Team:CSMU Taiwan/Test
Proposing Solution
Selecting Sample + Marker
Jau Song, Yu is a professor in Molecular Medicine Research Center, Chang Gung University. He has dedicated in various biomarker research for decades, and his team has developed an immuno-based oral cancer detection kit,
since their vision was to assist in current detection method also.
The first important step in designing our device was to choose the biomarker and biological sample. Upon facing several biomarkers to choose from, we had a hard time narrowing down. Professor Yu shared his
experience of choosing biomarkers for his PNAS article, which inspired our biomarker and biological sample selection. He also shared insights on conducting research projects with international teams and how their team
gradually self-learned on the field of entrepreneurship.
After this interview, we not only successfully picked out our biomarkers, but also learned important lessons on team work and self-learning, which we firmly believe are extremely crucial factors that supported
our team all the way long through the whole process.
High Risk Groups' Attitude
High-Risk Groups of Oral Cancer include people who often consume alcohol, betel nut and smoke. Instead of merely understanding this disease from articles and statistics, we visited Sunshine Social Welfare Foundation to gain insights from patients'
and high-risk groups' perspectives. It is a non-government organisation which aims to change the lives of people with facial disfigurement, as well as raising social awareness about the issue. In other words, the oral cancer patients they
are assisting are actually those individuals who experience the most severe condition.
Manager Chuang shared how oral cancer would greatly affect individuals and their families in several cases. She also revealed obstacles such as inconvenience, financial loss due to work hours taken off, and lack of awareness. These
factors prevent those high-risk groups from attending HPA's oral cancer screening program. For example, they often mistook OPMD (Oral Potentially Malignant Disorders) lesions as wounds caused by betel nut residue or weak immunity, which they
thought were trivia that shouldn't be paid too much attention to. What's more, Oral Submucous Fibrosis that is often seen in lots of betel nut chewers hold them back from visual examinations, because they aren't able to open their mouth.
After the visit, we realized that an accessible detection method would be a blessing to lots of high-risk individuals. We were also touched by the social barriers behind this issue. Though there are problems we might not be able
to solve in the short term, our team began to think about what kind of impact and engagement we students can make toward society .
Modification
Alexander Green has pursued research in diverse areas ranging from synthetic biology to self-assembly to carbon nanomaterials with applications in flexible electronics, energy, and low-cost diagnostics. He also conducted
postdoctoral research at the Wyss Institute at Harvard regarding toehold switches and was the lead author for several types of research on toehold switches.
When designing our initial set of toehold switches, we were struggling to understand some basic structures of the toehold switch such as the common linker and the loop structure. Therefore, we reached out to
Dr. Alexander Green and he kindly offered to video chat with us to answer some of our inquiries. From the video session, he answered a lot of our concerns regarding designing a toehold switch such as designing our target
binding site. He even explained to us the effect of the loop structure to us - the larger the loop the more leakage problem will occur. The loop contains the ribosomal binding site (RBS) in which ideally the ribosome
would not bind to unless the toehold switch structure is unlocked by the target RNA. However, realistically, the ribosome would still be able to bind to the RBS randomly which causes the leaking of the protein. Green
told us in order to minimize the leakage, a smaller loop is more ideal and he recommended us to use one of his more recent design as a general guideline for the loop design. Furthermore, he reassured us that changing
the protein from GFP to our desired invertase would not have a huge impact on the functionally of the toehold switch as he has done it several times without any issues. Lastly, when we brought up that the use of the
common linker he provided in his initial toehold switch paper would bind to our target binding site and create undesired secondary structures, he explained to us that the linker sequence can be randomized as long as
it helps the toehold switch maintain its stability. He has introduced a common linker for everyone else to use in his initial paper for the sake of simplicity and easy replications of his steps. The linker sequence
is just there to simply provide space to prevent domain interference between the loop and the protein sequence, so the sequence itself can be modified.
After the video session, we were able to drastically modify our initial sets of toehold switches based on his feedback and fix a lot of uncertainties to create our final versions.
Yang, Shun Fa is the head of the department of research and development of Chun Shan Medical University. He has published over 500 researches and has conducted research on cancer detection and researches on miRNA.
During the time period where we started conducting our experiment, we have reached out to him for help regarding our experimental design as he has expertise in the field of miRNA and cancer detection. He started
by giving us feedback regarding our current research design and gave some positive feedback regarding the choice of biomarkers. He also gave us alternative options for biomarkers such as lnRNA and provided us with several
different research papers on the topic of choosing miRNA. Afterwards, he carefully went over our experimental design and gave us advice on how to make the research more conclusive. For instance, he recommended us to
use a separate negative control to test out the specificity of our toehold switches.
The negative control miRNA 191 was chosen as a response to his recommendation as it is also present in saliva and is quite stable across all individuals. Therefore, to ensure the specificity of our miRNA is
not interfered with other present miRNAs in saliva.
EPFL 2019 iGEM team was the grand prize winner of 2019. They used toehold switches as the detection method to identify Flavescence Dorée , a disease that infects grapevines.
During the initial testing stage of testing our cell-free system with the application of toehold switch, we were encountering low production of signal levels from our reporter protein, green fluorescence protein
(GFP). We tried to manipulate the different conditions and adjust the volumes of each material to attempt fixing the problem. We tried to find experts in the field of toehold switch, but we could not find anyone to
help us debug the persisting low production. While searching for help, we stumbled upon EPFL 2019 iGEM and their project which also utilizes a cell-free system with production of toehold switches. We sent an email and
documented our current situation and tried to see if they could give us any guidance. Luckily, they have also encountered the same problem previously and were able to fix the problem with the simple addition of an RNAse
inhibitor. The RNA produced in the cell-free system can be easily degraded by environmental RNAse and therefore, would result in a low yield.
Reification and Evaluation
After the hardware design, we approached dental clinics around CSMU in order to see whether there are considerations or feedback on our device. Though financial factors are still concerned, they said that installing our device in clinics would undoubtedly achieve earlier diagnosis, and clinics can be more engaged in oral cancer detection.
