Team:BIT-China/Description

Inspiration

        The members of our team come from all over the world and alwaysstruggle with taste differences when we get together. And several members always feel that a dish at school is not as good as the same dish back home. Does this indicate that the problem of ourfavorite tastes not being satisfied objectively exists? In addition, we also learned from a bilibili blogger's channel that some people whocannot eat meat for physical reasons are eager to try the taste of meat. Does this indicate that the taste needs of certain specific groups of people have not been satisfied? After tasting the food we always thinking of making the same deliciousness at home, but always failing to do so.We are thinking whether we can record the heritage of Chinese food in a quantitative way and more convenient way.

Background

        The 2021 Nobel Prize in Physiology or Medicine was awarded to David Julius and Ardem Patapoutian, two scientists who discovered temperature and tactile receptors. Their achievements have led to an understanding of how cold, heat, and mechanical forces trigger nerve impulses and the mechanisms by which humans perceive and adapt to external stimuli. This shows thatthe study of receptors is currently a hot topic in physiological research and our research is close to the current mainstream direction of scientific research.

        At the same time, with the development and progress of society and the improvement of people's living standard, people are more eager toget the taste enjoyment after fully solving the problem of food and clothing, and they also want to know more about their differences in the trend of popularization and are eager toexplore the unknown self further.

Quantifying Taste-enables food companies to develop more innovative products and improve their competitiveness

The Problem: Food companies currently lack an objective, accurate and less costly means of taste testing for quantifying flavor.

        The current means of taste detection is mainlymanual tasting, supplemented by theelectronic tongue.Manual tasting has the advantage of perfectly matching the actual human feelings and can give feedback on taste.

        However, training a food sensory researcher is veryexpensive, and its long training period and small scope of work after a single culture undoubtedly make it even more costly. Manual tasting is alsosubjective and poses food safety risks.

        The electronic tongue, as an auxiliary tool, canonly measure the taste of a substance based on human tasting results, but cannot evaluate the overall taste of food. At the same time, one electronic tongue can only be used to measure the taste of one certain food, theapplication is limited and the detection time is long. Therefore, there is a lack of an objective, accurate and low cost taste detection tool for quantifying taste in the market.

Current solution: Food sensory researcher tasting evaluation-based, electronic tongue-assisted measurement

        Food sensory researcher is an emerging profession. Through long-term training, food sensory researchers can objectively and precisely quantify the taste of food by tasting it. Because the evaluation is done manually, the value of the evaluation is completely in line with human perception. Also after understanding, food companies are assigned to appropriate positions to evaluate food based on the researcher's selection and assessment. Evaluation of food is used a group of a total of more than 20 researchers to evaluate together, each researcher does not interfere with each other, using the form of light to score. If there are serious errors in several data, the results of this evaluation are discarded. Therefore, the food sensory researchers are very efficient and relatively accurate in evaluating food.

        However, sensory researchers are human beings after all, and are inevitably influenced by subjective factors. Moods, preferences, and other factors potentially influence the outcome of a tasting. Also, according to the above description, it is very costly and labor intensive to train a food sensory researcher, so quantifying taste through a food sensory researcher is a very costly and lengthy process.

        This is why many food companies around the world are looking for ways to quantify flavors instead of sensory researchers. Thus the electronic tongue was conceived. As a new modern intelligent sensory instrument, the electronic tongue is a detection technology based on a multi-sensor array with low selectivity, non-specificity and interaction sensitivity, which detects the overall characteristic response signal of a liquid sample and combines a chemometric approach to pattern recognition processing of the sample for qualitative and quantitative analysis, as well as an intelligent detection technology that simulates the taste assessment process.

However, according to discussions with experts from the COFCO Health and Nutrition Research Institute and literature research, the electronic tongue is currently used as a tool to assist in manual evaluation and quantification of taste. Because the electronic tongue can only measure the "taste" by measuring the content of a specific substance, for example, the electronic tongue can measure the sugar content by detecting the content of sucrose, but not the quantification of sweetness.

And electronic tongue data analysis is based on manual evaluation of the data database,computing, and the real manual evaluation there is still not a small gap. At the same time an electronic tongue can only detect the taste of similar food, but can not detect the taste of various foods, does not have universal applicability, such as tea evaluation electronic tongue can only quantify the taste data of tea, but can not detect the taste data of coffee, so it will also make the cost of detection higher.

Our solution: Tasting Officer - human-derived taste receptors heterologously expressed in Saccharomyces cerevisiae.

        We learned that there is almost a gap in the current biological ways to quantify taste. Therefore, we addressed the shortcomings of current ways to quantify taste and designed human-derived taste receptors heterologously expressed in Saccharomyces cerevisiae as Tasting Officer to amplify the signal by modifying the MAPK pathway to quantify taste using fluorescence intensity.

        The basic human tastes are sour, sweet, bitter, salty and umami. Since cells will die of dehydration in solutions with too much salt, while sour tastes are mainly related to pH, which is easier to measure. Thus, we mainly plan to construct a bioassay system using cells for sweet, umami and bitter tastes.

