Team:GDSYZX/Contribution
CONTRIBUTION
Add New Documentation to An Existing Part
We will artificially synthesize the codon-optimized HQT gene and assemble it with a constitutive promoter and GluD-1 promoter. GluD-1 is a specific and highly expressed promoter in rice seed cells. We are going to transfer the two recombinant-plasmids into rice protoplasts. We hope to achieve high expression of exogenous HQT only in rice seeds. However, due to technical constraints, we can only use rice leaves to collect protoplasts. Therefore, we also constructed a plasmid containing a constitutive promoter to test whether exogenous HQT can be expressed in rice cells. The ideal result is that the group transferred into the constitutive promoter has a higher expression level of HQT, while the group transferred into GluD-1 does not express.
Add New Parts
In our 2020 project, we provide 5 new parts:
1.HQT(BBa_K3458001)
Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene encodes a protein of 439 amino acids from L. japonica. It can catalyze the reaction of caffeoyl-Coenzyme-A and quinic acid to produce chlorogenic acid.
http://parts.igem.org/Part:BBa_K3458001
2.GluD-1 Promoter（BBa_K3458002）
The promoter of the Oryza sativa L. glutelin gene (GluD-1), due to the deviation in the GCN4 motif, this pomoter has the characteristics of being specifically expressed in the endosperm of Oryza sativa L.
http://parts.igem.org/Part:BBa_K3458002
3.GluD-1 Promoter+HQT( BBa_K3458004)
This part is the L. japonica. hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) coding region, with a composite GluD-1 promoter element. Since our goal is to increase the content of chlorogenic acid in rice seeds specifically, we chose the 1.2kb GluD-1 promoter.
http://parts.igem.org/Part:BBa_K3458004
4.35s Promoter+HQT(Part:BBa_K3458003)
The GluD-1 promoter cannot express the gene in rice protoplasts due to its specific expression in Oryza sativa L. endosperm. To verify this, we designed the composite part that contains HQT and constitutive promoter 35s as a control and used western blot to compare GluD-1 promoter and 35S constitutive promoter.
http://parts.igem.org/Part:BBa_K3458003
5.Apis Mellifera Silk Fibroin 3 With Codon Optimization（ Part:BBa_K3458005）
We also improve part BBa_K1763000 (2015 UCLA), to let Apis mellifera Silk Fibroin 3 express in Arabidopsis thaliana better.
Troubleshooting
1.Because of the COVID-19, we have no condiction to finish the whole experiment, expressing the exogenous HQT in rice seed.
We artificially synthesized the codon-optimized HQT gene and assembled it with a constitutive promoter 35s and GluD-1 promoter. But we transferred the two sets of combined genes into rice protoplasts extracted from rice stems and leaves, hoped to test whether exogenous HQT can be expressed in rice cells. Because of the spatial specificity of GluD-1, the group transferred into GluD-1 does not express theoretically. Through Western blot test, the result demonstrates that 35s promoter can initiate the expression of HQT gene in Oryza sativa L. protoplasts while the GluD-1 promoter cannot.This is in accord with our expected results and proves the success of our ideas.
2.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.
3.Besides, 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. Except 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.