Team:UESTC-China/design

• ACHIEVEMENT

• PROJECT ∨

• MODELING

• ATTRIBUTIONS

• HUMAN PRACTICE ∨

• TEAM ∨

• NOTEBOOK ∨

Part

Demonstrate

Design

Introduction

Engagement

Supporting Research

Our Story

Collaborations

Team Introduce

Interlab

Safety

Protocal

Plant

Day Note

• Overview
• Construct the pathway
• Improve the function
• References

Overview

As early as in 2013, iGEM team of our university (UESTC-Life) have built the plasmid carrying both DhaA31 and HheC-W249P for the degradation of 1,2,3-TCP in E. coli.. Subsequent studies have shown that the three-enzyme cascade pathway consisting of DhaA31, HheC and EchA can efficiently convert 1,2,3-TCP into non-toxic glycerol[1]. In our project this year, we improved our 2013-project by constructing a powerful three-enzyme system that included two excellent mutants DhaA31[2], HheC-W249P[3], together with the wild type EchA. The system was transformed into tobacco in which we can achieve the efficient phytoremediation pathway of 1,2,3-TCP (Fig. 1).

Figure 1. The key strategies used in our project.

Pathway construction of phytoremediation

Three enzymes DhaA, HheC, EchA are the key enzymes to degrade 1,2,3-TCP into glycerol (Fig. 2). Their expression efficiency in tobacco determines the degradation efficiency of 1,2,3-TCP. For this, we selected pCaMV35s, a common constitutive promoter in plant for the expression of these three genes in tobacco. The 2A peptide strategy was adopted to achieve the stable expression of three enzymes in tobacco[4]. Three plasmids carrying each of these three genes (as controls) were firstly constructed respectively.

Figure 2. The degradation pathway of 1,2,3-TCP in tobacco.

Then, a plasmid module containing three enzymes of DhaA31, HheC-W249P and EchA was constructed. Considering that the plant may produce endogenous EchA[5], in order to avoid too many exogenous enzymes to affect plant metabolism, a plasmid with only the first two enzymes was also designed by us (Fig. 3).

 

Figure 3. The construction of phytoremediation system converted 1,2,3-TCP to Glycerol.

Function improvement of our tobacco

Considering the fact that the roots of tobacco are in direct contact with groundwater or soil containing 1,2,3-TCP, we designed the plant root-specific expression promoter pYK10 helping enzymes enriched at the root to improve our biodegradation system [6]. Besides, the development of super tobacco roots is particularly important, in order to improve the biomass of roots to increase the yield of enzymes, the cytokinin oxidase gene, CKX3 was selected by us[7]. Finally, in order to make the three enzymes more susceptible to 1,2,3-TCP, we constructed vectors which fulfilled the fusion expression of cell wall localization signal peptide AO-S together with three enzymes (at the same time AO-S also has the role of stabling enzyme expression)[8]. By this way, we got our 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. Like the design of UESTC-China in 2014 iGEM project, we transferred AdCP gene into our plants because of its capability to lead to pollen abortion[9]. 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 (Fig. 4).

 

Figure 4. The strategies to improve the function of tobacco.

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.
9. Shukla, P., et al., Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco. Funct Integr Genomics, 2014. 14(2): p. 307-17.