        At the same time, we thought that human taste receptors have the ability to detect taste, so we wanted to build taste detection cells by heterologous expression of human-derived taste receptors, so that we can solve the problem of detecting specific taste, and our iGEM2017_BIT-China team had experience in building sweet taste detection yeast in the iGEM competition in 2017, so we chose this as the basis for developing "Tasting Officer "-a biosensor system for heterologous expression of human-derived taste receptors.

Step1. Heterologous expression of human-derived taste receptors

        In order for Saccharomyces cerevisiae to help us detect umami, bitterness, and sweetness, we need to introduce genes encoding human umami receptors, human bitterness receptors, and human sweetness receptors into Saccharomyces cerevisiae, of which the genes encoding umami receptors are T1R1 and T1R3, and the genes encoding sweetness receptors are T1R2 and T1R3, and the proteins encoding bitterness receptors are 25 kinds after literature research. We selected three of them: T2R1, T2R4 and T2R46 (these three genes belong to three different chromosomes) and introduced them into Saccharomyces cerevisiae for expression.

        Taking the construction of plasmids expressing the umami receptor as an example, the first step we must complete the acquisition, optimization and synthesis of the umami receptor genes.We first obtain the sequences of T1R1 and T1R3 from NCBI, and then use Snap Gene for sequence optimization.

        The sequence optimization mainly includes the modification of the signal peptide of the heterologous receptor, i.e., adding the signal sequence of Ste2, the purpose of which is to increase the content of T1R1 and T1R3 in the endoplasmic reticulum of Saccharomyces cerevisiae, thus improving their localization to the cell membrane. Finally, the codon optimization and gene synthesis were entrusted to Genewiz Company.

Subsequently, we planned to integrate T1R1 and T1R3 into pESC-LEU, and to be able to determine whether the umami taste receptor is localized on the cell membrane, we added the linker sequence after T1R1 and attached mRFP, so that we could observe whether the receptor is localized on the membrane by fluorescence microscopy, and then because we were going to introduce the plasmid expressing the umami taste receptor, the plasmid expressing the sweet taste receptor, and the plasmid expressing the bitter taste receptor into Saccharomyces cerevisiae at the same time, which may cause difficulties in the growth of Saccharomyces cerevisiae.

After discussion and literature reading, we decided to add different inducible promoters to the plasmids expressing taste receptors so that Saccharomyces cerevisiae can express different taste receptor proteins when different inducers are added, thus detecting different flavors. At the same time, different fluorescent protein sequences are connected, so that the expression position of different receptors can be detected by fluorescence microscope after receptor expression, so as to confirm the transformation of yeast into a specific Tasting Officer.

        We chose three inducible promoters, Modified Gal promoter (linked before T1R1), PADH7 (linked before T1R2), and PCUP1 (linked before T2R1.) Modified Gal promoter is induced by estradiol, PADH7 is induced by vanillin, and PCUP1 is induced by copper ions induction.

        With the above idea, we successfully constructed a plasmid for detecting umami, a plasmid for detecting sweetness, and a plasmid for detecting bitterness by using OE-PCR, Gibson ligation, and enzyme ligation.

Step2.Modification of MAPK pathway

        Referring to our team's project in 2017, we found that in order to enable signal amplification after binding of different substances to taste receptors, a modification of the natural MAPK pathway in yeast is required. referring to their design, it was decided to knock out three genes in the signaling pathway, namely Ste2, Far1 and Sst2. knocking out Ste2 is to allow T1R1 and T1R3 to bind to the previously modified Gpa1 protein, the knockdown of Ste2 is to allow T1R1 and T1R3 to bind to the previously modified Gpa1 protein, thus converting the MAPK pathway that binds pheromone α to the MAPK pathway that detects umami.

        The knockdown of Far1 gene is necessary because its expression can stall the cell cycle in G1 phase and affect the growth of the bacterium. And Sst2 is a negative feedback regulatory protein that specifically hydrolyzes GTP bound to Gpa1, resetting the signaling pathway from an activated state to a resting state to avoid infinite transmission of signals, so its gene is knocked down with the aim of amplifying the signal output. Therefore, the Saccharomyces cerevisiae strain used in our experiments is the one that has knocked out these three genes - Cen.PK2-1C.

Step3. Detection of the signal

        We finally need to visualize the signal generated by the receptor protein after binding different substances, and we also intend to use fluorescence to achieve this step, following the design of our team in 2017, linking the promoter Pfus (which will be activated by Ste12 in this MAPK pathway) with the reporter gene green fluorescent protein eGFP, and CYC1T to form the detection gene line, and the shuttle vector pRS42K was carried into Saccharomyces cerevisiae cells to complete the construction of the detection line.

Step4.Improvment

        During the project discussion, we thought that if the sample to be tested is hot soup, it may make the Saccharomyces cerevisiae inactive, but if we wait for the sample to become cold before testing, it will not only take too long, but also the flavor of the sample may change. We intend to overexpress PHO3 gene in Saccharomyces cerevisiae to improve its heat tolerance and make it possible to detect a wider range of samples.

