Team:TU Dresden/Spirulina
Beaming BioBricks from Space...
Our other project...
A global Problem
Overweight and obesity are important clinical diseases that affect to all the world (Figure 1) and are increasing at an alarming rate.
The World Health Organization (WHO) states that obesity has tripled in the last 50 years.
One of the main factors related to overweight is the increase of sugar intake via processed foods of the modern food industry.
Sugar that is used in the food industry to sweeten many different foods and drinks. Since breakfast cereals to mayonnaise and hamburgers. The interest in the search for sugar-free and low caloric sweetener to replace sugar has increased in the last years due to this in order to find a product that can replace sugar, and thus, contribute to the reduction of the growth of obesity.
In this context we found the so-called miraculin berry, which was discovered in the 18th century and is growing on the Synsepalum dulcificum plant. Miraculin (Figure 2) is a protein flat in taste at neutral pH which exhibits a taste-modifying activity under acidic conditions that convert sour stimuli to sweetness (pH 6.5 to 4.8). This modification of the taste occurs, because the protein activates the hT1R2-hT1R3 receptor (Figure 3) (Koizumi et al., 2011).
However, Synsepalum dulcificum (Figure 4) has a low productivity and is difficult to cultivate outside of its natural environment (Ezura, H., & Hiwasa-Tanase, K., 2018). Therefore extracting the protein from the plant would end up in an expensive product. In order to increase the production, we decided to produce the miraculin in a different organism: we chose Spirulina.
Spirulina (Figure 5) is a common name used to define the biomass of the cyanobacteria Arthrospira platensis and Arthrospira maxima. Cultivated worldwide, spirulina is used as a dietary supplement or even as food itself. Spirulina has a very fast growing rate and can be easily cultivated in open ponds or in closed bioreactors.
Dried it contains about 60% of proteins, including all the essential amino acids. It also has a high mineral content, especially iron, magnesium, potassium and phosphorous. Therefore, expressing miraculin in spirulina will not only result in a higher production of miraculin, but also in a healthy and sweet food supplement.
However, despite the great advantages of spirulina, there are limited successful strategies for genetic engineering of spirulina because of some complications that have been reported. The biggest challenge when trying to engineer spirulina is the presence of multiple strong nucleases in its cytoplasm (Kawamura et al., 1986; Tragut et al., 1995).
Fortunately, in 2018 a new system that allows the engineering of Arthrospira platensis with good results was performed by Dehghani et al. They used Agrobacterium tumefaciens, a gram-negative bacterium that causes gall tumours in plants, as a vector for the transformation of spirulina.
Our approach consists on using pCAMBIA1304 (Figure 6), a plasmid that can be introduced in spirulina via the tDNA of A. tumefaciens, including on it two homology regions with the genome of spirulina flanking the insert composite to increase the specificity of the recombination. These are used to achieve a better recombination of miraculin and incorporate different promoters, terminators and marker genes. We also have included a strep-tag sequence in the miraculin gene in order to facilitate the purification of the protein after the expression, so we can easily check if the spirulina is producing the protein.
In addition, we get in contact with Dr. Marko Brankatschk who agreed to perform a feeding assay with Drosophila melanogaster to test if the flies can feel the sweetness of the miraculin and the engineered cyanobacteria.
With this project we expected to create not only a way to increase the production of miraculin but also participate in the establishment of new standards for the engineering of spirulina.
However, at some point during the competition, due to the lack of resources and manpower we had to set aside this project and focus completly in DipGene.
Feeding Assay
This experiments were performed by Dr. Marko Brankatschk.
In this experiments miraculin pills were used. The pills (0.38 g) were diluted in 1600 μL of H2Odd.
Each assay plate was equipped with two different food-bait. The miraculin suspension was always loaded on top. In the bottom, depending on the experiment, 20% soy-protein, 20% fructose or 20% glucose (20 μL) were applied.
In the videos the behaviour of adult flies (9-12 days old wild type, cantonS at 23-25ºC) can be observed. One frame corresponds to 5 s. Each video has 1500 frames.
Miraculin - Soy Protein
The fly prefers the soy protein
Miraculin - Fructose
The flies prefer the miraculin suspension
Miraculin - Glucose
No food preference
Conclusion:
As the miraculin used is a commercial one, the protein is not pure, but a mixture of the berries with cornstarch. Therefore, the interpretation of the data is rather difficult.
With these results, it looks like the flies prefer the soy protein more than the miraculin. This can have two reasons: either the miraculin sweet taste is too strong or the miraculin is not very attractive to flies. Given the choice between sugars and miraculin, the latter seems to be preferred with respect to fructose, and they can’t decide between the glucose and miraculin. It looks like the animals have hard time to decide between miraculin and others. However it has to be take into account that the miraculin is a protein.
Soy protein – Glucose
<
>Flies prefer the protein
Soy protein – Fructose
Flies prefer the protein
Conclusion:
Both experiments demonstrate that pure carbons are not preferred for the flies. Then, as the animals prefer proteins rather than carbons and miraculin is a protein, the sweet taste could not be the reason of the preference. More experiments were performed:
Soy Protein (top) – Soy Protein + Fructose
The mixture is preferred by the flies
Soy Protein (top) – Soy Protein + Glucose
The mixture is preferred by the flies
Conclusion:
These experiments show the modulating capacity of sugars (2%). The results show that the flies prefer the proteins rather than sugars, but they prefer proteins with sugar more then just proteins.
In summary, miraculin seems to be a protein source attractive to flies compared to sugars (fructose or glucose). They prefer the taste of the soy protein. However, we don’t know the real composition of the miraculin pills we can’t take definitive conclusions. Further experiments will have to be performed with a purification of miraculin.
Behbood Moeini Chaghervand
Baron of the plasmid design
- Age: 25
- City + country of origin: Tehran, Iran
- Bachelors: Biotechnology. Tehran University of Medical Sciences
- Interests: Reading, Collection making, Bioinformatics, Working on new ideas, Movies/Series, Music and symphonies
- Peculiarities:
I consider myself an introverted person who enjoys to be alone. I gain energy from working on my plans/goals in my cave. Besides that, I am quite intuitive
and don’t really care about reality. I just follow my dream. The other part of my personality is my judging character. I prefer to tell the truth
and taking serious action instead of postponing decisions and appeasement with wrong ideas. Finally I am typical turbulent person who can’t tolerate non-functional
and traditional trends without a reason.
- What do you like about iGEM?
In spite of mainstream competitions which focus on theoretical tasks , iGEM has both theoretical and practical part which is interesting for me.
Apart from that , iGEM also has extra criteria, like poster making ,wiki making, human engaging and finding sponsors, which in my opinion
prepares competitors for their own real professional life and establishing businesses . In addition to that,
iGEM requires you to provide it with the same requirements which startups want you to have for promoting an idea or invention,
according to this I can say that engaging in one of the biggest startups across the world would be unavoidable for those who finally want to
establish their own business one day .
our great sponsors
We are proudly sponsored by the following partners in science:
