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

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<!--************下面为正文******************-->
 
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<p><img src="https://static.igem.org/mediawiki/2017/3/32/T--UESTC-China--leaf.png" alt="" class="hhh" /></p>
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<p><img src="https://static.igem.org/mediawiki/2017/f/fe/T--UESTC-China--placeholder_description.jpg" alt="" class="hhh" /></p>
  
 
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<ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="400">
 
<ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="400">
 
<li class="active">
 
<li class="active">
<a href="#Pathway" style="font-size: 14px;">Pathway construction</a>
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<a href="#Overview">Overview</a>
 
</li>
 
</li>
 
<li>
 
<li>
<a href="#Tobacco" style="font-size: 13.5px;">Tobacco Transformation</a>
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<a href="#Harm">Harm of 1,2,3-TCP</a>
 
</li>
 
</li>
 
<li>
 
<li>
<a href="#Positive" style="font-size: 14px;">Positive validation</a>
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<a href="#Technology">Technology to treat</a>
 
</li>
 
</li>
 
<li>
 
<li>
<a href="#Construct" style="font-size: 14px;">Construct validation</a>
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<a href="#Strategy">Our strategy</a>
 
</li>
 
</li>
 
<li>
 
<li>
<a href="#Enzyme" style="font-size: 14px;">Enzyme activity assay</a>
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<a href="#Conference">Conference</a>
</li>
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<li>
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<a href="#Activity" style="font-size: 14px;">Activity determination</a>
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</li>
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<li>
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<a href="#Work">Work going on</a>
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</li>
 
</li>
 
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<div class="col-lg-10" id="new-style">
<div id="Pathway">
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<div id="Overview">
<h2>Pathway construction for degrading 1,2,3-TCP </h2>
+
<h2>Overview</h2>
 
+
<p>The progress of the chemical industry and agriculture has brought great convenience to our lives. But a large number of chemical pollutants have been discharged through various means, most of them difficult to degrade and accompanied by toxicity, seriously polluting our environment. Among them, Organochlorine compounds is occupy a large part of the proportion. Through collecting questionnaire and soil research in most parts of China, our project is directed against 1,2,3-Trichloropropane - an organic chloride which is a less concerned and insecurity pollution. In this summer, we decided to use synthetic biology methods to achieve plant degradation of 1,2,3-trichloropropane by transferring three enzymes to tobacco to produce glycerol, which is environmentally friendly and recyclable.</p>
<p class="sub-title"> 1.1 Degrading Pathway construction based on the expression of DhaA31, HheC-W249P & EchA in tobacco </p>
+
<p>In order to express the three enzymes (DhaA31 isolated from Rhodococcus rhodochrous, HheC-W249P and EchA isolated from Agrobacterium radiobacter AD1) in tobacco, we firstly did the codon-optimized of the three enzymes in dicotyledonous plants. And then the genes of DhaA31, HheC-W249P and EchA were commercially synthesized. Finally, with the application of 2A peptide and Golden Gate strategy, we successfully introduced DhaA31, HheC-W249P and EchA into the plant expression vector piGEM2017-backbone which contained the resistance of kanamycin (NptII) (Fig 1). The expression of these three enzymes were driven by the common constitutive promoter CaMV35s in plants.</p>
+
 
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/c/c3/T--UESTC-China--demo_21.jpg" style="width: 95%;" /></p>
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<p class="mid">Figure 1. The introduction of piGEM2016-001 to piGEM2016-005</p>
+
 
+
<p>Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful (Fig 2).</p>
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/f/f5/T--UESTC-China--demo_1.png" style="width: 80%;" /></p>
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<p class="mid">Figure 2. The image of agarose gel electrophoresis by double enzyme digestion. </p>
+
 
+
<p class="mid">(a)piGEM2017-001 (Line1,enzyme digested by PstI+BamHI; Line2,enzyme digested by ScaI) </p>
+
 
+
<p class="mid">(b)piGEM2017-002 (Line1,enzyme digested by PstI+BamHI; Line2, enzyme digested by ScaI) </p>
+
 
+
<p class="mid">(c) piGEM2017-003 (Line1,enzyme digested by EcoNI+BamHI; Line2,enzyme digested by ScaI) </p>
+
 
+
<p class="mid">(d) piGEM2017-004 (Line 1, enzyme digested by EcoRI+BamHI; Line 2, enzyme digested by PstI+BamHI)</p>
+
 
+
<p class="mid">(e) piGEM2017-005 (Line 1, enzyme digested by EcoRI+BamHI; Line 2, enzyme digested by ScaI) </p>
+
 
+
<p class="mid">M: DNA marker</p>
+
 
+
<p class="sub-title">1.2 Degrading Pathway construction based on the root expression of DhaA31, HheC-W249P & EchA in tobacco </p>
+
<p>Considering that TCP mainly existed in the soil and groundwater, we chose the plant root specific expression promoter pYK10 from Arabidopsis thaliana to replace the constitutive promoter pCaMV35s in order to obtain better degradation efficiency of TCP by enriching these three enzymes at the root of the plants. </p>
+
 
+
<p>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 piGEM2017-backbone after the codon optimization of dicotyledonous plants (Fig 3).</p>
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/8/8e/T--UESTC-China--demo_22.jpg" style="width: 95%;" /></p>
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<p class="mid">Figure 3. The introduction of piGEM2017-008, piGEM2017-021 and piGEM2017-024</p>
+
 