Do we really know ourselves?

The problem: Losing yourself in the trend of popularization

        The perception of taste is a delicate process, and people often face difficulties in choosing food and struggle with what they really want to eat. Especially for condiments, currently people mostly just passively accept the taste of condiments that can be purchased in the market, without really knowing what they really like the taste of. Especially in today's popular era, the fast-paced life makes us tired of adapting to the intense work and study, often neglecting who we are and what we like.

Current solution: Using applets to answer questions and explore yourself

        With the progress of life, all kinds of technology floods our lives, and applets can be found everywhere testing people's character or uniqueness. Discovering the unknown self is something people love to do, especially in such a popular era, people are always used to follow the trend to do something and lose themselves in many cases. The shopping platform currently introduces the function of recommending products you may like based on browsing, so that consumers can find their favorite products more conveniently. But for food, there is not yet a applets with a similar function.

Our solution: You Are Who You Are

        We designed the Flavor Card website with Chinese dishes divided into vegetarian and meat lover, taking into account various people. We hope that in this way, we can help the public understand their own tastes and promote Chinese cuisine at the same time.

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        In the website customers choose the dishes they want to try in the menu according to their mood or thoughts at that moment, and also choose their favorite level. The back office will process the data according to the customer's choice, get the value of the customer's preference of the five basic flavors, and form a report with unique color and taste to the customer, and then our "Creative Food seasonings" will be customized according to the data in the Flavor Card. At the same time, our Flavor Card website can collect people's favorite flavors from all over the world and compile them into a flavor database, which can be a useful reference for food companies.

        In this way, we want people to explore themselves and find themselves in the midst of popular trends, to know themselves better today than they did yesterday, to understand more about what they like, and to feel that you are the only you at this moment.

Seasoning is a necessary part of people's enjoyment of flavor

The problem:People generally choose seasonings based on experience, and it is difficult to buy the most suitable seasonings for their tastes.

        Flavor presentation is an intriguing process, and seasonings are essential in this process to improve the flavor of food. Modern condiments include soy sauce, vinegar, salt, monosodium glutamate, yeast extracts, chicken essence, spices and compound condiments. Nowadays, there is a wide variety of condiments and the condiment market is developing rapidly.

        However, the types and functions of many condiments are similar, and the differences between flavors are difficult to quantify, so individual users generally need to choose the right condiment based on experience, and it is difficult to buy the most suitable condiment for their taste. At the same time, the flavor customization of condiments is still mainly for the condiment manufacturers, but for customers they can only buy the existing condiments on the market to determine the flavor, and it is difficult to realize the independent customization of condiment flavor.

Current solution: Market-oriented research and development of compound seasonings

        In life, people may not have time to cook a delicious meal or to taste something delicious due to many reasons, such as busy work schedule and different food culture choices. By talking with the COFCO Nutrition and Health Research Institute and Beijing Liu Bi Ju Food Co., Ltd., we found that food companies are actively innovating and developing seasonings for young people. Because people are more likely to accept a variety of flavors and nutritious seasonings, and want to be able to consume them after simple processing, compound seasonings.

Our solution：Creative Food Seasonings-meet individual umami taste needs and be nutritious

        After learning about your taste through the Flavor Card website , we will customize Creative Food seasonings based on the data. After communicating with Beijing Liu Bi Ju Food Co., Ltd., we understand that there is a great demand for umami, so our customized seasoning for umami is also in line with the social demand.

        Firstly, the soybean isolate protein is enzymatically digested by specific types and ratios of enzymes to obtain peptides with different umami, sweet and bitter flavors, and after testing, the appropriate sample of them is selected for subsequent rationing. We complete our private customization by building a mathematical model to calculate the ratios and make the blending based on the data of user preferences obtained from the Flavor Card.

        Because of the complexity of human tastes, we intend to customize the seasoning for regional differences in taste, and then gradually add certain flavors and substances to make the seasoning taste richer, while the color of the seasoning jar matches the color of the color taste report, presenting your own exclusive seasoning.

        Our seasoning ingredients are plant-derived soybean isolate protein, and the enzymes used are food-grade commercial enzymes, all of which meet national food safety standards ,so that we can end up with a custom-flavored and nutrient-rich seasoning. And we take into account the differences in eating habits between vegetarians and meat-lover by treating them separately, which also helps vegetarians to enhance the richness of their daily flavors.

        At the same time, our Creative Food Seasonings may provide a umami taste experience that has never been seen in the market and is unique to our own tastebuds. In addition, our seasonings are rich in all essential amino acids and have a milk-like nutritional profile.

        Creative Food Seasonings have been successfully added to the existing products of Beijing Liu Bi Ju Food Co., Ltd., and can be added to other products of food companies.We strive to create a branded product with corporate characteristics,which not only improves the competitiveness of food companies, but also satisfies individuals the demand for taste.

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[10] iGEM2017 BIT-China https://2017.igem.org/Team:BIT-China