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

 
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<p><img src="img/222.png" alt="" class="hhh" /></p>
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<a href="#Pathway" style="font-size: 14px;">Pathway construction</a>
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<a href="#Overview">Overview</a>
 
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<li> <a href="#Harm">Distributions of TCP</a> </li>
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<li> <a href="#Technology">Ways to solve </a> </li>
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<li> <a href="#Strategy">Our super tobacco</a> </li>
 
<li>
 
<li>
<a href="#Tobacco" style="font-size: 14px;">Tobacco Transformation</a>
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<a href="#References">References</a>
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<li>
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<a href="#Positive" style="font-size: 14px;">Positive validation</a>
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<a href="#Construct" style="font-size: 14px;">Construct validation</a>
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<a href="#Enzyme" style="font-size: 14px;">Enzyme activity assay</a>
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<a href="#Activity" style="font-size: 14px;">Activity determination</a>
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<h2>Pathway construction for degrading 1,2,3-TCP </h2>
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<h2>Overview</h2>
 
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<p>The progress of the chemical industry and agriculture has brought great convenience to our lives. But a large number of chemical pollutants which were toxic and difficult to degrade have been discharged to the rivers and soils, seriously polluting our environment. Xenobiotic organohalognse played a large part among all the chemical pollutants. After collecting a questionnaire for industrial and agricultural producers and doing a soil research in most areas of China, our team aimed at degrading an organic chloride, 1,2,3-Trichloropropane (1,2,3-TCP), which was less concerned by environmental protection department. In this summer, we decided to use synthetic biology to achieve plant degradation of 1,2,3-Trichloropropane by transferring three enzymes into tobacco which is environmentally friendly and the product, glycerol, is recyclable in tobacco.</p>
<p class="sub-title"> 1.1 Degrading Pathway construction based on the expression of DhaA31, HheC-W249P & EchA in tobacco </p>
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<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>
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<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>
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<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>
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<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>
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<p class="mid">(a)piGEM2017-001 (Line1,enzyme digested by PstI+BamHI; Line2,enzyme digested by ScaI) </p>
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<p class="mid">(b)piGEM2017-002 (Line1,enzyme digested by PstI+BamHI; Line2, enzyme digested by ScaI) </p>
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<p class="mid">(c) piGEM2017-003 (Line1,enzyme digested by EcoNI+BamHI; Line2,enzyme digested by ScaI) </p>
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<p class="mid">(d) piGEM2017-004 (Line 1, enzyme digested by EcoRI+BamHI; Line 2, enzyme digested by PstI+BamHI)</p>
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<p class="mid">(e) piGEM2017-005 (Line 1, enzyme digested by EcoRI+BamHI; Line 2, enzyme digested by ScaI) </p>
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<p class="mid">M: DNA marker</p>
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<p class="sub-title">1.2 Degrading Pathway construction based on the root expression of DhaA31, HheC-W249P & EchA in tobacco </p>
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<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>
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<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>
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<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>
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<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>
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/7/76/T--UESTC-China--demo_2.png" style="width: ;" /></p>
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<p class="mid">Figure 4. The image of agarose gel electrophoresis by double enzyme digestion. </p>
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<p class="mid"> (a) piGEM2017-008 (b) piGEM2017-021 (c) piGEM2017-024</p>
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<p class="mid">(M: DNA marker; Line 1, enzyme digested by PstI; Line 2, enzyme digested by EcoRI +BamHI)</p>
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<p class="sub-title">1.3 Degrading Pathway construction based on the extracellular expression of DhaA31, HheC-W249P & EchA in tobacco </p>
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<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>
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<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>
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<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>
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<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>
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<p class="pic"><img src=" https://static.igem.org/mediawiki/2017/d/d6/T--UESTC-China--demo_3.png" style="width: ;" /></p>
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<p class="mid">Figure 6. The image of agarose gel electrophoresis by double enzyme digestion. </p>
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<p class="mid">(a) piGEM2017-022 (b) piGEM2017-023 (c) piGEM2017-025 (d) piGEM2017-026 (e) piGEM2017-027</p>
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<p class="mid">(M: DNA marker; Line 1, enzyme digested by PstI; Line 2, enzyme digested by EcoRI +BamHI)</p>
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</div>
 
