SWEETNESS AND US
Sweetness, a pleasurable taste associates with happiness and satisfaction.
For most animals including human, it seems innate to be addicted to sugars and some other sweeties.
When we were just babies in the warm embrace of moms, breast milk came firstly as a gift containing galactose which also survived us.
In the Victorian era, some British people ate excessive sweet food thus their tooth were decayed, which was regarded as a symbol of high status in society etcetera.
In some tribes nowadays, people still managed to get honey under the risk of being stung.
As a matter of fact, modern science has demonstrated that our love to sweetness is due to our genes.
Our brain will release serotonin when we take sugar, which makes us feel delightful.
Sugar is essential nutrient for everyone.
However, according to the World Health Organization (WHO), having excessive sugar is even more harmful than smoking.
Having excessive sugar may not only cause obesity, but also trigger diseases, such as diabetes and gout.
We need to satisfy our brain with sweet things, nevertheless, metabolizable sugar with enough sweetness usually has high calories at the same time.
Therefore, the demand for seeking sweeteners containing both high sweetness and low calories is quite urgent.
To detect sweetness of substances and find new ideal sweeteners, people have done a lot.
At the very beginning, saccharometer was used to physically measure the refraction simplicity, but it is not accurate and can only work on the carbohydrate.
Then some people were hired and trained as professional tasters to tell the sweetness of each particular sugar. But it’s really costly and time consuming.
Although there are several other current methods to test how sweet the sugars are, there remains limitations from labs to society.
Thus, it is really urge for us to create a new convenient and effective way to test the sweetness and find more available sweeteners.
With the development of technology, artificial sweeteners are becoming better choices. The production technology of artificial sweeteners is becoming more mature. Meanwhile ,as a food additive, artificial sweeteners are also more widely used in the field of food production. But the potential harm of artificial sweeteners to humans is still a controversial issue.
Therefore, it is imperative to detect the ideal sweetener to tackle the problems we mentioned above as well as satisfy our gustation.
We hope to design a bio-system via synthetic biology methods which can measure the sweetness of various sugars and identify them by corresponding signal expression in microbe.
First and foremost, we found the mechanism why people can feel sweetness: the human sweet G protein-coupled receptor T1R2-T1R3.
It’s a pair of extracellular proteins with homodimer, which has six structural domains.
Different sweet substances combine with different regions of T1R2-T1R3, then triggering intracellular G protein-coupled pathway and leading to downstream signal expression.
After complex nerve transduction, a "sweet" feeling was led at last.
At the same time, we chose Saccharomyces cerevisiae, a kind of yeast as our host strain with the function of glycosylation.
In the design, the yeast original G protein-coupled receptor Ste2, which is related to yeast mating, will be replaced by the T1R2-T1R3.
Then, to amplify our signal, some side effects genes will be knocked out by homologous recombination technology.
After building a completed system, we will take some kinds of sugar to judge their sweetness according to the strength signal.
Our project is divided into three subsystems:
Receptor Expression system: Using OE-PCR, and other methods to achieve the synthesis of T1R2-T1R3 G protein-coupled receptors T1R2-T1R3.
Confirming the expression and position(if on the cell membrane) of T1R2/T1R3 in our engineered yeast by immunofluorescence.
Pathway Optimization system: Using trp, his, and ura as screening marker fragments with homologous arms to knock out ste2, sst2, and far1 genes by homologous recombination in yeast.
* ste2, sst2 gene can inhibit the signal transduction of Gpa1 protein.
*far1 gene can cause interference signal for sweet yeast normal physiological function after transformation.
Siginal Output system: Constructing the red fluorescent protein circuit and improving the promoter Pfus to detect the sweetness through the expression of the fluorescent signal.