Human Practices

Achievements

• ...designed a biobrick consisting of the yeast surface anchoring protein Alpha-Agglutin 2 (Aga2) fused to GFP.
• ...successfully implemented an alpha pheromone-inducible expression system for myrosinase in S. cerevisiae. We showed that the yeast produces myrosinase when grown together with alpha pheromone and demonstrated the functionality of the target molecule.
• ...considered how to design a pill that best delivers the yeast to the colon of a patient. This helped us evaluate the feasability of our project.
• ...implemented a kinetic model of the alpha pheromone pathway and coupled it to the production of p28 and myrosinase in order to demonstrate and analyze the function of the alpha pheromone-inducible expression system.
• ...used kinetic modeling to compare the efficiencies of p28 and myrosinase. Based on the results we decided to move on with only myrosinase as the target molecule.
• ...implemented a community of genome-scale models and analyzed it with a dynamic flux balance analysis approach. With the help of the community dFBA model we evaluated the effect of S. boulardii on key species of the gut microbiota. We demonstrated that can establish and cooperate with the gut microbiota without dramatically changing the composition of it. We also found a lower bound on the concentration of S. boulardii needed for the yeast to grow.
• ...used genome-scale modeling to show that S. boulardii can produce myrosinase without depleting its amino acid resources.
• ...evaluated the need for and use of our product on the market with an experimental survey. The survey showed that people seemed positive towards using our yeast for detection and treatment of colon cancer.
• ...reached out to the community and conducted interviews with several experts and got valuable feedback on our product. We used the feedback to improve our project design.
• ...analyzed the project from ethical and environmental points of views. Based on the analysis we considered how we in the future can avoid some of the ethical and environmental risks that come with our product.

Medal requirements



• We registered for iGEM, had a rewarding summer and all of our team members will attend the Giant Jamboree.
• We have or will complete the following deliverables: Document the project on our Wiki page, Design a poster and display it during the Poster Session at the Giant Jamboree, Hold a presentation of our project on the Giant Jamboree, Fill out the Judging Form.
• We have correctly attributed all of the work that was done during the course of the project on our Attributions page.
• We have successfully completed the InterLab Measurement Study and the data has been accepted.



• We have designed a new BioBrick Part that has been characterized, documented and submitted to the Registry. LINK TO PART
• We have exchanged knowledge and collaborated with several other iGEM teams. For more information on our collaborations, see the Collaborations page.
• We have considered ethical and environmental aspects of our project as well as evaluated the need and use of our product in society. Visit our Human Practices page for more information.



• We have taken the knowledge gained from our human practices investigations as well as interviews with experts and let it guide us in the development of our project. Take a look at our Human Practices page to find out more.
• We have modeled our system and used the results from simulations to make decisions regarding the path of our work. We have also used the modeling results to demonstrate the basic principles of our system and to make inferences about how our final product will work in practice. Please visit our Modeling page for details.










The aim of our human practices work was to spread the word about synthetic biology and to consider our project in ethical and environmental aspects. We also wanted to get valuable feedback from people outside of the team, both experts and the general public, in order to further develop the project. During the course of our work, we got interviewed by the local newspaper Göteborgs-Posten, held a presentation about our project at the university and had meetings with several experts. We met with the oncology professor Mef Nilbert that gave us advice on how we could improve our project. Mef Nilbert made us consider alternative detection methods and broadened our perspective on the potential uses of our product. A meeting with the philosophy professor Karl de Fine Licht eventually led to the design of an experimental survey. The survey helped us to evaluate our product from a patient point of view and to understand how to best reach out with our project to society. Carl Johan Franzén, professor in bioreaction engineering, gave us guidance regarding ethical and environmental issues. Based on his guidance, we evaluated the effects that our yeast could have on patient health and the environment. In turn, this made us consider how we could avoid possible negative effects on the environment.

Outreach

In order to reach out with our project and to spread the knowledge of synthetic biology, we had an article published in a local newspaper and held a presentation at our university.

