Overview

   Since the twenty-first century, people in today's society are often plagued by insomnia and anxiety due to the rapid development of various industries, accelerated pace of life and increased competitive pressure. iGEM team of FAFU-China discovered that GABA is a substance that improves human sleep quality, lowers blood pressure and relieves anxiety, which helps to relieve modern people's stress. After brainstorming and focusing on the topic, the team discussed and finally decided to modify the biological metabolic system of longan: screen key genes in the GABA synthesis pathway, use synthetic biology and molecular biotechnology as the core tools for experimental implementation, and maximize the GABA content through genetic engineering modification and CRISPR-Cas9 technology without affecting the normal developmental development of longan cells . Subsequently, we will promote longan GABA to the food processing and medical fields for the benefit of mankind by using high-yield cell lines for fermenter production in order to create high-yield GABA cell factories and collaborate with related companies to extract GABA and use it in the pharmaceutical industry.

Inspiration

   In recent years, we have been inspired by the deletion of the CaMBD gene from the tomato genome in Japan through gene editing techniques, resulting in tomatoes with high GABA content, which is 5-6 times higher than that of ordinary tomatoes.

Ideas

   Fujian is rich in longan, and longan fruit trees are abundant in resources. Longan fruit pulp is rich in nutrients and high in functional metabolites, with considerable GABA content. Genetic modification of longan is targeted through synthetic biology to enable it to produce more GABA substances as the team wishes.
Feasibility
   The autonomic regulation of the sleep-wake cycle is disturbed by a significant decrease in the centrally important neurotransmitter GABA and a significant increase in Glu in patients with endogenous insomnia. In plants and animals, GABA is synthesized mainly by the conversion of Glu by the enzyme GAD, a glutamic acid decarboxylase. Although the human brain can convert Glu to GABA, the accumulation of GABA is extremely difficult under conditions such as aging and increased mental stress. GABA supplementation in the daily diet is necessary for effective improvement.
Intention
   Local people solve local problems
   High yield GABA for the Benefit of Humanity

Experimental design

   The screening of metabolic pathways in Lobelia was carried out at the front stage, targeting at the GABA branch: glutamate is decarboxylated by glutamate decarboxylase to produce GABA. overexpression vectors were constructed for GAD genes to improve the efficiency and total amount of glutamate conversion. Knock out the CaM gene which has a negative correlation with GAD. Achieve the effect of genetic modification. The later stage relies on the factory cell culture technology.

Technology Route

Route
   CRISPR-Cas9, genetic engineering, factory cultured cells
Technical Support
     1. Longan embryonic callus, fast-growing succession-ready cell clusters
     2. Rapid transformation gene system
     3. Gene Editing Related Academic Resources
     4. Longan Whole Genome
   5. Researchers, MS students, PhD students, and an outstanding group of undergraduate students with a passion and enthusiasm for synthetic biology, collaborating across disciplines.

Specific implementation

Target Users

   With a large base of insomnia groups and depression groups, we are mainly targeting users who can accept gene editing and genetically engineered organisms designed to produce GABA. We mainly target young student groups and high-pressure white-collar groups.
User Instructions:
1、The recommended additional daily intake of GABA should not exceed 100mg;
2、Use GABA and any drugs affecting the GABA pathway with caution;
3、GABA may cause drowsiness, and an overdose is not recommended.

Products

Product Showcase
Weak points
   Gene edited foods are only genetically edited by means of gene editing of the scientific species itself without transferring genes from other scientific species and no cancer or other safety problems have been found to be induced by gene edited foods. Although no foreign genes are added during the gene editing process, and gene edited plants are considered as safe as new varieties improved by traditional methods, the general public is generally unable to properly distinguish them from genetically modified foods. Consumers do not know enough about gene edited foods, and people have mixed feelings about them, making it more difficult to bring them to market.

Outlook

Technical improvements
   We have great interest in exploring more factory modules and more ideas for the design of genetic circuits for the Longan cells and discussing the direction of upgrading in all aspects. However, due to the short duration of the competition, the path of exploring the nature of life science of longan in this project will end at the end of the season. In the follow-up, we expect to make creative changes to metabolic pathways and use longan healing tissues to produce more functional metabolites for the benefit of humanity.
Expected products
   Our project wanted to establish a high GABA producing Lobelia cell line by. To simulate the working of this cell line, we performed experiments in suspension culture to simulate the production of GABA in small light-protected fermenters. Our modeling was achieved in part by experimenting with expanded cultures under ideal conditions that are difficult to perform in the standard model of a factory. Our cell line culture is renewed in weeks of time and a basic model of GABA content variation is simulated by measuring GABA content in batches of suspension material over a week. Based on this model, we perform another modeling to find out the most suitable time for GABA extraction and the corresponding number of days and batches of culture to propose a reliable production model for factory-scale production. The basis for standardized production was provided.
Plant design and implementation
Site selection
   Considering the extremely high market potential of GABA-related products, we used the distance between superior and inferior solutions (TOPSIS) method based on the simulated annealing projection tracing method (SA-PP) to select the best sites for the production of GABA-related products among nine prefecture-level cities in Fujian Province, with respect to the conditions suitable for the growth and production and transportation of longan. Suitable sites for the production of GABA products can effectively reduce the production cost of the products and improve certain support for the promotion of regional economic development. In the results of the SA-PP-TOPSIS model, if the proximity of each location to the optimal location is higher, it means that the sample is more suitable for building a factory. We used matlab to solve the model and found that Putian City is the best city for building a factory.
Core mechanical construction
   There are more fermentor designs on strains with mature industrial technology, but few fermentor designs based on plant cell factories have been reported.
Costs
   Since the team does not include economic management-related personnel this year, this aspect has not yet been extended to the cost and profitability of establishing cell factories.