicole C, Austin H, and Emily T)
Updating Health Curriculum
In our school, all ninth grade students are required to take a health class which already discusses the negative effects of alcohol consumption. We felt it was also important for students to be aware of the increased cancer risks and the large ALDH2 deficient population in Taiwan, so we spoke about this in several health classes and class meetings. (Presented by Yvonne W, Justin Y, Catherine Y, Austin H, Catherine C, and Justin W)
Spreading Public Awareness
To interact with our community on a more personal level, we went around Taipei city to pass out fliers about ALDH2 deficiency, ask people to fill out our survey, and perform free bandage tests to determine if people are ALDH2 deficient. We followed the procedures from a “DIY ALDH2 Deficiency Testing Kit” given to us by TAIES. This simple test just requires placing a bandage soaked with 75% ethanol on one’s forearm for 7 to 10 minutes. If the skin under the bandage turns red, the person is tested positive for the deficiency (this test is about 70% accurate).
We went around Taipei to pass out our fliers about ALDH2 deficiency, perform free bandage tests, and ask people to fill out our survey (we even went to the Apple store and put our survey up on their display phones!).
Spreading Public Awareness
To raise awareness of ALDH2 deficiency to the general public, we reached out to media outlets. In our immediate community, we published an article with Blue and Gold, our school newspaper. To extend our impact outside our school community, we reached out to Today News, the Global Views Commonwealth Publishing Group, and The News Lens International. After corresponding with them, these news outlets published articles about ALDH2 deficiency and our team. Through International Community Radio Taiwan (ICRT), we were also able to speak about the common misconceptions surrounding ALDH2 deficiency over the radio!
Click here to read the article in Today News
Click here to read the article in The News Lens International
Click here to listen for a recording of the our ICRT segment
Taiwan Alcohol Intolerance Education Society (TAIES)
TAIES, a non-profit organization founded by Dr. Che-Hong Chen, aims to educate the general public about ALDH2 Deficiency. In March 2018, TAIES and the Taiwan Federation of Medical Students co-hosted a 2-day workshop for over 80 student participants from different universities.
There, we learned about ALDH2 deficiency, including information such as alcohol consumption rates, the prevalence of ALDH2 deficiency throughout Asia, the origins of this disorder, the toxicity of acetaldehyde and the health risks that come with the deficiency, and the limits of alcohol labeling (warning labels). We heard from many experts, both scientific and political, on talks related to alcohol and ALDH2 deficiency, and we had the opportunity to learn from former alcoholics about their experiences and views of the deficiency. With this information, we developed various ideas for our human practices. For example, hearing about the limits of current alcohol warning labels, we composed a policy brief to encourage the development of more stringent labeling regulations. (Attended by Emily T, Charlotte C, Longan S, Yvonne W, and Justin W)
Click here to learn more about the workshop!
FUNDRAISING FOR TAIES
We held multiple sales in our school throughout the year to help raise funds for TAIES. At these sales, we also performed simple bandage tests to test people for ALDH2 deficiency. Through these sales and donations, we raised and donated a total of 15,000 NTD to TAIES.
We have continued our long standing collaboration with NYMU-Taipei. Several of our members have attended weekly meetings with the team and discussed our projects together. They have been very generous in lending us chemicals and reagents (PEG 8000, MR17 broth). Additionally, they have provided access and instruction for several instruments. Specifically they provided protocols, cuvettes, and access to an electroporation machine so that we could attempt electroporation into Lactobacillus casei and Lactococcus lactis. They also provided access and instruction to a 96-well plate reader so that we could test the efficiency our alcohol induced promoter (BBa_K2539550). In return we have helped NYMU with the cloning of biobrick parts. Lastly, TAS and NYMU teams worked together to help troubleshoot the interlab study and measurements using their plate reader.
Near the DNA submission deadline, we assisted NYMU by cloning their ALB-mCherry (BBa_K2751012) and mEGFP (BBa_K2751011) part into the standard BioBrick backbone pSB1C3. We did this for them while they were also trying to clone the same part, just in case their ligation and transformation was unsuccessful. Both teams were successful and this helped them verify that ligation and transformation of their part was successful.
PCR check results for AMC and MEGFP constructs using VF2 and VR primers. The expected size of both PCR products are 1 kb (yellow box). (Cloning & Figure: Jake Y, Catherine C)
We also have a long standing collaboration with Lambert iGEM team. This year we were excited to test their prototype of an ElectroPen. They shipped their pen to us along with videos and a protocol. After going over specific details of the protocol over Skype, we then used their ElectroPen to electroporate a GFP expression construct into both E. coli K-12 DH5a and E. coli Nissle 1917. We validated that the ElectroPen works! It successfully helped transform both E. coli strains!
Using Lambert_GA’s ElectroPen and following their protocol, we successfully transformed GFP into E. coli Nissle 1917 and E. coli K-12 DH5a, verifying that their ElectroPen works!