News article

The concept of synthetic biology, and the many possibilities it offers, is commonly unknown to many who are not involved within the area. To share our knowledge of synthetic biology and with a hope to increase interest in the subject, we contacted Göteborgs-Posten, known as GP. GP is the biggest newspaper in Gothenburg with around 1,000,000 daily readers in 2018 (GP, 2018). We asked if they were interested in doing a reportage about iGEM and our project, to which they were positive. One week later the reporter Madeleine Johansson and the photographer Olof Ohlsson visited us in the lab. A news article was published, both in paper format and on the web, two days after their visit. In the article the concepts of synthetic biology, the iGEM competition as well as our project were explained.



Something unexpected came out of the GP article, as the research institute at Sahlgrenska University Hospital in Gothenburg contacted our supervisors and expressed interest in the project. It is now likely that our research will continue beyond iGEM. We want to thank GP, Madeleine Johansson and Olof Ohlsson for this opportunity.

The news article can be found here.

Project presentation at Chalmers

To increase the interest in iGEM and in synthetic biology at Chalmers, we did a lunch seminar collaboration with the Chalmers Student Chapter of Society for Biological Engineering (SBE) at the university. We reached out to all disciplines with an invite to a presentation about our project. We want to thank SBE Student Chapter Chalmers for this collaboration opportunity.

Current Methods and Research

In order to evaluate the need for our yeast pill in health care, we considered the most common detection and treatment options for colon cancer available today. We also looked into current research. Based on our findings we concluded that there is a need for an easy and non-invasive method for colon cancer detection as well as more efficient treatment options. In this section, we have summarized the information that we found during our background search.

Detection

One of the most common methods for detecting colon cancer is colonoscopy. Colonoscopy is an invasive method that requires the patient to adapt food intake and to use laxatives prior to the examination (Simon, 2016). As an alternative to colonoscopy, the colon can be examined by x-ray with CT colonography. The colonography is semi-invasive but still requires the patient to prepare for the examination as for the colonoscopy (Simon, 2016).

Non-invasive detection methods where blood or abnormal DNA is detected in the stool are available. FOBT or fecal immunochemical tests can be used at home and detects hemoglobin in the stool (Simon, 2016). The drawbacks of these tests are that they have a low detection rate of precancerous lesions and that they can give false results as a consequence of diet (Simon, 2016). Multi-targeted stool DNA tests, mt-sDNA, that combines the detection of blood together with abnormal DNA has shown to have a higher sensitivity compared to FOBT and FIT (Simon, 2016).

To find non-invasive detection methods that are more specific and sensitive than those that are available today, research is put into the development of nanosensors. Nanosensors could for example detect volatile compounds that are indications of colon cancer in the breath (Peng et al., 2010) or urine (Arasaradnam et al., 2014). Since nanosensors only need a small amount of sample, they would be both fast and easy to use (Salvati, Stellacci, & Krol, 2015).

Another field of research revolves around the microbiota and its connection to the presence of cancer in the colon. It has been found that the relative abundance of species in the gut differs between healthy individuals and individuals with colon cancer (Zackular, Rogers, Ruffin, & Schloss, 2014). This could be exploited to detect this type of cancer in an early stage (Zackular et al., 2014).

Early detection has a key role in survival of colon cancer patients. Regular screening of people in the risk group of developing colon cancer is therefore of importance (National cancer institute, 2018). Factors that can contribute to low screening rates are complications around the screening procedure such as fear and lack of resources (National cancer institute, 2018). Research is put on how to increase the screening rates with one of the option being patient-reach out in order to increase awareness (National cancer institute, 2018). In addition to this, cheaper, easier and faster detection methods might encourage more people to do regular check-ups.

Treatment

Today, surgery is commonly the first choice in the treatment of colon cancer. However, in 1 out of 2 patients undergoing surgery, the cancer recurs (PDQ Adult treatment editorial board, 2018). Other treatment options are radiation therapy and chemotherapy which can also be combined with surgery (PDQ Adult treatment editorial board, 2018). Both of the latter treatment options cause side effects such as weakness and diarrhea (Cancer research UK, 2015).

Research is put into the field of personalized oncology in order to personalize cancer treatment based on genomic profiling. In this way, the treatment becomes more effective while side effects are reduced (Rosenblum & Peer, 2014). In the field of personalized oncology, omics data describing for example enzymatic activity is used to decide which therapy to employ. The data can also be used to monitor drug response and adjust drug dosage (Rosenblum & Peer, 2014).

