Difference between revisions of "Team:UESTC-China/design"

Overview

As early as 2013, for the degradation of TCP, 2013iGEM team of our school had built the plasmid of DhaA31 and HheC-W249P , and used E. coli as a chassis biology where the two enzymes achieved the purpose of degradation. Subsequent studies have shown that the triple enzyme cascade pathway consisting of DhaA31, HheC and EchA can convert TCP into non-toxic glycerol[1]. In order to increase degradation efficiency, we used the excellent mutant DhaA31[2], HheC-W249P[3], together with the wild type EchA to realize gene stacking. We used synthetic biological means to construct a plasmid carried three genes firstly. Compared to physical and microbial degradation, phytoremediation is more sustained and efficient by which we can achieve the degradation pathway. Considering that the plant is likely to express EchA under stress, we will build a plasmid containing only the first two genes, and finally get a highly efficient phytoremediation pathway of TCP. In the process of degradation, toxic effects have decreased as well as the growth cycle of plants has extended.

Construct the pathway of phytoremediation

DhaA, HheC, EchA these three enzymes are the key enzymes to degrade TCP and produce glycerol. Their expression activity in tobacco determines the degradation efficiency of TCP. First, we selected pCaMV35s, a commonly used promoters in plant and the application of 2A peptide strategy to achieve the stable expression of three enzymes in tobacco[4]. In order to realize the degradation of TCP and realize the production of beneficial glycerol, we constructed a plasmid model containing three enzymes of DhaA31, HheC-W249P and EchA. When DhaA31 works independently in tobacco, 1,2,3-TCP can be degraded to 2,3-DCP. When HheC-W249P works independently in tobacco, 2,3-DCP can be degraded to ECH, and CPD is degraded to GDL, when EchA work independently in tobacco, it can degrade ECH into CPD and degrade GDL into glycerol. In 2013, UESTC-China induced DhaA31and HheC-W249P in E.coli, and this year we induced the two enzymes together with EchA in tobacco. In this way, 1,2,3-TCP can be degraded into glycerol, so we first designed a three-gene plasmid. Considering the plant itself may exist EchA[5], in order to avoid too many exogenous enzymes and affect plant metabolism, so we designed a plasmid with only the first two enzymes.

Improve the function of our tobacco

Only degrading 1,2,3-TCP is not enough, we also pursue a more efficient way. Considering that the three enzymes expressed mainly in the roots where tobacco are in direct contact with wastewater or soil containing 1,2,3-TCP, we designed the plant root-specific expression promoter pYK10 helping enzymes fixed at the root[6]. Thus the development of super-tobacco roots is particularly important, in order to improve the biomass of roots to increase the yield of enzyme, the CKX gene is selected by us[7]. Finally, in order to make the three enzymes more susceptible to TCP, we chose the cell wall localization signal peptide AO-S to achieve fusion expression with three enzymes (at the same time AO-S also has the role of stabling enzyme expression)[8]. Through the improvements above, we get more powerful super tobacco at last.

We plan to apply super-tobacco to real life in the future, and for this purpose we should take full account of biosafety and maneuverability. We will induce AdCP gene into our plans in the future because of its capability to lead to pollen abortion. At the same time, chloroplast transformation will be taken into consideration to avoid gene flow and improve gene expression. Thus, we can both meet the actual needs and ensure the biosafety.

References

1. Dvorak, P., et al., Immobilized synthetic pathway for biodegradation of toxic recalcitrant pollutant 1,2,3-trichloropropane. Environ Sci Technol, 2014. 48(12): p. 6859-66.
2. Pavlova, M., et al., Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nat Chem Biol, 2009. 5(10): p. 727-33.
3. Wang, X., et al., Improvement of the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by engineering C-terminal amino acids. J Biotechnol, 2015. 212: p. 92-8.
4. Buren, S., et al., Use of the foot-and-mouth disease virus 2A peptide co-expression system to study intracellular protein trafficking in Arabidopsis. PLoS One, 2012. 7(12): p. e51973.
5. Guo, A., J. Durner, and D.F. Klessig, Characterization of a tobacco epoxide hydrolase gene induced during the resistance response to TMV. Plant J, 1998. 15(5): p. 647-56.
6. Nitz, I., et al., Pyk10, a seedling and root specific gene and promoter from Arabidopsis thaliana. Plant Sci, 2001. 161(2): p. 337-346.
7. Werner, T., et al., Root-specific reduction of cytokinin causes enhanced root growth, drought tolerance, and leaf mineral enrichment in Arabidopsis and tobacco. Plant Cell, 2010. 22(12): p. 3905-20.
8. Nanasato, Y., et al., Biodegradation of gamma-hexachlorocyclohexane by transgenic hairy root cultures of Cucurbita moschata that accumulate recombinant bacterial LinA. Plant Cell Rep, 2016. 35(9): p. 1963-74.