Team:UESTC-China/Plant
Overview
Our team produced a Super Tobacco which could degrade 1,2,3-TCP into glycerol this year. We introduced three key enzymes, DhaA31, HheC-W249P, EchA into tobacco by Agrobacterium mediated transformation with the application of synthetic biology of plants. In order to obtain better degradation efficiency of TCP by enriching these three enzymes at the root of tobacco, we also used a plant root specific expression promoter YK10 to replace the constitutive promoter CaMV35s. At the same time, a gene encoding the expression of cytokinin, CKX3, which could improve the biomass of roots as well as a plant cell wall localization signal peptide AO-S were also introduced into tobacco. All the genes we used have been codon-optimized for dicotyledonous plants and were submitted as BioBricks to iGEM Registry. Our results provided a potential approach for degrading 1,2,3-TCP by plants and we hoped that our Biobricks could be helpful to other iGEMers who worked with synthetic biology of plants in the future!
Synthetic Biology in plants
1. Plasmid construction for working in tobacco
In order to express the DhaA31, HheC-W249P and EchA in tobacco, we firstly did the codon-optimized of the three enzymes in dicotyledonous plants. With the application of 2A peptide and Golden Gate strategy, we successfully introduced DhaA31, HheC-W249P and EchA into the tobacco. The expression of these three enzymes were driven by the common constitutive promoter CaMV35s in plants.
Considering that TCP mainly existed in the soil and groundwater, we chose the plant root specific expression promoter YK10 to replace the constitutive promoter CaMV35s in order to obtain better degradation efficiency of TCP by enriching these three enzymes at the root of the plants.
At the same time, in order to strengthen the root growth of tobacco, a gene that coded the expression of the cytokinin, CKX3, was also introduced into tobacco after the codon optimization of dicotyledonous plants.
Finally, in order to transport the three enzymes out of tobacco to play their functions, we chose AO-S, a plant cell wall localization peptide, to help achieve this goal. Besides, AO-S can also stabilize the expression of the proteins. Based on these, the vectors which can fulfil the fusion expression of AO-S together with the three enzymes were constructed.
See more details in our Demonstrate.
2. Tobacco Transformation
In order to introduce foreign genes into tobacco and achieve the stable expression of the exogenous proteins in tobacco, Agrobacterium mediated transformation which was a common transgenic method for dicotyledonous plants was used.
For obtaining the transgenic tobacco, the whole steps can be divided into the following eight parts. Seed germination, pre-culture, infection, co-culture, resistance screening, calli induction, root culture, transplanting. The cycle of obtaining a transgenic tobacco was about ten weeks.
3. Positive validation and Enzyme activity assay of transgenic plant
See more details in our Demonstrate.
Plant parts submitted to the Registry
We have submitted five parts connected to plants synthetic biology to the Registry this year.
| Parts number | Type | Composition | Length |
|---|---|---|---|
| BBa_K2286000 | Coding | Halohydrin dehalogenase mutant | 762bp |
| BBa_K2286002 | Coding | ascorbate oxidase signal peptide and haloalkane dehalogenase mutant | 981bp |
| BBa_K2286003 | Coding | epoxide hydrolase | 882bp |
| BBa_K2286004 | Coding | Cytokinin-degrading Cytokinin Oxidase 3 | 1569bp |
| BBa_K2286005 | Regulatory | a seedling and root specific promoter | 1448bp |
See more details in our Parts.
Unforgettable photos
This year we UESTC-China try our best to produce the Super Tobacco.