In relation to genome profiling, better understanding of mutations that could cause cells to become cancerous has led to the development of targeted therapies (Rosenblum & Peer, 2014). An example of a targeting therapy is use of epidermal growth factor receptor (EGFR) antibodies that inhibit EGFR signaling (Martinelli, De Palma, Orditura, & De Vita, 2009). EGFR is involved in important cellular transduction pathways (Martinelli et al., 2009) and has found to be overexpressed in colorectal cancer (Rosenblum & Peer, 2014).

Research is also put into the development of new immunotherapies that can be used for colorectal cancer (Kalyan, Kircher, Shah, Mulcahy, & Benson, 2018). Immunotherapy focuses on stimulating the immune response to fight the cancer and has shown to be successful in the treatment of other cancer types (Kalyan et al., 2018). There are several kinds of immunotherapies, including therapeutic vaccines and oncolytic virus therapy, that are under review for treatment of colon cancer (Kalyan et al., 2018).

Alternative Treatments

Meeting with professor in oncology

Mef Nilbert is a professor in oncology at Lunds University and at Copenhagen University. She has worked with inheritable cancer tumors and has a deep knowledge of colon cancers and their behaviour. We first came in contact with Mef Nilbert thanks to Olle Bergman, one of the judges at the Nordic iGEM conference in Lund. Olle Bergman is a Swedish journalist that is specialized in different types of communications. He was a great asset for many of the teams participating in the Nordic iGEM conference, providing us with advice on how to communicate through text, for example in a poster, and connected teams with relevant people. Olle Bergman was kind to connect us with Mef Nilbert so that we could discuss our project and its possibilities with her.

The meeting with Mef Nilbert was very helpful. We had a chance to ask an expert all kinds of question related to colon cancer. Gladly, Mef Nilbert was overall positive to our project and our idea. She had a strong belief that there is a need for fast, easy and accurate detection and treatment for colon cancer since this type of cancer is generally diagnosed at a late stage. Mef Milbert had ideas about how we could develop our project. As an example, she suggested that the yeast could be used as a preventative measure and not only for detection and treatment. Unfortunately, Mef Nilbert did not think that our detection method would work. She said that it most certainly would be hard to distinguish the gas vesicles, with all the movement and gas that already is in the colon, using only ultrasound. She suggested that we should look for a marker that could be used to detect colon cancer, for example some secreted molecule that could be detected in the stool. With this new information from Mef Nilbert, we decided to see if we could find some possible alternatives to the gas vesicles that could be coupled to our system.

Alternative treatments

So what other methods would be applicable to our product? For diagnosis and localization of the cancer, the most favorable approach would be expression of an MRI detectable reporter gene in the yeast. These give accurate indication of the location of the host cell, and the depth of tissue is not an issue (Srivastava & Bulte, 2014). There are several possible candidate genes for this, as described in the review “Seeing Stem Cells at Work In Vivo” by Srivastava and Bulte (Srivastava & Bulte, 2014). With this approach, the final product would work in the same way as our previously designed system, but with MRI used for screening instead of ultrasound.

Another promising approach that allows for detection, but not localization, is through the use of DNA-recording devices. More specifically, DNA-recording devices in which a specific cell stimulus of choice induces permanent changes in the host genome, which can then be read through DNA sequencing at a later date (Seth & Wang, 2018). One such method, Temporal recording in arrays by CRISPR expansion (TRACE), performs recording through CRISPR-mediated sequential spacer sequence integration into a genomic locus (Seth & Wang, 2018). In our system, induction of TRACE by the pFUS promoter would allow for quantitative DNA encoding of yeast cell density (Seth & Wang, 2018). By sequencing the integration locus of Saccharomyces boulardii from fecal samples obtained from the patient, it would then be possible to determine whether pFUS expression occurred, and to which extent. This would not only provide information about the presence of cancer, but could also indicate treatment progression and has a possibility of predicting tumor size, since the size of the tumor correlates with pFUS expression.