</div>
  
<div id="Tobacco">
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<div id="Harm">
<h2>Tobacco Transformation</h2>
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<h2>The distributions of pollutant 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-TCP is an emerging organic pollutant which is usually been used as industrial solvents[1] and raw materials for producing 1,1,2,3-Tetrachloropropene or other chemicals substances[2]. In agriculture, it has been used as one of the ingredients of soil fumigants and it is also a harmful byproduct of other pesticides. The global production of 1,2,3-TCP reached about 50,000 tons annually[3]. Because 1,2,3-TCP is hard to degrade naturally in the environment, it would cause a huge damage to the groundwater and the soil if discharged casually.</p>
+
                                          <p>One of the most serious pollution of 1,2,3-TCP was discovered in California. It has spread to all over the California since the 1940s when Dow Chemical and Shell started selling two soil fumigants (D-D and Telone) which include 1,2,3-TCP. Although 1,2,3-TCP was banned to use as soil fumigants in the 1990s, there was still a large amount of 1,2,3-TCP remained, to be a threat to the environment and people's lives seriously[4].</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>
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  <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="pic"><img src="https://static.igem.org/mediawiki/2017/8/82/T--UESTC-China--demo_4.jpg" style="width: 60%;" /></p>
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  <p class="mid">Figure 1. Water systems where significant levels of the 1,2,3-TCP have been detected in California</p>
<p class="mid">Figure 7. The steps for obtaining transgenic tobacco</p>
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<p>Dr. Yong Qian from University of Geosciences of China has explored the behavior of 1,2,3-TCP in the groundwater. He found 1,2,3-TCP still huge concentration (3890mg / L) underground in an abandoned factory that was only run from 1976 to 1979 in 2016 [5], which showed the great stability of 1,2,3-TCP in the groundwater and soil (Fig. 2). </p>
 
+
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/8/8b/T--UESTC-China--222.png" style="width: 60%;"/></p>
 +
  <p class="mid">Figure 2. The distribution of underground 1,2,3-TCP pollution in this factory [5].</p>
 +
<p>Meanwhile, some studies showed that the adhesion coefficient of 1,2,3-TCP is very low[5]. It means that 1,2,3-TCP can be easily diffused into our daily use water. Its potential carcinogenicity and damage to kidney will threaten the health of human beings. In the recent past 10 years, more and more tests showing the existence of 1,2,3-TCP among worldwide drinking water is a sound proof (Fig. 3). </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. 1,2,3-TCP has been detected in hundreds of surface water and drinking water sources </p>
 +
<p>Based on the above information, 1,2,3-TCP is absolutely a threatening pollutant. However, attentions are far more than enough to be paid on 1,2,3-TCP; and a lot of countries have not put 1,2,3-TCP as the test list of water quality. Since the current situation has been known to us, our team hopes to find a feasible way to stop 1,2,3-TCP by attracting the attentions of the whole society through doing advertisement and iGEM competition before it might cause great damage to the health of human beings. </p>
 
