IMPLEMENTATION

To combat COVID-19, medical workers in China have combined western and traditional Chinese medicine with satisfactory results. In traditional Chinese medical treatment of COVID-19 , Lonicera japonica played a very important role. It's main active ingredient, chlorogenic acid (CGA), has been shown to have anti-inflammatory effects. In Lonicera japonica cell, a hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene encods an enzyme that turn quinic acid and caffeoyl-CoA into CGA. As both quinic acid and caffeoyl-CoA are naturally exist in Oryza sativa L, the most common staple food in China, we are planning to transfer the HQT into Oryza sativa L. This can help people intake the anti-inflammatory ingredient they need during their daily diet.

Engineering

To verify our imagine, we tried to proof our concept by engineering in 3 steps:

STEP 1: Expression of Key Enzymes for Chlorogenic Acid Synthesis In Rice
STEP 2: HQT Expression Increases The Content of Chlorogenic Acid
STEP 3: Specific Expression of HQT In Rice Seeds

In our project, we planned to use two engineering systems:

SYSTEM A: p35S::HQT
SYSTEM B: pGluD-1::HQT

To form the engineering system designed 2 basic parts and 2 composite parts:

PART 1: GluD-1 Promoter(BBa_K3458002)
PART 2: HQT (BBa_K3458001)；
PART 3: 35S Promoter + HQT (BBa_K3458003)
PART 4: GluD-1 Promoter + HQT (BBa_K3458004)

(more information on the engineering success)

Implementation

Proposed End Users

Oryza sativa L. which is usually called rice, is not only a native species, but also the most common food crop in China. IGEM advocates "local people slove local problems". Starting from the local eating habits, we are trying to provide a brand new low-cost medicinal diet option.

What's more, as a staple food, rice has been widely accepted in the world, not only in Asia. Due to the planting of honeysuckle is regional, expressing the active ingredient in the rice can benefit more people in the world.

Implement In The Real World

Our Work

After engineering design and proof a series of concepts, we are confident that our project may have the potential to work in the real world. In our project, we successfully expressed the HQT gene heterologously in rice protoplasts, and verified that the expression of the HQT gene can indeed increase the content of chlorogenic acid in rice. In order to express HQT specifically in rice seeds, we introduced the endosperm-specific promoter GluD-1 and confirmed the specificity of GluD-1 promoter. (more information on the design and results)

Further Work

Regrettably, due to the COVID-19, the laboratory has temporarily closed. Also, considering the safety of GMO crops, our experimental plan stops at the stage of protoplasts. To turn our project into product, we definitelly need further tests including but not limited to verify our ideas in rice seeds conduct clinical tests. Here are some tests that may still be needed:

Use pGluD-1::HQT transfected protoplasts to obtain transformation seedlings;
Detect the expression in other stages and parts of the transformed seedlings to verify the specificity of expression;
Cultivate transformed seedlings to obtain rice seeds;
Test the HQT expression level and chlorogenic acid content in rice seeds;
Testing medicinal value;
Edible safety test;
…………

Safety

When conducting an experiment or developing a new product, our first cosideration is always the biosafety. It indicates whether the product may be at risk in the experimental design, process and subsequent development. Therefore, we consulted many infenrences to evaluate the safety of CGA, HQT and rice to ensure the overall safety of our project. At the same time, in the course of experiments and human practice, we have taken various measures to ensure that all actions are based on safety. (more information on the safety)

In The Project

As both Lonicera japonica and Oryza sativa L.are both native nontoxic species in China, there is no healthy risk to people. On the other hand, it does have the risk of gene flow which may pollute the gene pool of wild Oryza sativa L. However, we are going to use only the protoplast of Oryza sativa L. as our chassis and we won't culture them into whole plants. This should minimize the risk of gene flow.

Chlorogenic acid is widely distributed in plants. Furthermore, it is one of the main polyphenols in the human diet. After years of testing, chlorogenic acid is a common active ingredient in Traditional Chinese medicine. Therefore, it has a wide range of health-promoting properties and has been widely studied. Many drugs that contain chlorogenic acid are approved by the Food and Drug Administration(FDA). At the same time, we did not find any references that mentioned chlorogenic acid was harmful to humans. Thus, chlorogenic acid is safe.

Laboratory

Before entering the laboratory, our team conducted a course on life science laboratory safety and the use of conventional instruments.

Human Practices

All our human practice activitices strictly abide by the safety rles. Especially considering the special situation of COVID-19, we adopted an online qestionnaire survey. In other offline activities, we have also taken sufficient safety measures to maintain the distance between peoplle.

Other Challenges

1.The exist of corona virus limit the range of our human practice, and hinder our activities-- people will go to crowded places less often, and most of them would prefer staying at home. In addition, the relevent principal of different sites we can hold our activities in will approve we use the sites more strictly.

In our original plan, we will hold our presentation in library or local Children's Palace， but the principals do not allow as use these places. To solve the problem about site, we talk with the principal of a member's community, and gain the permission to use the place of the community. What's more, to attract people to join in our activities, we prepare some poster and flyer pasted on the wall in the community.

2.Beasides, we had a interview with two doctors of traditional Chinese medicine--Mis.Jiangning and Mr. Hupeng. For our project, they gave various evaluations and suggestions. Expect for agreement of our idea, they also questioned for the choice of medicinal materials.

" According to the view of traditional Chinese medicine, actually, honeysuckle is not the best material to raise immunity, since it's more suitable to clean away heat and toxic material." This means that it can not be taken by all people. For this reason, we adjusted the range of the people that can eat the rice of our project and the best condition that use our rice.

Although there are still some drawback in our project, we improve our project constantly. The problems that we met can also provide some experience for other teams, convenient for others to perpare their projects.

Team:GDSYZX/Implementation