Experimental Survey

Meeting with lector in practical philosophy

In order to get insight into the ethical prospects of our project, we met with Karl de Fine Licht, a lector within practical philosophy at both Gothenburg University and Sahlgrenska Hospital University. The reason why we wanted to have a meeting with Karl de Fine Licht is that he has worked with bioethics, and we thought he could give us some important views on our project and hopefully some good advice, which was also the case. The meeting with Karl de Fine Licht was very informative and we were able to go into some really interesting ethical aspects of our project. We came to the conclusion that we basically have two ethical approaches to consider. Firstly, we had to consider how we wanted to reach out with our product to the society; how we would present it in order to reach out to as many people as possible. We wanted to describe our project transparently in a way that makes people understand how our product works, without provoking preconceptions.

Together with Karl de Fine Licht, we discussed our plan of making some kind of a survey to detect common fears of GMO. Our plan was that we could use this when reaching out with our product. Karl de Fine Licht suggested that we should do an experimental survey instead of a regular one to get clearer results. In an experimental survey, the respondents are handed a scenario in which they have to take different stand points. By creating the same scenario but describing it in two separate ways, it is possible to see if people react differently depending on how the scenario is described. For example, the same scenario can be described, but the difference is that the main character is a man in one scenario and a woman in the other. Do people think of the main character in different ways depending on if it is a man or a woman? By doing an experimental survey, it is possible to detect psychological mechanisms in the society rather than peoples opinion. The reason for this is that in this kind of survey you get a context to every question. Karl de Fine Licht informed us that the psychological mechanisms within a society tend to be less varied than the opinions, which would make it easier for us to interpret the tendencies in the result. Karl de Fine Licht suggested that we could do an experimental survey where our project is described in two separate ways. The first description could be non-academic and describe our project in an easy but correct way while the other could describe our project in a more academic way and use words such as genomic modifications and mutations. We want to thank Karl de Fine Licht for giving us an opportunity to discuss our project with him and for giving us input on the ethical aspects of our project, and for the great idea about how we could improve our survey.

Experimental survey

To get a sense of how people would react to our product if it was out on the market, we decided to make an experimental survey based on the advice from Karl de Fine Licht. We created two different versions of a scenario where we described our product and how it potentially could be used, to see how people would react to it. In the first version of the scenario, which we called the academic version, we described our product in a way that may be interpreted as controversial. In this version, words like gene modification, mutation and generally more academic terms were used. In the other version we tried to avoid academic terms as much as possible and to describe our product and its use in an easy way. The aim with the survey was to see if there were any trends or differences in the reactions to the two versions. The survey was written in three languages; Swedish, Italian and English in order to get as varied target group as possible. The English version of the survey can be found here. Below, a summary of the answers to the survey are presented in pie charts where all answers have been translated into English. The blue pie charts present the answers to the academic versions of the scenario and the green charts present the answers to the non-academic versions.



We received a total of 145 answers to the survey and approximately half of the respondents answered to the academic version and half to the non-academic version. We were hoping to get at least 100 respondents per scenario version in order to be able to statistically verify our results. However, we think that 145 answers can still provide some guidance regarding what people think of a product like ours. When looking at the summarized results we can see that the respondents have answered similarly to the two different versions. We can see a slightly higher acceptance of our product in the academic version, which is opposite to what we expected. Since we do not have enough answers, we cannot establish that there is a significant difference. However, we might be able to say that people seems to be positive towards this kind of treatment even though it includes eating gene modified yeast. It might even be the case that people are more positive towards the treatment when the information about it seems more professional and scientific. From the written answers in the survey it appears to be the easiness and the immediate start of the treatment that convinced most people to take the yeast pill both for detection and treatment and as a preventative measure. For the respondents that did not want to use the yeast treatment, the reason appears to be lack of testing and risk information about the product. How could they know that it was trustworthy? The written answers have been summarized here.

Product Safety and Ethical Aspects

Meeting with associate professor in biolotechnology field

Carl Johan Franzén is an associate professor in bioreaction engineering at Chalmers University of Technology. He has not only experience of working with yeast, but he has also been a part of the ethical committee at Chalmers University. We thought that it was a great opportunity to discuss our project with Carl Johan Franzén to hopefully get an expert’s view on our idea. The meeting with Carl Johan Franzén was rewarding, and it resulted in a discussion about the risks that comes with our product. He reminded us that it is not only important to look at the risks of the yeast being ingested by the patient, but to also consider what will happen when it leaves the body. Is it for example possible that the inserted genes will spread in nature and what will happen if they do? We made our best to do an unbiased and probable evaluation of the potential risks with our product.