</div>
 
</div>
  
<div id="Positive">
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<div id="Technology">
<h2>Positive validation of transgenic plants</h2>
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  <h2>Ways to solve the pollution of 1,2,3-TCP</h2>
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  <p>Then the question is how to solve the problem of 1,2,3-TCP pollution? Traditional methods to deal with 1,2,3-TCP includes granular activated carbon (GAC)、hydrogen release compound (HRC)、reductive dechlorination by zero valent iron(ZVI) and son on[6]~[8]. However, some of these methods are either too inefficient or too expensive to be used within natural conditions (Fig. 4). </p>
<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>
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  <p class="pic"><img src="https://static.igem.org/mediawiki/2017/3/34/T--UESTC-China--description_4.jpg" style="width: 95%;"/></p>
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  <p class="mid">Figure 4. Treatability tests with 1,2,3-TCP-contaminated groundwater/soil</p>
<p class="pic"><img src="https://static.igem.org/mediawiki/2017/2/25/T--UESTC-China--demo_5.png" style="width: 80%;" /></p>
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  <p>Since 1,2,3-TCP can hardly be dealt with traditional ways, Microbial remediation of 1,2,3-TCP is getting praised. Early studies have shown that 1,2,3-TCP can be converted into CO<sub>2</sub>、H<sub>2</sub>O and HCl by biological catalysis through Oxidation of metabolic mechanism with O<sub>2</sub>. So people are trying to find degradation method by using aerobe. But until now, all the tests to gather and filtrate aerobe to degrade 1,2,3-TCP have failed. However, scientists have found a few categories of bacteria that can degrade 1,2,3-TCP in absolute anaerobic environment. However, due to its strict requirement, the conversion efficiency is not high enough to get popular. In recent years, some studies have started to apply genetic engineering method to inject some enzymes in the seek of 1,2,3-TCP degradation. Some good results have been obtained but with some limitations. Firstly, these bacteria require very strict nutrition requirement and there only exits weak competition among them. Secondly, the diffusion of antibiotic resistance gene can be easily triggered. Finally, this method rely heavily on some specific induction condition[9]. We are not satisfied with these disadvantages and thus we hope to find a 1,2,3-TCP degradation method that is energy-efficient and sustainable. The burgeoning “Phytoremediation” has come into our attention. </p>
<p class="mid">Figure 8. The image of agarose gel electrophoresis by PCR detection</p>
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<p class="mid">a.piGEM2017-001    b.piGEM2017-002 </p>
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<p class="mid">c.piGEM2017-003    d.piGEM2017-004</p>
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<p class="mid">e. piGEM2017-004    f.piGEM2017-005  </p>
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<p class="mid"> ( PC, positive control (plasmid),WT, negative control (wild-type tobacco))</p>
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<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>
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<p class="pic"><img src="" style="width: ;" /></p>
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<p class="mid">Figure 9. The image of agarose gel electrophoresis by RT-PCR detection</p>
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<p class="mid">a.piGEM2017-001  b.piGEM2017-002  c.piGEM2017-003</p>
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<p class="mid">d.piGEM2017-004  e.piGEM2017-005  f.piGEM2017-008</p>
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<p class="mid">(PC, positive control (plasmid),WT, negative control (wild-type tobacco), WT-L(leaves of </p>
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<p class="mid">wild-type tobacco),WT-R(roots of wild-type tobacco),Tubulin was an internal control in tobacco)</p>
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</div>
 
</div>
  
<div id="Construct">
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<div id="Strategy">
<h2>Construct validation of multi-gene expression system </h2>
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  <h2>Our super tobacco</h2>
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  <p>As one of the new “Green remediation” strategy,phytoremediation shows enormous potential. Compared to microbial remediation, the most important advantage of phytoremediation is that phytoremediation comes with the potential to dispose pollutants by small amount of nutrition input. This is due to its unique feature of photosynthetic autotrophs system. By doing pollutant disposition, at the same time, plants can also help stabilize the soil, purify the water and clear air pollution[10]~[11]. There are four different ways of “Phytoremediation”: phytoextraction、phytostabilization、phytovolatilization and phytodegradation (Fig. 5). </p>
<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>
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/f/f6/T--UESTC-China--plrecover.jpg" style="width:80% ;padding-top: 7%;"/></p>
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  <p class="mid">Figure 5. The main models of phytoremediation strategy.</p>
<p class="pic"><img src="" style="width: ;" /></p>
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  <p>By understanding the physical and chemical properties of 1,2,3-TCP, we know that 1,2,3-TCP normally can not gather in living things and is easily to migrate due to its low adhesion. This makes it hard to deal with 1,2,3-TCP using the strategy of extraction and fixation. Moreover, phytostabilization and phytovolatilization requires frequent disposition and change of plants. This furthers requests to build another complete time and labor consuming system. phytoextraction won’t be taken into our consideration since 1,2,3-TCP can cause severe damage to human health by breathing. Therefore, we choose to use the strategy of phytodegradation to degrade 1,2,3-TCP into glycerol by creating super tobacco.</p>
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</div>
 