+
<p>Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful (Fig 4). </p>
+
 
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/7/76/T--UESTC-China--demo_2.png" style="width: ;" /></p>
+
<p class="mid">Figure 4. The image of agarose gel electrophoresis by double enzyme digestion. </p>
+
 
+
<p class="mid"> (a) piGEM2017-008 (b) piGEM2017-021 (c) piGEM2017-024</p>
+
 
+
<p class="mid">(M: DNA marker; Line 1, enzyme digested by PstI; Line 2, enzyme digested by EcoRI +BamHI)</p>
+
 
+
<p class="sub-title">1.3 Degrading Pathway construction based on the extracellular expression of DhaA31, HheC-W249P & EchA in tobacco </p>
+
<p>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. </p>
+
 
+
<p>Finally, in order to intuitively confirm whether the multi-gene plant expression vectors we designed can play their roles, GUS reporter gene was also introduced into the piGEM2017-backbone (Fig 5).</p>
+
 
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/9/91/T--UESTC-China--demo_23.jpg" style="width: 95%;" /></p>
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<p class="mid">Figure 5. The introduction of piGEM2017-022 023 025 026 and 027</p>
+
 
+
<p>Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful (Fig 6). </p>
+
 
+
<p class="pic"><img src=" https://static.igem.org/mediawiki/2017/d/d6/T--UESTC-China--demo_3.png" style="width: ;" /></p>
+
<p class="mid">Figure 6. The image of agarose gel electrophoresis by double enzyme digestion. </p>
+
 
+
<p class="mid">(a) piGEM2017-022 (b) piGEM2017-023 (c) piGEM2017-025 (d) piGEM2017-026 (e) piGEM2017-027</p>
+
 
+
<p class="mid">(M: DNA marker; Line 1, enzyme digested by PstI; Line 2, enzyme digested by EcoRI +BamHI)</p>
+
 
+
 
</div>
 
</div>
  
<div id="Tobacco">
+
<div id="Harm">
<h2>Tobacco Transformation</h2>
+
<h2>Harm of 1,2,3-TCP</h2>
<p>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. </p>
+
 
 
<p>We firstly transferred our plasmids into Agrobacterium EHA105 and then the T-DNA fragment which contained the target genes of the Agrobacterium EHA105 was integrated into the chromosome of tobacco through the Agrobacterium mediated transformation.</p>
+
<p>1,2,3-Trichloropropane(TCP), an emerging organic pollutant, usually formed as industrial solvents[1] and also is a raw material for the production of 1,1,2,3-Tetrachloropropene and other chemical substances[2]. In agriculture, it has been one of the ingredients of soil fumigants, as well as a harmful byproduct for the production of other pesticides. The global yield of TCP reached about 50,000 tons annually[3] at present. Because it is biodegradation-recalcitrant[3] and will cause groundwater pollution and the damage of soil if it is discharged without treatment. </p>
 
 
<p>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 (Fig 7).</p>
+
<p class="mid">Table 1. Lethal concentration data. These experiments show that TCP is a DNA reactive carcinogen. [4]~[6]</p>
 +
<p class="mid">Lethal concentration data:</p>
 +
<table border="" cellspacing="" cellpadding="" class="form-hover">
 +
<thead>
 +
<tr>
 +
<th>Species</th>
 +
<th>Reference</th>
 +
<th>LC50(ppm)</th>
 +
<th>LCLo(ppm)</th>
 +
<th>Time</th>
 +
<th>Adjusted 0.5-hrLC (CF)</th>
 +
<th>Derived value</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>Mouse</td>
 +
<td>Izmerov et al. 1982</td>
 +
<td>555</td>
 +
<td>\</td>
 +
<td>2 hr</td>
 +
<td>888 ppm (1.6)</td>
 +
<td>89 ppm</td>
 +
</tr>
 +
<tr>
 +
<td>Mouse</td>
 +
<td>McOmie & Barnes 1949</td>
 +
<td>\</td>
 +
<td>5,000</td>
 +
<td>20 min</td>
 +
<td>4,350 ppm (0.87)</td>
 +
<td>435 ppm</td>
 +
</tr>
 +
<tr>
 +
<td>Rat</td>
 +
<td>McOmie & Barnes 1949</td>
 +
<td>LC100: 700</td>
 +
<td>\</td>
 +
<td>4 hr</td>
 +
<td>1,400 ppm (2.0)</td>
 +
<td>140 ppm</td>
 +
</tr>
 +
<tr>
 +
<td>Mouse</td>
 +
<td>McOmie & Barnes 1949</td>
 +
<td>LC100: 700</td>
 +
<td>\</td>
 +
<td>4 hr</td>
 +
<td>1,400 ppm (2.0)</td>
 +
<td>140 ppm</td>
 +
</tr>
 +
<tr>
 +
<td>Mouse</td>
 +
<td>McOmie & Barnes 1949</td>
 +
<td>LC100: 340</td>
 +
<td>\</td>
 +
<td>4 hr</td>
 +
<td>680 ppm (2.0)</td>
 +
<td>68 ppm</td>
 +
</tr>
 +
<tr>
 +
<td>Rat</td>
 +
<td>Smyth et al. 1962</td>
 +
<td>LC83: 1,000</td>
 +
<td>\</td>
 +
<td>4 hr</td>
 +
<td>2,000 ppm (2.0)</td>
 +
<td>200 ppm</td>
 +
</tr>
 +
<tr>
 +
<td>Rat</td>
 +
<td>UCC 1973</td>
 +
<td>LC83: 5,600</td>
 +
<td>\</td>
 +
<td>1 hr</td>
 +
<td>7,000 ppm (1.25)</td>
 +
<td>700 ppm</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="mid">Human data: It has been reported that objectionable ocular and mucosal irritation were experienced after 15 minutes of exposure to 100 ppm </p>
 +
<p>One of the most serious pollution incidents was happened at Californians. TCP was spread to all over California because the agricultural divisions of Dow Chemical and Shell started selling two soil fumigants (D-D and Telone) including TCP from the 1940s. Although TCP was banned from use in soil fumigants in the 1990s,there was a large amount of TCP remained and it was frequently detected in drinking water, threating to people's lives seriously[7]. </p>
 +
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/7/7c/T--UESTC-China--description_1.jpg" style="width: 55%;"/></p>
 +
<p class="mid">Figure 1 This map was produced by KQED[7], drawing on information from the State Water Resources Control Board. It shows water systems where significant levels of the 123-TCP have been detected. Image courtesy KQED, whose reporter, Sasha Khokha, recently found her own water supply to be contaminated as part of a story on this issue.</p>
 +
<p>What’s more, Dr. Qian Yong from China University of Geosciences has studied the behavior and relevant mechanism of TCP. At the ruins of a factory which was running from 1976 to 1979, he found TCP in high concentration(3890mg / L) underground in 2016[8]. </p>
 +
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/7/70/T--UESTC-China--description_2.jpg" style="width: 60%;"/></p>
 +
<p class="mid">Figure 2. The distribution of TCP contamination underground at this factory[8].</p>
 
