Our approach to the iGEM project is the following:
- First, find a problem
- Know your tools
- Design a solution for the problem by using the tools
- Execute your design in the lab
- Have fun :)
1. The first days of our new iGEM group were all about finding problems. Finding a problem is not easy as it sounds, we must keep in mind that most of the problems that we want to solve have already been partially solved, or are well underway as we speak. For example, there are many kits and chemicals available in the market that are designed to solve the problem of wine spoilage. The key to choosing the right problem for an iGEM project is to filter out the problems that their solution is good but not good enough. In other words, we can find a better solution using our new tools thanks to recent advancements in synthetic biology.
After we devoted a few team meetings to find a problem, we decided to focus on the world of wine. Wine is more than a regular drink, it has been meaningful to human society for over 7000 years and remains prominent in religious and cultural practices to this day. No less important is the simple yet elegant biological process that produces wine. A simplicity we found perfect for our goals, with one main organism to focus on and manipulate genetically.
The next step was to investigate the world of wine through people. Please read more about this in our Human Practice section.
In conclusion, the list below describes the problems we wish to tackle in our iGEM project:
- How can we increase the health attributes of the wine?
- How can we design a product that will make wine accessible to consumers with diabetes and/or overweight?
- How can we help wineries avoid wine spoilage without using SO2 (sulfur dioxide), which is known to cause asthmatic reactions in certain individuals?
- How can we alter fermentation to decrease ethanol production?
2. The problems mentioned above can be solved by synthetic biology just like a “jigsaw puzzle” game. The key for the solution is to identify the parts (our tools) and assemble them for the desired function.
We chose three main genes parts that we would like to express into a protein in S. cerevisiae:
Resveratrol: a polyphenol present in grapes and some other foods, which increases the life expectancy of numerous species and is considered to mimic calorie restriction, though this point is still controversial. Furthermore, resveratrol has shown some anticancer properties, it has antioxidant and anti-inflammatory properties, it can helps protect against ischemia-reperfusion, neurodegenerative processes and metabolic diseases. Based on animal and in vitro studies, resveratrol reverts these risk factors via stimulation of silent mating type information regulation 2 homolog 1 (SIRT1).
Miraculin: a homodimeric protein isolated from the fruits of Richadella dulcifica, a shrub native to West Africa. Although it is flat in taste at neutral pH, Miraculin has taste-modifying activity in which sour stimuli produce a sweet perception. Once Miraculin enters the mouth, strong sweetness can be detected.
KP6: a fungal killer toxin protein, secreted by the fungi Ustilago maydis. KP6 consists of two small polypeptides (alpha and beta) that are not covalently linked. KP6 have been shown to inhibit the growth of Brettanomyces bruxellensis (The wine spoilage yeast) under winemaking conditions.
Glycerol: redirecting the metabolic pathway away from its “normal” endproduct, ethanol, towards an alternative metabolite, glycerol, has the benefit of lowering the alcohol content of wine without damaging its desired characteristics.
In addition, we chose other parts for regulatory, signaling and purification reason:
- ADH1: used as a strong constitutive promoter.
- ADH1 terminator: used for improving mRNA half-life and gene expression.
- Alpha factor signal peptide: used for the secretion of the expressed proteins.
- His tag: used for protein purification.
Please read more about our parts here
3. Time to assemble all the parts in our puzzle - Every gene was fitted with a promoter that would express the protein at the right moment, and in the right amount. other parts, like a secretion sequence and a terminator sequence ware added to optimize protein expression and location. ‘His tags’ ware added in order to purify the proteins after expression.
Please read more about our project design here
4. After finding a problem to solve, getting to know the tools and planning the work in a way that will allow us to build the puzzle, it’s time to work in the lab.
Please read more about our notebook and results here
5. Have fun!!!