</div>
 
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<div id="Enzyme">
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  <div id="References">
<h2>Enzyme activity assay for the validation of transgenic tobacco</h2>
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    <h2>References</h2>
 +
    <ol>
 +
    <li>EPA. Technical Fact Sheet – 1,2,3-Trichloropropane (1,2,3-TCP), 2017.</li>
 +
    <li>Liu FS. The comprehensive utilization of 1,2,3-Trichloropropane. Speciality Petrochemicals, 1995;2:11-4.</li>
 +
    <li>Samin G, Janssen DB. Transformation and biodegradation of 1, 2, 3-trichloropropane (TCP). Environmental Science and Pollution Research, 2012. 1;19(8):3067-78.</li>
 +
 
 
 
<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>
+
    <li>Sasha Khokha . California Finally Begins Regulating Cancer-Causing Chemical Found in Drinking Water. KQED Science Menu, 2017.</li>
 +
  <li>Qian Yong. Research on Environment Behavior of 1,2,3-Trichloropropane in Groundwater of a Contaminated Site with Chlorinated Pollutants. China University of Geosciences(Beijing). 2016</li>
 +
 
 +
  <li>Tratnyek PG, Sarathy V, Fortuna JH. Fate and remediation of 1, 2, 3-trichloropropane. InInternational Conference on Remediation of Chlorinated and Recalcitrant Compounds, 6th, Monterey, CA 2008.</li>
 +
 +
  <li>Sarathy V, Salter AJ, Nurmi JT, O’Brien Johnson G, Johnson RL, Tratnyek PG. Degradation of 1, 2, 3-trichloropropane (TCP): hydrolysis, elimination, and reduction by iron and zinc. Environmental science & technology, 2009. 14;44(2):787-93.</li>
 +
 +
  <li>Sarathy V, Salter AJ, Nurmi JT, O’Brien Johnson G, Johnson RL, Tratnyek PG. Degradation of 1, 2, 3-trichloropropane (TCP): hydrolysis, elimination, and reduction by iron and zinc. Environmental science & technology, 2009. 14;44(2):787-93.</li>
 +
  <li>Kang JW. Removing environmental organic pollutants with bioremediation and phytoremediation. Biotechnology letters, 2014. 1;36(6):1129-39.</li>
 +
  <li>Cherian S, Oliveira MM. Transgenic plants in phytoremediation: recent advances and new possibilities. Environmental science & technology, 2005. 15;39(24):9377-90.</li>
 +
  <li>Kang JW. Removing environmental organic pollutants with bioremediation and phytoremediation. Biotechnology letters. 2014. 1;36(6):1129-39.</li>
 +
 +
    </ol>
 
 
<p class="sub-title">5.1 Measure the standard curves of reaction products</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 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>
 
 
 
 
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<h2>Activity determination of multi-enzyme system in hydroponic solutions</h2>
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<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>
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/5/5c/T--UESTC-China--demo_14.jpg" style="width: ;" /></p>
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<p class="mid">Figure 19. The concentration of 2,3-DCP within 3 days. </p>
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<p class="mid">Figure 20. The concentration of CPD within 3 days.</p>
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Latest revision as of 03:45, 2 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 which were toxic and difficult to degrade have been discharged to the rivers and soils, seriously polluting our environment. Xenobiotic organohalognse played a large part among all the chemical pollutants. After collecting a questionnaire for industrial and agricultural producers and doing a soil research in most areas of China, our team aimed at degrading an organic chloride, 1,2,3-Trichloropropane (1,2,3-TCP), which was less concerned by environmental protection department. In this summer, we decided to use synthetic biology to achieve plant degradation of 1,2,3-Trichloropropane by transferring three enzymes into tobacco which is environmentally friendly and the product, glycerol, is recyclable in tobacco.