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/8/82/T--UESTC-China--demo_4.jpg" style="width: 60%;" /></p>
+
<p>That tell us TCP can keep in the soil and groundwater for years, showing the great stability of TCP in the groundwater and soil. By the way, some researches show the adhesion of 1,2,3-TCP is very low[8], which means that it can easily spread into people's living area and threaten people's health because of  its potential carcinogenicity and the huge damage to the kidney. The best evidence is that TCP has been detected around the world more and more frequently in the past decade. </p>
<p class="mid">Figure 7. The steps for obtaining transgenic tobacco</p>
+
 
+
</div>
+
 
+
<div id="Positive">
+
<h2>Positive validation of transgenic plants</h2>
+
 
 
<p>After Agrobacterium mediated transformation, we successfully obtained the callis which have the resistance of kanamycin from the leaves of tobacco. The callis began to sprout in about 3 weeks. Another 3 weeks later, we obtained several transgenic tobacco. In order to verify whether the transgenic tobacco was false positive or not, we extracted the genomic DNA of T0 generation tobacco and designed pairs of primers to do PCR amplification of DhaA31, HheC-W249P& EchA. Compared with wild type, we successfully amplified the target bands in T0 transgenic tobacco, which indicated that our multi-gene expression system worked successfully and we obtained the positive transgenic tobacco (Fig 8). </p>
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/4/4b/T--UESTC-China--description_3.png" style="width: 90%;"/></p>
+
<p class="mid">Figure 3. The important report about TCP contamination[3]. We can learn about that the contamination of TCP are becoming more serious.  </p>
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/2/25/T--UESTC-China--demo_5.png" style="width: 80%;" /></p>
+
<p class="mid">Figure 8. The image of agarose gel electrophoresis by PCR detection</p>
+
<p class="mid">a.piGEM2017-001    b.piGEM2017-002 </p>
+
<p class="mid">c.piGEM2017-003    d.piGEM2017-004</p>
+
<p class="mid">e. piGEM2017-004    f.piGEM2017-005  </p>
+
<p class="mid"> ( PC, positive control (plasmid),WT, negative control (wild-type tobacco))</p>
+
<p>In order to confirm whether DhaA31, HheC-W249P, EchA and CKX3 can be normally transcribed in tobacco, and determine the transcription level of these genes in tobacco, we extracted the RNA from the leaves and roots of the transgenic tobacco. And then RT- PCR was used to detect. Tubulin, a common used endogenous gene of the tobacco was selected as internal reference. Compared with the wild type, the cDNA of transgenic tobacco amplified the corresponding band of target genes, indicating that the above four exogenous genes were successfully transcribed in tobacco. At the same time, the cDNA of genes expressed by pYK10 promoter amplified a clearly band in root tissue than in leaves or other tissues, indicating that pYK10 promoter could successfully drive DhaA31, HheC-W249P, EchA and CKX3 these four exogenous genes expressed in the root of tobacco (Fig 9). </p>
+
+
<p class="pic"><img src="" style="width: ;" /></p>
+
<p class="mid">Figure 9. The image of agarose gel electrophoresis by RT-PCR detection</p>
+
<p class="mid">a.piGEM2017-001   b.piGEM2017-002  c.piGEM2017-003</p>
+
<p class="mid">d.piGEM2017-004  e.piGEM2017-005  f.piGEM2017-008</p>
+
<p class="mid">(PC, positive control (plasmid),WT, negative control (wild-type tobacco), WT-L(leaves of </p>
+
<p class="mid">wild-type tobacco),WT-R(roots of wild-type tobacco),Tubulin was an internal control in tobacco)</p>
+
 
