Motivation

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  The World Health Organization estimates that, around the world, there were 4 million new cancer cases and 8.2 million cancer-related deaths in 2012.
  In Taiwan, according to cancer statistics released by the Health Promotion Administration (HPA), one is diagnosed with cancer every 5 minutes and 26 seconds in 2012, and the record was 14 seconds faster than the previous year. The major cancers in Taiwan includes colon, lung, liver, breast, oral cancers, however, while the other cancers are decreasing their influences remarkably, oral cancer remains the highest mortality rate changes from 2002 to 2012!
  There are many causes of oral cancer, and 90% of the oral cavity cancer is due to chewing betel nuts. Up to200 million people have this bad oral habit around the world, and the population centers in the Southeastern Asia. We think this is why the Southeastern Asia includes the most oral cancer patient worldwide.
  Oral cancer was not so successfully controlled, owing to it is mostly detected by direct observation in Taiwan, which can easily leads to error detection, also, lots of patients refuse further evaluation for fearing the invasive biopsy. Consequently, oral cancer is mostly diagnosed when it is already at 4th stage, and this significantly reduces the surviving rate.
  For that oral cancer is such a severe and important sickness in Taiwan and in Asia, we want to put efforts in improving the condition by constructing an objective and noninvasive early detector, which can sense the potential overexpressed salivary biomarkers simply by patients’ saliva. Also, by helping establish correct health concept through health education, we want to raise public’s awareness of oral cancer.
  Through the project, we hope we can do some changes and make this world a better place with less pain and less late stage oral cancer diagnosis.

Yeast With IL-8 Receptor

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Toehold Switches as RNA sensors

Background

Toehold switch is a hairpin structure RNA. The reporter gene is also assembled downstream to the hairpin, and helps indicate the result of the experiment. The hairpin structure hides the ribosome binding site and the starting codon, so the translation of the reporter gene is repressed. The toehold switch structure is designed to detect the target RNAs, it contains the sequences perfectly complementary to the target RNA in the former half of the hairpin. When the target RNA is present, the toehold switch binds to it and this binding opens the hairpin. As a result, the initially hidden ribosome binding site and the starting codon will be exposed. The reporter gene at downstream of the switch can now be expressed.

Methods

  Toehold switch sequence design
Seven oral cancer biomarkers are found in a previous research [3], and four of them are chosen to be the target of our project, which are IL-8, IL-1β, dual specificity phosphatase 1(DUSP1), and spermine N1-acetyltransferase(SAT). We design our toeholds by getting our target sequences from the NCBI website, and then we define and design our target region into 30 base pairs. It is very important that it contains about equal amounts of ATCGs, and we need to make sure they don’t match with human genome; also, they cannot contain restriction enzyme sites in the sequences. When we complete the stem loop, we add luciferase, which is our reporter, at the end of the sequences. Finally, predict the structure with RNA Structure website (http://rna.urmc.rochester.edu/RNAstructureWeb) to confirm the secondary structure of our toehold switches.
  Biobricks assemble
To construct the sensor and test its quality, we built two plasmids which contain toehold switches and triggers respectively. In the toehold switch, we sent the designed sequences to the IDT Company, and had the toehold switch synthesized with a T7 promoter (BBa_R0085) upstream to it. In order to quantify our detection, we assemble the toehold switch and reporter protein, luciferase. The T7-toehold part is digested with EcoRI and SpeI, then ligated to the luciferase (BBa_I712019) provided by iGEM, which is digested with EcoRI and XbaI. The T7-toehold switch-luciferase is later subcloned to pSB1C3 backbone. As for the trigger part, we construct it to validate the function of toehold switch. The designed trigger is synthesized by the IDT Company, and digested with XbaI and PstI. It then ligate downstream to the T7 promoter (BBa_R0085) digested by SpeI and PstI.






Sunshine Social Welfare Foundation

Prototype Design

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Future Work

Given the fact that chewing betel nuts, smoking and drinking are the most significant causes of oral cancer and that people who keep these bad habits are labor workers that need refreshment, there are certain occupations that cover the most consumers; for instance, cab drivers, truck drivers and constructional workers. To approach these high-risk population with our early detection devices, we want to establish the detector-buses that tour around communities, especially to sites such as cab stands, travel centers, constructional cites, and even rehabs where we can encounter people that have ever been or still are consumers. After getting the results, we can then inform the subject if he or she should get further examination. The advantage of our detector-buses are that their mobility offers chances to benefit people all over the country and that they are able to provide a moderate environment for the devices equipped on them so that the results wouldn’t be affected by conditions like temperature or humidity. Apart from the detector-buses that are mainly for the specific groups of people mentioned above, we also want to set up our early detection devices in clinics or community health centers so that the general public can also gain access to them. By having our devices popularized, we can achieve our ultimate goal of point-of-care testing, which enables everyone to check on his/her own conditions conveniently and instantly. We strongly hope that our idea can be implemented in the near future, not only in Taiwan, but also in the world.