The distributions of pollutant 1,2,3-TCP

1,2,3-TCP is an emerging organic pollutant which is usually been used as industrial solvents[1] and raw materials for producing 1,1,2,3-Tetrachloropropene or other chemicals substances[2]. In agriculture, it has been used as one of the ingredients of soil fumigants and it is also a harmful byproduct of other pesticides. The global production of 1,2,3-TCP reached about 50,000 tons annually[3]. Because 1,2,3-TCP is hard to degrade naturally in the environment, it would cause a huge damage to the groundwater and the soil if discharged casually.

One of the most serious pollution of 1,2,3-TCP was discovered in California. It has spread to all over the California since the 1940s when Dow Chemical and Shell started selling two soil fumigants (D-D and Telone) which include 1,2,3-TCP. Although 1,2,3-TCP was banned to use as soil fumigants in the 1990s, there was still a large amount of 1,2,3-TCP remained, to be a threat to the environment and people's lives seriously[4].

Figure 1. Water systems where significant levels of the 1,2,3-TCP have been detected in California

Dr. Yong Qian from University of Geosciences of China has explored the behavior of 1,2,3-TCP in the groundwater. He found 1,2,3-TCP still huge concentration (3890mg / L) underground in an abandoned factory that was only run from 1976 to 1979 in 2016 [5], which showed the great stability of 1,2,3-TCP in the groundwater and soil (Fig. 2).

Figure 2. The distribution of underground 1,2,3-TCP pollution in this factory [5].

Meanwhile, some studies showed that the adhesion coefficient of 1,2,3-TCP is very low[5]. It means that 1,2,3-TCP can be easily diffused into our daily use water. Its potential carcinogenicity and damage to kidney will threaten the health of human beings. In the recent past 10 years, more and more tests showing the existence of 1,2,3-TCP among worldwide drinking water is a sound proof (Fig. 3).

Figure 3. 1,2,3-TCP has been detected in hundreds of surface water and drinking water sources

Based on the above information, 1,2,3-TCP is absolutely a threatening pollutant. However, attentions are far more than enough to be paid on 1,2,3-TCP; and a lot of countries have not put 1,2,3-TCP as the test list of water quality. Since the current situation has been known to us, our team hopes to find a feasible way to stop 1,2,3-TCP by attracting the attentions of the whole society through doing advertisement and iGEM competition before it might cause great damage to the health of human beings.

Ways to solve the pollution of 1,2,3-TCP

Then the question is how to solve the problem of 1,2,3-TCP pollution? Traditional methods to deal with 1,2,3-TCP includes granular activated carbon (GAC)、hydrogen release compound (HRC)、reductive dechlorination by zero valent iron(ZVI) and son on[6]~[8]. However, some of these methods are either too inefficient or too expensive to be used within natural conditions (Fig. 4).

Figure 4. Treatability tests with 1,2,3-TCP-contaminated groundwater/soil

Since 1,2,3-TCP can hardly be dealt with traditional ways, Microbial remediation of 1,2,3-TCP is getting praised. Early studies have shown that 1,2,3-TCP can be converted into CO2、H2O and HCl by biological catalysis through Oxidation of metabolic mechanism with O2. So people are trying to find degradation method by using aerobe. But until now, all the tests to gather and filtrate aerobe to degrade 1,2,3-TCP have failed. However, scientists have found a few categories of bacteria that can degrade 1,2,3-TCP in absolute anaerobic environment. However, due to its strict requirement, the conversion efficiency is not high enough to get popular. In recent years, some studies have started to apply genetic engineering method to inject some enzymes in the seek of 1,2,3-TCP degradation. Some good results have been obtained but with some limitations. Firstly, these bacteria require very strict nutrition requirement and there only exits weak competition among them. Secondly, the diffusion of antibiotic resistance gene can be easily triggered. Finally, this method rely heavily on some specific induction condition[9]. We are not satisfied with these disadvantages and thus we hope to find a 1,2,3-TCP degradation method that is energy-efficient and sustainable. The burgeoning “Phytoremediation” has come into our attention.