 
 +
<p>To sum up, we know that TCP is a very dangerous contaminant. However, governments don't pay enough attention to 1,2,3-TCP.Most counties even don't include 1,2,3-TCP into the pollutant detection list. Under these circumstances, we hope that we can attract attention of the society and contain the spread of 1,2,3-TCP pollution through this project.</p>
 
</div>
 
</div>
  
<div id="Construct">
+
<div id="Technology">
<h2>Construct validation of multi-gene expression system </h2>
+
<h2>Technology to treat 1,2,3-TCP</h2>
 
 
<p>β-D - glucuronidase (encoding GUS gene) was an acidic hydrolase isolated from the E. coli K-12 strain that catalyzed the hydrolysis of many beta-glucoside esters. In the histochemical staining with X-gluc, the plants containing the GUS gene will appear blue. Based on this, transgenic tobacco (piGEM2017-026) which had GUS gene and wild type tobacco were identified after the histochemical staining with X-gluc for 24h at the same time. Compared with wild type tocacco, the transgenic tobacco was dyed blue, proved that the multi-gene expression system in tobacco worked as we expected again. (Fig 10).</p>
+
<p>How do people solve TCP? Traditional remediation technology to treat 1,2,3-TCP includes granular activated carbon (GAC), dechlorination by hydrogen release compound (HRC®), reductive dechlorination by zero valent iron(ZVI) and so on[10]~[12]. </p>
 
 
<p class="pic"><img src="" style="width: ;" /></p>
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/8/85/T--UESTC-China--description_5.png" style="width: 95%;"/></p>
 +
<p class="mid">Figure 4. Result of treatability tests with TCP-contaminated groundwater/soil[10].</p>
 +
<p>Most of them are inefficient and impractical. There are just a few methods such as ZVI that has a good efficiency. But all of them are cost and hard to large-scale deal with TCP in nature. In that scenario, the concept of " Microbial remediation " began to be respected by people. Some studies have shown that 1,2,3 trichloropropane may be converted to CO2, H20 and HCl by biocatalytic action under the oxidative co-metabolism of O2 as the electron acceptor, so that people are mainly seeking to degrade in aerobic microorganisms method. Unfortunately, scientists have failed to enrich and screen aerobe which can degrade 1,2,3 - trichloropropane. However, they found several strains could degrade TCP in absolutely anaerobic environment . But this method, anaerobic microorganism degradation , hasn't been spread because of its harsh condition and low conversion efficiency. So there are some studies that want to introduce a series of enzyme genes into microorganisms such as Escherichia coli and Pseudomonas putida to degrade TCP. This method is efficient. But it has some limitations. First, these microoganisms have strict nutrient demand and weak competitiveness. Second, they may cause antibiotic resistance gene. Third, they usually depend on special inductions so that they can work. So, we hope to find a better method that can degrade TCP for a long time without extra resource[3]. In this time,Phytoremediation, a safe and long-lasting remediation strategy,go into our field of vision</p>
 
 
 
</div>
 
</div>
  
<div id="Enzyme">
+
<div id="Strategy">
<h2>Enzyme activity assay for the validation of transgenic tobacco</h2>
+
<h2>Our strategy</h2>
 
 
<p>In order to detect that the transgenic tobacco can work properly, the concentration of the reaction products was measured by gas chromatography (GC) as described in our protocol. Gas chromatograph Network GC System (Fuli 9750, China) equipped with a capillary column ZB-FFAP 30 m x 0.25 mm x 0.25 µm (Phenomenex, USA) was used to the quantitative analyses of all metabolites of the TCP degradation pathway except for GLY. The concentration of GLY was determined by GC (Fuli 9790) equipped with a DB-5 column (L60 m×i.d. 0.25 mm and df 0.25μm thin coating film) (Supelco, Bellefonate, PA, USA).</p>
+
<p>As an emerging "Green remediation" technology, phytoremediation shows its own great potential. Compared with "Bioremediation", its advantages are very obvious. The most amazing one is that plants have a set of photosynthetic autotrophic system which means they can degrade TCP in a long time and just need a little nutrition input, This method is easier and cheaper. Plants can also stabilize soil and absorb CO2 while cleaning the environment.</p>
 
 
<p class="sub-title">5.1 Measure the standard curves of reaction products</p>
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/3/34/T--UESTC-China--description_4.jpg" style="width: ;"/></p>
 +
<p class="mid">Figure 5.The main models of phytoremediation strategy.</p>
 
