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

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<h2>Overview</h2>
 
<h2>Overview</h2>
  
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  <p>As earlier as in 2013, 2013iGEM team of our university (UESTC-Life) had built the plasmid carrying both DhaA31 and HheC-W249P for the degradation of TCP in E. coli.. Subsequent studies have shown that the triple enzyme cascade pathway consisting of DhaA31, HheC and EchA can efficiently convert TCP into non-toxic glycerol[1]. In our project this year, we improved our 2013-project by constructing a powerful three-enzyme system that includes 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 TCP (Fig 1). Compared to physical and microbial degradation methods, phytoremediation is more sustained and efficient and can be used widely in the treatment of polluted soil and underground water. </p>
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/1/1a/T--UESTC-China--design_1.jpg" style="width: 95%;" /></p>
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/1/1a/T--UESTC-China--design_1.jpg" style="width: 95%;" /></p>
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<p>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.</p>
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<p class="mid">Figure1. The key strategies used in our project.</p>
 
</div>
 
</div>
  
 
<div id="Construct-the-pathway">
 
<div id="Construct-the-pathway">
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<h2>Construct the pathway of phytoremediation</h2>
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  <h2>Pathway Construction of phytoremediation</h2>
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  <p>DThree enzymes DhaA, HheC, EchA are the key enzymes to degrade TCP into glycerol (Fig 2). Their expression efficiency in tobacco determines the degradation efficiency of TCP. For this, we selected pCaMV35s, a commonly used promoters in plant as a promoter 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. </p>
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/c/cc/T--UESTC-China--design_2.png" style="width: 75%;" /></p>
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/c/cc/T--UESTC-China--design_2.png" style="width: 75%;" /></p>
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<p class="mid">Figure2. The degradation pathway in tobacco.</p>
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  <p>Then, a plasmid model containing three enzymes of DhaA31, HheC-W249P and EchA was constructed. Considering that the plant may produce endogenous EchA[5], a plasmid with only the first two enzymes was also designed(Fig 3).</p>
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  <p class="pic"><img src="https://static.igem.org/mediawiki/2017/d/d1/T--UESTC-China--design_3.jpg" style="width: 100%;" /></p>
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/d/d1/T--UESTC-China--design_3.jpg" style="width: 100%;" /></p>
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/d/d1/T--UESTC-China--design_3.jpg" style="width: 100%;" /></p>
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<p>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.</p>
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<p class="mid">Figure3. The construction of phytoremediation system conversion TCP to Gly.</p>
 
</div>
 
</div>
  
 
<div id="Improve-the-function">
 
<div id="Improve-the-function">
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<h2>Improve the function of our tobacco</h2>
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<h2>Function improvement of our tobacco</h2>
  
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<p>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.</p>
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<p>To improve our biodegradation system, we designed the plant root-specific expression promoter PYK10 helping enzymes enriched at the root by considering the fact that the roots in tobacco are in direct contact with wastewater or soil containing 1,2,3-TCP [6]. Besides, the development of super-tobacco’s 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]. By this way, we get our super tobacco at last.</p>
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<p>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. </p>
+
<p>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-Life in 2014iGEM, we induce 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). </p>
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/a/ac/T--UESTC-China--design_4.jpg" style="width: 90%;" /></p>
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/a/ac/T--UESTC-China--design_4.jpg" style="width: 90%;" /></p>
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<p class="mid">Figure4. The strategies to improve tobacco’s function.</p>
 
</div>
 
</div>
  
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<li>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.</li>
 
<li>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.</li>
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<li>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.</li>
 
</ol>
 
</ol>
  

Revision as of 15:00, 31 October 2017

Team:UESTC-China/Design - 2017.igem.org

Overview

As earlier as in 2013, 2013iGEM team of our university (UESTC-Life) had built the plasmid carrying both DhaA31 and HheC-W249P for the degradation of TCP in E. coli.. Subsequent studies have shown that the triple enzyme cascade pathway consisting of DhaA31, HheC and EchA can efficiently convert TCP into non-toxic glycerol[1]. In our project this year, we improved our 2013-project by constructing a powerful three-enzyme system that includes 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 TCP (Fig 1). Compared to physical and microbial degradation methods, phytoremediation is more sustained and efficient and can be used widely in the treatment of polluted soil and underground water.

Figure1. The key strategies used in our project.

Pathway Construction of phytoremediation

DThree enzymes DhaA, HheC, EchA are the key enzymes to degrade TCP into glycerol (Fig 2). Their expression efficiency in tobacco determines the degradation efficiency of TCP. For this, we selected pCaMV35s, a commonly used promoters in plant as a promoter 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.

Figure2. The degradation pathway in tobacco.

Then, a plasmid model containing three enzymes of DhaA31, HheC-W249P and EchA was constructed. Considering that the plant may produce endogenous EchA[5], a plasmid with only the first two enzymes was also designed(Fig 3).

Figure3. The construction of phytoremediation system conversion TCP to Gly.

Function improvement of our tobacco

To improve our biodegradation system, we designed the plant root-specific expression promoter PYK10 helping enzymes enriched at the root by considering the fact that the roots in tobacco are in direct contact with wastewater or soil containing 1,2,3-TCP [6]. Besides, the development of super-tobacco’s 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]. By this way, we get 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-Life in 2014iGEM, we induce 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).

Figure4. The strategies to improve tobacco’s function.

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.