Our super tobacco

As one of the new “Green remediation” strategy,phytoremediation shows enormous potential. Compared to microbial remediation, the most important advantage of phytoremediation is that phytoremediation comes with the potential to dispose pollutants by small amount of nutrition input. This is due to its unique feature of photosynthetic autotrophs system. By doing pollutant disposition, at the same time, plants can also help stabilize the soil, purify the water and clear air pollution[10]~[11]. There are four different ways of “Phytoremediation”: phytoextraction、phytostabilization、phytovolatilization and phytodegradation (Fig. 5).

Figure 5. The main models of phytoremediation strategy.

By understanding the physical and chemical properties of 1,2,3-TCP, we know that 1,2,3-TCP normally can not gather in living things and is easily to migrate due to its low adhesion. This makes it hard to deal with 1,2,3-TCP using the strategy of extraction and fixation. Moreover, phytostabilization and phytovolatilization requires frequent disposition and change of plants. This furthers requests to build another complete time and labor consuming system. phytoextraction won’t be taken into our consideration since 1,2,3-TCP can cause severe damage to human health by breathing. Therefore, we choose to use the strategy of phytodegradation to degrade 1,2,3-TCP into glycerol by creating super tobacco.

References

  1. EPA. Technical Fact Sheet – 1,2,3-Trichloropropane (1,2,3-TCP), 2017.
  2. Liu FS. The comprehensive utilization of 1,2,3-Trichloropropane. Speciality Petrochemicals, 1995;2:11-4.
  3. Samin G, Janssen DB. Transformation and biodegradation of 1, 2, 3-trichloropropane (TCP). Environmental Science and Pollution Research, 2012. 1;19(8):3067-78.
  4. Sasha Khokha . California Finally Begins Regulating Cancer-Causing Chemical Found in Drinking Water. KQED Science Menu, 2017.
  5. Qian Yong. Research on Environment Behavior of 1,2,3-Trichloropropane in Groundwater of a Contaminated Site with Chlorinated Pollutants. China University of Geosciences(Beijing). 2016
  6. Tratnyek PG, Sarathy V, Fortuna JH. Fate and remediation of 1, 2, 3-trichloropropane. InInternational Conference on Remediation of Chlorinated and Recalcitrant Compounds, 6th, Monterey, CA 2008.
  7. Sarathy V, Salter AJ, Nurmi JT, O’Brien Johnson G, Johnson RL, Tratnyek PG. Degradation of 1, 2, 3-trichloropropane (TCP): hydrolysis, elimination, and reduction by iron and zinc. Environmental science & technology, 2009. 14;44(2):787-93.
  8. Sarathy V, Salter AJ, Nurmi JT, O’Brien Johnson G, Johnson RL, Tratnyek PG. Degradation of 1, 2, 3-trichloropropane (TCP): hydrolysis, elimination, and reduction by iron and zinc. Environmental science & technology, 2009. 14;44(2):787-93.
  9. Kang JW. Removing environmental organic pollutants with bioremediation and phytoremediation. Biotechnology letters, 2014. 1;36(6):1129-39.
  10. Cherian S, Oliveira MM. Transgenic plants in phytoremediation: recent advances and new possibilities. Environmental science & technology, 2005. 15;39(24):9377-90.
  11. Kang JW. Removing environmental organic pollutants with bioremediation and phytoremediation. Biotechnology letters. 2014. 1;36(6):1129-39.