 
<p>For quantitative assay, standard curves of 1,2,3-TCP, 2,3-DCP, ECH, CPD, GDL, GLY ranging from 0-5 mM in 200 mM of Tris-SO4 buffer(pH=8.5) were measured (Fig 11).</p>
+
<p>"Phytoremediation"[13] mainly contains four models: Phytoextraction, Phytostabilization, Phytovolatilization and Phytodegradation. Through analyzing physical and chemical properties of 1,2,3-TCP, we know that 1,2,3-TCP is unlikely to become concentrated in plants and aquatic organisms because it has a low estimated bioconcentration factor (BCF) and sticking coefficient. So it is difficult to degrade 1,2,3-TCP with phytoextraction and phytostabilization. Furthermore, it needs a complex system to deal with these plants if we choose phytoextraction and phytostabilization, which requires much time and effort. By the way, phytovolatilization is more unsuitable because 1,2,3-TCP , inhaled by human body, would create more damage in the gas. Thus, we finally identified the strategy of phytodegradation. We introduce the gene of three enzyme-haloalkane dehalogenase(DhaA31), haloalcohol dehalogenase(HheC) and Epichlorohydrin epoxide hydrolase (EchA) into model plant- Nicotiana tabacum and transform 1,2,3-TCP into glycerol</p>
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/0/08/T--UESTC-China--demo_6.jpg" style="width: 80%;" /></p>
+
<p class="mid">Figure 11. Standard curves of reaction products</p>
+
+
+
<p class="sub-title"></p>
+
<p></p>
+
+
<p class="sub-sub-title">5.2.1 Activity determination of DhaA31 (piGEM2017-001) in transgenic tobacco</p>
+
<p></p>
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/4/41/T--UESTC-China--demo_7.png" style="width: ;" /></p>
+
<p class="mid">Figure 12. The Concentration of 2,3-DCP produced within 7 hours. The activity of DhaA31 was </p>
+
+
<p class="mid">determined in 6.5 mL supernatant of tobacco carrying piGEM2017-001 (2 g leaves (FW) were </p>
+
+
<p class="mid">rinsed thoroughly with 6 mL 200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and </p>
+
<p class="mid">6.5mL supernatant was stored) and 5 mM 1,2,3-TCP. Each data represented the mean value ± standard deviation from three independent experiments.</p>
+
+
+
+
<p class="sub-sub-title">5.2.2 Activity determination of HheC-W249P (piGEM2017-002) in transgenic tobacco </p>
+
<p> Haloalcohol dehalogenase (HheC) could degrade 2,3-DCP and CPD to ECH and GDL, respectively. So we detected its activity with 2,3-DCP and CPD as substrates in piGEM2017-002, respectively. While no ECH or GDL were detected at the selected time in wild-type or piGEM2017-002 (data not shown), indicating that no active HheC-W249P was expressed in transgenic tobacco carrying piGEM2017-002. This could be either due to the fact that there was no HheC-W249P expression or the expressed enzyme was less stable in plants. Among these three enzymes, HheC-W249P was the only enzyme that was active as a tetramer, while the other two enzymes were monomer. This could also explain why the activity of HheC-W249P was not detected in transgenic tobacco carrying piGEM2017-002. Due to the limitation of time, we could’t further optimize the expression of HheC-W249P.  </p>
+
+
<p>Instead, we tested the activity of HheC-W249P by mimicking the environment of tobacco in vitro. For this, HheC produced by recombinant E.coli was added into the supernatant of wild-type tobacco, rinsed in Tris-SO4 buffer. As shown in Figure 13, the concentration of ECH in the supernatant of tobacco and Tris-SO4 buffer was similar, indicating that components existed in tobacco did not show a significant effect on the activity of HheC-W249P. In the following experiments, HheC produced by E.coli was added in the TCP degradation pathway. </p>
+
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/1/1a/T--UESTC-China--demo_8.png" style="width: ;" /></p>
+
<p class="mid">Figure 13. Concentration of ECH within 7 hours. 2 g leaves (FW) were rinsed thoroughly with 6 mL </p>
+
+
<p class="mid">200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and 6.5mL supernatant was </p>
+
+
<p class="mid">stored for the determination of HheC enzymatic activity with 5 mM ECH . 6.5mL 200mM Tris-SO4 </p>
+
+
<p class="mid">buffer (pH=8.5) with same amount HheC was used as control. Each data represents the mean value ± standard deviation from three independent experiments.</p>
+
+
<p class="sub-sub-title">5.2.3 Activity determination of EchA (piGEM2017-003) in transgenic tobacco</p>
+
<p></p>
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/0/0a/T--UESTC-China--demo_9.png" style="width: ;" /></p>
+
<p class="mid">Figure 14. Concentration of CPD within 7 hours. 2 g leaves (FW) were rinsed thoroughly with 6 mL </p>
+
+
<p class="mid">200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and 6.5mL supernatant was </p>
+
+
<p class="mid">stored for the determination of EchA enzymatic activity with 5 mM ECH. Each data represents the </p>
+
<p class="mid">mean value ± standard deviation from three independent experiments.</p>
+
+
+
<p class="sub-sub-title">5.2.4 Work validation of multi-enzyme system (piGEM2017-004, piGEM2017-005) in transgenic tobacco </p>
+
<p>DhaA31, HheC-W249P and EchA catalyzed five-step reactions converting toxic TCP to harmless glycerol. To test whether this multi-enzyme conversion system could work in tobacco successfully or not, we detected the time consumption curve of 6 mM TCP by using the grinded leaves. The concentrations of TCP, GLY and other intermediates were monitored. In this case, HheC produced by E.coli was added in the reaction mixture. The conversion of TCP to GLY within 30 h was determined as shown in Figure 15. Glycerol produced gradually with the consumption of 1,2,3-TCP. After 30 h, less than 1 mM of TCP was left, indicating that our system worked well in tobacco. </p>
+
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/b/b0/T--UESTC-China--demo_10.jpg" style="width: 70%;" /></p>
+
<p class="mid">Figure 15. Time courses of conversions of 1,2,3-TCP with enzymes extracted from leaves of </p>
+
+
<p class="mid">transgenic tobacco carrying piGEM2017-004 and HheC produced by E.coli . 4 g leaves(FW) were </p>
+
+
<p class="mid">rinsed thoroughly with 8 mL 200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and </p>
+
+
<p class="mid">10 mL supernatant was stored for the determination of multi-enzyme activity with 6 mM 1,2,3-TCP.</p>
+
+
<p>As described above, tobacco contained endogenous epoxide hydrolase so as shown in Figure 16, the efficiency of converting TCP to GLY by piGEM2017-005 was detected and showed a similar degradation efficiency as the one shown in Figure 15.</p>
+
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/9/94/T--UESTC-China--demo_11.jpg" style="width: 70%;" /></p>
+
<p class="mid">Figure 16. Time courses of multi-enzyme conversions of 1,2,3-TCP with enzymes extracted from </p>
+
+
<p class="mid">leaves of piGEM2017-005-7 and added HheC. 4 g leaves(FW) of piGEM2017-005-7 were rinsed </p>
+
+
<p class="mid">thoroughly with 8 mL 200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and10 mL </p>
+
+
<p class="mid">supernatant was stored for the determination of multi-enzyme activity with 6 mM 1,2,3-TCP. </p>
+
+
<p class="sub-sub-title">5.2.5 Glycerol measurement using gas chromatography–mass spectrometry (GC-MS)</p>
+
<p>The glycerol production was monitored by periodically taking samples from the supernatant of piGEM2017-004 and piGEM2017-005. The final extract was used for the detection of glycerol by GC-MS (Fig 17). As shown in Figure 17, the peak of glycerol after the pre-column derivatization was observed from samples with 0h and 20h incubation. Most importantly, the peak of glycerol after the pre-column derivatization increased within 20 hours while glycerol after the pre-column derivatization was not found in sample of wild-type tobacco. </p>
+
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/0/04/T--UESTC-China--demo_12.jpg" style="width: 70%;" /></p>
+
<p class="mid">Figure 17. Chromatogram of glycerol and internal 1,2,3-butanetriol derivatives of supernatant of </p>
+
+
<p class="mid">transgenic tobacco carrying piGEM2017-004 and wild-type tobacco after the pre-column derivatization. Samples were taken from reaction mixture at 0h, 20h.</p>
+
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/5/52/T--UESTC-China--demo_13.jpg" style="width: 70%;" /></p>
+
<p class="mid">Figure 18. Mass spectrums of tris (trimethylsilyl) glycerol ether and 1,2,3-tris (trimethylsilyl) butanetriol ether</p>
+
 