Patient safety

Since the final product, a pill containing freeze-dried Saccharomyces Boulardii, is ingested by the patient it is important that the safety of the patient is considered. In our case this mainly concerns the relationship between the yeast and the gut, in terms of the probability of the yeast outgrowing the gut bacteria and colonizing the gut. From literature covering the subject of S.boulardii as a probiotic, we can conclude that this is not likely. In medical studies it has been shown that in patients that ingest S. boulardii continuously over a period of time, the probiotic yeast concentration in the colon reaches a steady state after 3 days (Czerucka, D. et al. 2007).  Furthermore, it was found that in 96 % of the patients, the fecal concentration of S.boulardii was undetectable 3 days after dosing had stopped (Elmer et al., 1999) and that the yeast remained in the gut at most 4 days after stopped dosing (Czerucka, D. et al. 2007). This, together with the GRAS status of S. boulardii, let us assume that the yeast is unable to colonize the gut on its own, and that it will not outgrow the gut microbiota.

With the safety of the unmodified S. boulardii confirmed, the next question to address is if our system could affect patient safety once implemented in the yeast. The probability of this is low, considering that the pheromone sensing system in some cases causes cell cycle arrest (Williams et al., 2013), and thus inhibits the growth of the yeast to some extent. The risk of gut colonization by yeast overgrowth is thereby not likely caused by our system. If we next consider the anchor binding, our goal is to make the yeast bind specifically to the cancer cells and not offsite to the healthy gut cells. Based on this, we can conclude that with a functioning system the yeast should only remain in the gut if the patient has cancer. Of course, extensive preclinical testing of the probiotic would be necessary to confirm this.

Environmental safety

One commonly used argument against genetically modified organisms is that they can spread in the ecosystem and, with them, their genetic material. There is a possibility that our genetically modified S. boulardii will survive in the sewage system and eventually end up in nature. Therefore, we considered what will happen with our yeast once it leaves the body of the patient together with the feces. One of the main risks with our modified yeast being released into the sewage system is the risk of spreading the ampicillin resistance gene, present in the plasmids used for transformation, to bacteria. However, transfer of genetic material from yeast to bacteria has not been observed (Czerucka, Piche, & Rampal, 2007). In addition to this, the genetic material transformed into S. boulardii would optimally be implemented into the yeast genome in the final product, thereby eliminating the risk of spreading ampicillin resistance.

We asked ourselves if our yeast could harm the nature by release of target molecule. We deemed this unlikely since the probability that our S. boulardii will accumulate in nature is expected to be low. The reason for this is the low probability of the yeast having cells to bind to, combined with the low probability of several yeast cells binding to the same cell, or cells in proximity to each other. As a result of this, it is not likely that the FUS1 promoter will be activated by the alpha pheromone sensing system. This means that our yeast would behave as normal, unmodified, S. boulardii (see the section on patient safety). Consider the possibility that the FUS1 promoter is activated by some other means than by the alpha pheromone system. In this scenario, the amounts of anti-cancer agent released are expected to be low and the resulting concentrations in nature are assumed to be neglectable. The reason for this is the small probability of several yeast cells, expressing anti-cancer agent, accumulating in one place. We therefore consider the risk of our yeast harming nature via release of anti-cancer agent to be small.

In a future prospect, a kill switch could be introduced into our S. boulardii in order to make sure that it will not make any harm to the environment after it has left the body of the patient. One possibility is to combine a temperature sensor with a phosphate sensor, killing the yeast when the low temperature outside of the body is combined with the lower concentrations of phosphate in the wastewater (iGEM NUS Singapore, 2017).

