Applied Design
This is a prize we believe our team deserves for developing a reactive strip as a synthetic biology product which solves a real world problem, cancer risk detection, in the most elegant way. We have considered how well this product addresses the problem versus the potential solutions we already have in the market, how this product could be integrated and how it can broadly impact our lives and even change our way of seeing modified, engineered or even transgenic products.
Motivation
Cancers figure among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer related deaths in 2012 (World Cancer Report 2014). One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs, the latter process is referred to as metastasizing. Metastases are the major cause of death from cancer (World Health Organization). Why not all cancers end up metastasizing? Why from 14 million people, 5.8 million people are able to overcome this disease? One of the mains reasons is Cancer Screening. The Polybiome Project was born from the initiative that cancer fight can be answered with prevention, because in the end, cancer is fighting a countdown.
Many cancers can be treated at early stages due to the fact that they cause symptons or because of screenings for other diseases may uncover a growing tumor. The problem is that many cancers do not cause symptons until they are grown enough and therapies are much complicated and less effective. That is the reason why we thought, with all of the new home-technologies, why there is still not a screening method, friendly-user and still accurate that you can use at your house? What if you could sense cancer risk, with the same Roche urine strip you detect your pH, glucose, creatine that you can buy at any pharmacy? Well, the Polybiome Project found the solution.
Nowadays cancer detection methods vs the Polybiome strip
Most of current tumor markers work through a blood test, even though there are many industries trying to divert this trend into a non-invasive test like urinary samples. However, these last ones are usually cancer-type specific and still no such effective as blood tests.
Dr. Kazuei Igarashi, Dr. Masao Kawakita and Dr. K. Hiramatsu are the three most read authors about polyamines and cancer detection. They have shown that a polyamine excretion molecule called Diacetylspermine, can be found in increased levels in cancer patients. In 2013, Hee Park and Igarashi wrote a paper, Polyamines and their metabolites as diagnostic markers of human diseases in which they state that a marked increase in urinary Diacetylspermine is associated with many types of human cancers, including: colorectal cancer, prostate cancer, testicular cancer, breast cancer, renal cancer, pancreatic cancer, hepatocellular carcinoma, lung cancer and brain cancer. The sensitivity of urinary Diacetylspermine for malignant conditions was 75%, which was higher than CEA (Carcinoembryonic antigen) which was 39,5% with 5ng/ml as a cutoff value (P<0.0001) and CA19-9 (Cancer antigen 19-9) which was 14,1% with 37 units/ml as a cutoff value (P<0.0001).
Diacetylspermine is only present in 0.46% of all the polyamines found in urine, which is why they can not be detected through conventional methods. The good thing about these molecules is that variations in urine are very small from one individual to another, which implies that they are secreted in a highly controlled manner.
Reactive strip as a tumor risk marker: the idea
The Polybiome project, as the name properly announces, was born as an initiative to reduce polyamines by modifying the human microbiota. From this research, we discovered the fascinating world of Diacetylspermine, which was the key to a hole new idea in the prevention of cancer. The first thing we thought was on how we could make a modified organism sense this molecule. This was not very hard since we already were working on auxotrophic E. Coli which were going to be able to introduce polyamines in order to survive. We only needed a fast and efficient way to warn the user which was his cancer risk. By reading many papers, we saw a weird promoter called atoC (Filippou et al 2007). This promoter expressed a molecule called Antizyme, which binds to ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. So basically, this molecule is expressed depending on the amount of polyamines there are in the media. This promoter, atoC, was polyamine induced, which was a huge discover since we only needed a molecule with fast color expression. We found an amazing pigment called Violacein from iGEM Cambridge 2009. The genes necassary for the production of Violacein are vioABCDE. So we thought on expressing four of those genes constitutively and the last one, regulated by atoC. That way, as soon as polyamines entered the cell and atoC recognized them, violacein would be produced and color would rapidly appear.
The next problem was that urine has 11 different types of polyamine-like molecules that could enter into our bacteria and be sensed by the promoter. Those 11 molecules are: * Putrescine * Acetylputrescine * Cadaverine * Spermidine * Acetylcadaverine * N1-Acetylspermidine * N8-Acetylspermidine * Diacetylspermidine * Spermine * Acetylspermine * Diacetylspermine
We were only interested in the last of these molecules, which is why we came up with the idea of treating the urine before it was sensed by our bacteria. We created the “Reaction Mix”, which contains 4 different enzymes: Putrescine Oxidase, Acetylputrescine Deacetylase, Acetylspermidine Deacetylase and Polyamine oxidase.
Putrescine Oxidase:
Acetylputrescine Oxidase:
Acetylspermidine Deacetylase:
Polyamine Oxidase:
Knowing their catalitic constants and their Michaelis Menten constants, we were able to know how much enzyme we needed in order to degrade these molecules in a certain amount of urine.
From a recent paper, Takahashi et al 2015, we obtained cutoff values that were good indicaters for cancer risk: - Males: 200 nmol/g creatine of Daicetylspermine - Females: 250 nmol/g creatine of Daicetylspermine
This cutoff values were normalized with creatine values, which is why we only needed a small amount of urine to test our device. Our strip will then include two different markers, one for diacetylspermine and the other for creatine. Bacterias would be freezed dried in a strip with the creatine sensor (adding 3,5-Dinitrobenzoic acid to the strip will react with creatine).
This amazing idea is well characterized in an analitical way, we know the amount of enzymes we need for two drops of urine, the time for these enzymes to finish their function, the creatine colors well characterized (there are many comerciable strips that sense urinary creatine), but we only needed to characterize the atoC promoter. Unfortunately, we had little lab time and this was a project that merged once we were already working on the probiotic, so we could not give the experimental time it required. We still made some clonings and tried to obtain color from GFP, but we had some troubles with this last gene that was not correct when the sequencing results arrived. Still, we believe this idea, even though no experimental results have come out, is worthy of this prize since we developed a synthetic biology product which could help solve a real world problem as it is cancer prevention, and could bring closer many users to the amazing, creative and encouraging Synthetic Biology.