 
 
</div>
 
</div>
 
+
<div id="Activity">
+
<div id="Conference">
<h2>Activity determination of multi-enzyme system in hydroponic solutions</h2>
+
<h2>Conference</h2>
<p>To investigate whether our “super tobacco” could work or not when placed in the environment, hydroponics method was used to demonstrate. Plants were incubated in liquid nutrient solutions containing a certain concentration of substrate. Samples were taken from medium at selected time. Gas chromatography was used to detect the degradation products in periodic sampling. Figure 19 showed that DhaA31 and EchA could work successfully in the culture, which indicated that our transgenic tobacco could work properly in the hydroponic environment.</p>
+
 
 
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/5/5c/T--UESTC-China--demo_14.jpg" style="width: ;" /></p>
+
<ol>
 
+
<li>Technical Fact Sheet – 1,2,3-Trichloropropane (TCP). 2017.EPA</li>
<p class="mid">Figure 19. The concentration of 2,3-DCP within 3 days. </p>
+
<li>刘福胜. 1995. 1,2,3-三氯丙烷综合利用. 精细石油化工(2), 14-17.</li>
+
<li>Samin, G., & Janssen, D. B. (2012). Transformation and biodegradation of 1, 2, 3-trichloropropane (TCP). Environmental Science and Pollution Research, 19(8), 3067-3078.</li>
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/4/4d/T--UESTC-China--demo_15.jpg" style="width: ;" /></p>
+
 
+
<li>McOmie WA, Barnes TR .1949. Acute and subchronic toxicity of 1,2,3-trichloropropane in mice and rabbits. Fed Proc 8:319. </li>
<p class="mid">Figure 20. The concentration of CPD within 3 days.</p>
+
+
<li>UCC .1973. Toxicology studies: 1,2,3-trichloropropane. New York, NY: Union Carbide Corporation.</li>
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/4/48/T--UESTC-China--demo_16.jpg" style="width: ;" /></p>
+
 
+
<li>Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA .1962. Range finding toxicity data: list VI. Am Ind Hyg Assoc J 23:95-107.</li>
<p class="mid">Figure 21. The hydroponics devices designed by us </p>
+
+
<li>Sasha Khokha .2017. California Finally Begins Regulating Cancer-Causing Chemical Found in Drinking Water. KQED Science Menu</li>
</div>
+
 