References

Annonsera i Göteborgs-Posten, GP. Retrieved from: https://info.gp.se/annonseraforetag/. Date of retrieval: 2018-09-23

Arasaradnam, R. P., McFarlane, M. J., Ryan-Fisher, C., Westenbrink, E., Hodges, P., Thomas, M. G., Chambers, S., O'Connell, N., Bailey, C., HArmston, C., Nwokolo, C. U., Bardhan, K.D., & Covington, J.A. (2014). Detection of colorectal cancer (CRC) by urinary volatile organic compound analysis. PLoS One, 9(9), e108750. Retrieved from: https://doi.org/10.1371/journal.pone.0108750

Cancer research UK. (2015). Chemotherapy treatment. Retrieved from: https://www.cancerresearchuk.org/about-cancer/bowel-cancer/treatment/chemotherapy/chemotherapy-treatment

Cancer research UK. (2015). Radiotherapy treatment, Retrieved from: https://www.cancerresearchuk.org/about-cancer/bowel-cancer/treatment/radiotherapy/radiotherapy-treatment

Czerucka, D., Piche, T., & Rampal, P. (2007). Review article: yeast as probiotics - Saccharomyces boulardii, Alimentary Pharmacology & Therapeutics, 26(6), 767-778. Retrieved from: https://doi.org/10.1111/j.1365-2036.2007.03442.x

Douradinha, B., Reis V.C.B., Rogers M.B., Torres F.A.G., Evans J.D., & Marques Jr E.T.A. (2014). Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii, Bioengineered, 5(1), 21-29. Retrieved from: https://doi.org/10.4161/bioe.26271

iGEM NUS Singapore. (2017). Modelling a kill switch for probiotics. Retrieved from: https://static.igem.org/mediawiki/2017/4/4c/NUS_iGEM_2017_Modelling_a_Kill_Switch_for_Probiotics_Report.pdf

Kalyan, A., Kircher, S., Shah, H., Mulcahy, M., & Benson, A. (2018). Updates on immunotherapy for colorectal cancer, Journal of gastrointestinal oncology, 9(1), 160-169. Retrieved from: https://doi.org/10.21037/jgo.2018.01.17

Martinelli, E., De Palma, R., Orditura, M., De Vita, F., & Ciardiello, F. (2009). Anti-epidermal growth factor receptor monoclonal antibodies in cancer therapy, The journal of translational immunology, 158(1), 1-9. Retrieved from: https://doi.org/10.1111/j.1365-2249.2009.03992.x

National cancer institute. (2018). Interactive app improves colorectal cancer screening rates. Retrieved from: https://www.cancer.gov/news-events/cancer-currents-blog/2018/colorectal-cancer-screening-app-improves-rates

PDQ Adult treatment editorial board. (2018). Colon cancer treatment. PDQ Cancer information summaries. Retrieved from: https://www.ncbi.nlm.nih.gov/books/NBK65858/#CDR0000062687__125

Peng, G., Hakim, M., Broza Y.Y., Billan S., Abdah-Bortnyak, R., Kuten, A., Tisch, U., & Haick, H. (2010). Detection of lung, breast, colorectal, and prostate cancers from exhaled breath using a single array of nanosensors, British journal of cancer, 103(4), 542–551. Retrieved from: https://doi.org/10.1038/sj.bjc.6605810

Rosenblum, D., & Peer, D. Omics-based nanomedicine: The future of personalized oncology, Cancer letters, 352(1), 126-136. Retrieved from: https://doi.org/10.1016/j.canlet.2013.07.029

Salvati. E., Stellacci, F., & Krol, S. (2015). Nanosensors for early cancer detection and for therapeutic drug monitoring, Nanomedicine (Lond.), 10(23), 3495–3512. Retrieved from: https://doi.org/10.2217/nnm.15.180

Simon, K. (2016). Colorectal cancer development and advances in screening, Clinical Interventions in Aging, 11, 967-976. Retrieved from: https://doi.org/10.2147/CIA.S109285

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Williams, T. C., Nielsen, L. K., & Vickers, C. E. (2013). Engineered quorum sensing using pheromone-mediated cell-to-cell communication in Saccharomyces cerevisiae. ACS synthetic biology, 2(3), 136-149.

Zackular, J.P., Rogers, M.A.M., Ruffin IV, M.T., & Schloss, P.D. (2014). The Human Gut Microbiome as a Screening Tool for Colorectal Cancer, Cancer prevention research, 7(11). Retrieved from: https://doi.org/10.1158/1940-6207.CAPR-14-0129