+
<li>钱永. (2016). 1,2,3-三氯丙烷在地下水中的环境行为研究. 中国地质大学(北京)).</li>
<div id="Work">
+
<h2>Work going on</h2>
+
<li>Kang, J. W. (2014). Removing environmental organic pollutants with bioremediation and phytoremediation. Biotechnology letters, 36(6), 1129-1139</li>
 +
 +
<li>Tratnyek, P. G., Sarathy, V., & Fortuna, J. H. 2008. Fate and remediation of 1, 2, 3-trichloropropane. In International Conference on Remediation of Chlorinated and Recalcitrant Compounds, 6th, Monterey, CA.</li>
 +
 +
<li>Sarathy, V., Salter, A. J., Nurmi, J. T., O’Brien Johnson, G., Johnson, R. L., & Tratnyek, P. G. (2009). Degradation of 1, 2, 3-trichloropropane (TCP): hydrolysis, elimination, and reduction by iron and zinc. Environmental science & technology, 44(2), 787-793.</li>
 +
 +
<li>Sarathy, V., Salter, A. J., Nurmi, J. T., O’Brien Johnson, G., Johnson, R. L., & Tratnyek, P. G. (2009). Degradation of 1, 2, 3-trichloropropane (TCP): hydrolysis, elimination, and reduction by iron and zinc. Environmental science & technology, 44(2), 787-793.</li>
 +
 +
<li>Cherian, S., & Oliveira, M. M. 2005. Transgenic plants in phytoremediation: recent advances and new possibilities. Environmental science & technology, 39(24), 9377-9390.</li>
 +
 +
</ol>
 
 
 
<br /><br /><br /><br /><br />
 
<br /><br /><br /><br /><br />
 
</div>
 
</div>
 
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</div>
 
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Revision as of 03:03, 1 November 2017

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

Overview

The progress of the chemical industry and agriculture has brought great convenience to our lives. But a large number of chemical pollutants have been discharged through various means, most of them difficult to degrade and accompanied by toxicity, seriously polluting our environment. Among them, Organochlorine compounds is occupy a large part of the proportion. Through collecting questionnaire and soil research in most parts of China, our project is directed against 1,2,3-Trichloropropane - an organic chloride which is a less concerned and insecurity pollution. In this summer, we decided to use synthetic biology methods to achieve plant degradation of 1,2,3-trichloropropane by transferring three enzymes to tobacco to produce glycerol, which is environmentally friendly and recyclable.

Harm of 1,2,3-TCP

1,2,3-Trichloropropane(TCP), an emerging organic pollutant, usually formed as industrial solvents[1] and also is a raw material for the production of 1,1,2,3-Tetrachloropropene and other chemical substances[2]. In agriculture, it has been one of the ingredients of soil fumigants, as well as a harmful byproduct for the production of other pesticides. The global yield of TCP reached about 50,000 tons annually[3] at present. Because it is biodegradation-recalcitrant[3] and will cause groundwater pollution and the damage of soil if it is discharged without treatment.

Table 1. Lethal concentration data. These experiments show that TCP is a DNA reactive carcinogen. [4]~[6]

Lethal concentration data:

Species Reference LC50(ppm) LCLo(ppm) Time Adjusted 0.5-hrLC (CF) Derived value
Mouse Izmerov et al. 1982 555 \ 2 hr 888 ppm (1.6) 89 ppm
Mouse McOmie & Barnes 1949 \ 5,000 20 min 4,350 ppm (0.87) 435 ppm
Rat McOmie & Barnes 1949 LC100: 700 \ 4 hr 1,400 ppm (2.0) 140 ppm
Mouse McOmie & Barnes 1949 LC100: 700 \ 4 hr 1,400 ppm (2.0) 140 ppm
Mouse McOmie & Barnes 1949 LC100: 340 \ 4 hr 680 ppm (2.0) 68 ppm
Rat Smyth et al. 1962 LC83: 1,000 \ 4 hr 2,000 ppm (2.0) 200 ppm
Rat UCC 1973 LC83: 5,600 \ 1 hr 7,000 ppm (1.25) 700 ppm

Human data: It has been reported that objectionable ocular and mucosal irritation were experienced after 15 minutes of exposure to 100 ppm

One of the most serious pollution incidents was happened at Californians. TCP was spread to all over California because the agricultural divisions of Dow Chemical and Shell started selling two soil fumigants (D-D and Telone) including TCP from the 1940s. Although TCP was banned from use in soil fumigants in the 1990s,there was a large amount of TCP remained and it was frequently detected in drinking water, threating to people's lives seriously[7].

Figure 1 This map was produced by KQED[7], drawing on information from the State Water Resources Control Board. It shows water systems where significant levels of the 123-TCP have been detected. Image courtesy KQED, whose reporter, Sasha Khokha, recently found her own water supply to be contaminated as part of a story on this issue.

What’s more, Dr. Qian Yong from China University of Geosciences has studied the behavior and relevant mechanism of TCP. At the ruins of a factory which was running from 1976 to 1979, he found TCP in high concentration(3890mg / L) underground in 2016[8].

Figure 2. The distribution of TCP contamination underground at this factory[8].

That tell us TCP can keep in the soil and groundwater for years, showing the great stability of TCP in the groundwater and soil. By the way, some researches show the adhesion of 1,2,3-TCP is very low[8], which means that it can easily spread into people's living area and threaten people's health because of its potential carcinogenicity and the huge damage to the kidney. The best evidence is that TCP has been detected around the world more and more frequently in the past decade.

Figure 3. The important report about TCP contamination[3]. We can learn about that the contamination of TCP are becoming more serious.

To sum up, we know that TCP is a very dangerous contaminant. However, governments don't pay enough attention to 1,2,3-TCP.Most counties even don't include 1,2,3-TCP into the pollutant detection list. Under these circumstances, we hope that we can attract attention of the society and contain the spread of 1,2,3-TCP pollution through this project.

Technology to treat 1,2,3-TCP

How do people solve TCP? Traditional remediation technology to treat 1,2,3-TCP includes granular activated carbon (GAC), dechlorination by hydrogen release compound (HRC®), reductive dechlorination by zero valent iron(ZVI) and so on[10]~[12].

Figure 4. Result of treatability tests with TCP-contaminated groundwater/soil[10].

Most of them are inefficient and impractical. There are just a few methods such as ZVI that has a good efficiency. But all of them are cost and hard to large-scale deal with TCP in nature. In that scenario, the concept of " Microbial remediation " began to be respected by people. Some studies have shown that 1,2,3 trichloropropane may be converted to CO2, H20 and HCl by biocatalytic action under the oxidative co-metabolism of O2 as the electron acceptor, so that people are mainly seeking to degrade in aerobic microorganisms method. Unfortunately, scientists have failed to enrich and screen aerobe which can degrade 1,2,3 - trichloropropane. However, they found several strains could degrade TCP in absolutely anaerobic environment . But this method, anaerobic microorganism degradation , hasn't been spread because of its harsh condition and low conversion efficiency. So there are some studies that want to introduce a series of enzyme genes into microorganisms such as Escherichia coli and Pseudomonas putida to degrade TCP. This method is efficient. But it has some limitations. First, these microoganisms have strict nutrient demand and weak competitiveness. Second, they may cause antibiotic resistance gene. Third, they usually depend on special inductions so that they can work. So, we hope to find a better method that can degrade TCP for a long time without extra resource[3]. In this time,Phytoremediation, a safe and long-lasting remediation strategy,go into our field of vision

Our strategy

As an emerging "Green remediation" technology, phytoremediation shows its own great potential. Compared with "Bioremediation", its advantages are very obvious. The most amazing one is that plants have a set of photosynthetic autotrophic system which means they can degrade TCP in a long time and just need a little nutrition input, This method is easier and cheaper. Plants can also stabilize soil and absorb CO2 while cleaning the environment.

Figure 5.The main models of phytoremediation strategy.

"Phytoremediation"[13] mainly contains four models: Phytoextraction, Phytostabilization, Phytovolatilization and Phytodegradation. Through analyzing physical and chemical properties of 1,2,3-TCP, we know that 1,2,3-TCP is unlikely to become concentrated in plants and aquatic organisms because it has a low estimated bioconcentration factor (BCF) and sticking coefficient. So it is difficult to degrade 1,2,3-TCP with phytoextraction and phytostabilization. Furthermore, it needs a complex system to deal with these plants if we choose phytoextraction and phytostabilization, which requires much time and effort. By the way, phytovolatilization is more unsuitable because 1,2,3-TCP , inhaled by human body, would create more damage in the gas. Thus, we finally identified the strategy of phytodegradation. We introduce the gene of three enzyme-haloalkane dehalogenase(DhaA31), haloalcohol dehalogenase(HheC) and Epichlorohydrin epoxide hydrolase (EchA) into model plant- Nicotiana tabacum and transform 1,2,3-TCP into glycerol

Conference

  1. Technical Fact Sheet – 1,2,3-Trichloropropane (TCP). 2017.EPA
  2. 刘福胜. 1995. 1,2,3-三氯丙烷综合利用. 精细石油化工(2), 14-17.
  3. Samin, G., & Janssen, D. B. (2012). Transformation and biodegradation of 1, 2, 3-trichloropropane (TCP). Environmental Science and Pollution Research, 19(8), 3067-3078.
  4. McOmie WA, Barnes TR .1949. Acute and subchronic toxicity of 1,2,3-trichloropropane in mice and rabbits. Fed Proc 8:319.
  5. UCC .1973. Toxicology studies: 1,2,3-trichloropropane. New York, NY: Union Carbide Corporation.
  6. Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA .1962. Range finding toxicity data: list VI. Am Ind Hyg Assoc J 23:95-107.
  7. Sasha Khokha .2017. California Finally Begins Regulating Cancer-Causing Chemical Found in Drinking Water. KQED Science Menu
  8. 钱永. (2016). 1,2,3-三氯丙烷在地下水中的环境行为研究. 中国地质大学(北京)).
  9. Kang, J. W. (2014). Removing environmental organic pollutants with bioremediation and phytoremediation. Biotechnology letters, 36(6), 1129-1139
  10. Tratnyek, P. G., Sarathy, V., & Fortuna, J. H. 2008. Fate and remediation of 1, 2, 3-trichloropropane. In International Conference on Remediation of Chlorinated and Recalcitrant Compounds, 6th, Monterey, CA.
  11. Sarathy, V., Salter, A. J., Nurmi, J. T., O’Brien Johnson, G., Johnson, R. L., & Tratnyek, P. G. (2009). Degradation of 1, 2, 3-trichloropropane (TCP): hydrolysis, elimination, and reduction by iron and zinc. Environmental science & technology, 44(2), 787-793.
  12. Sarathy, V., Salter, A. J., Nurmi, J. T., O’Brien Johnson, G., Johnson, R. L., & Tratnyek, P. G. (2009). Degradation of 1, 2, 3-trichloropropane (TCP): hydrolysis, elimination, and reduction by iron and zinc. Environmental science & technology, 44(2), 787-793.
  13. Cherian, S., & Oliveira, M. M. 2005. Transgenic plants in phytoremediation: recent advances and new possibilities. Environmental science & technology, 39(24), 9377-9390.