Difference between revisions of "Team:ZJU-China/Safety"

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{{ZJU-China}}
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<html lang="zh-CN">
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<html>
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  <head>
  
  
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<div class="column full_size">
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<h1> Safety </h1>
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<script>
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<p>Please visit <a href="https://2017.igem.org/Safety">the main Safety page</a> to find this year's safety requirements & deadlines, and to learn about safe & responsible research in iGEM.</p>
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<p>On this page of your wiki, you should write about how you are addressing any safety issues in your project. The wiki is a place where you can <strong>go beyond the questions on the safety forms</strong>, and write about whatever safety topics are most interesting in your project. (You do not need to copy your safety forms onto this wiki page.)</p>
 
  
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<h5>Safe Project Design</h5>
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<p>Does your project include any safety features? Have you made certain decisions about the design to reduce risks? Write about them here! For example:</p>
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<ul>
 
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<li>Choosing a non-pathogenic chassis</li>
 
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<li>Choosing parts that will not harm humans / animals / plants</li>
 
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<li>Substituting safer materials for dangerous materials in a proof-of-concept experiment</li>
 
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<li>Including an "induced lethality" or "kill-switch" device</li>
 
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</ul>
 
  
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<div class="column half_size">
 
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<h5>Safe Lab Work</h5>
 
  
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<p>What safety procedures do you use every day in the lab? Did you perform any unusual experiments, or face any unusual safety issues? Write about them here!</p>
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                  <!-- <h1><a class="navbar-brand" href="index.html">MuMei Lab</a></h1> -->
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                  <a href="https://2017.igem.org/Team:ZJU-China">
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                  </a>
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                  <!-- <h1><a class="navbar-brand" href="index.html">My Design</a></h1> -->
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              </div>
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              <!-- Collect the nav links, forms, and other content for toggling -->
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                  <ul class="nav navbar-nav navbar-right cl-effect-15">
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                      <!-- Hidden li included to remove active class from about link when scrolled up past about section -->
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                      <li class="hidden"><a class="page-scroll" href="#page-top"></a> </li>
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                      <li class="m_nav_item dropdown">
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Overview<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Overview">Description</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Demonstrate">Demonstrate</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Applied_Design">Applied Design</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Achievements">Achievements</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Improve">Improve Parts</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/InterLab">InterLab</a></li>
 +
 +
                          </ul>
 +
                      </li>
 +
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                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Project<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/tp">Trichoderma Proof</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/voc">VOC sensors</a></li>
 +
                              <li><a style="font-size: 0.7em!important;" href="https://2017.igem.org/Team:ZJU-China/Project/st">Chemical Signal Transduction</a></li>
 +
                              <li><a style="font-size: 0.7em!important;" href="https://2017.igem.org/Team:ZJU-China/Project/mt">Medium Wave Transduction</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/Downstream">Downstream</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Project/conclusion">Conclusions</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Notebook">Notebook</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Protocols">Protocols</a></li>
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown" >
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Model<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <!--<li><a href="https://2017.igem.org/Team:ZJU-China/Model">Summery</a></li>-->
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Model">VOC analysis</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Model/Coculture">Coculture</a></li>
 +
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown">
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Parts<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Parts">All Parts</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Basic_Part">Basic Parts</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Composite_Part">Composite Parts</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Part_Collection">Part Collection</a></li>
 +
 +
 +
 +
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown" >
 +
                          <a href="#" class="dropdown-toggle link" data-toggle="dropdown">Hardware<b class="caret"></b></a>
 +
                          <ul class="dropdown-menu ">
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Hardware">Overview</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Hardware/Device">Device</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Hardware/Improvements">Improvements</a></li>
 +
                              <li><a href="https://2017.igem.org/Team:ZJU-China/Hardware/MediumWave">Medium Wave</a></li>
 +
                          </ul>
 +
                      </li>
 +
 +
                      <li class="m_nav_item dropdown" >
 +
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<h5>Safe Shipment</h5>
 
  
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<p>Did you face any safety problems in sending your DNA parts to the Registry? How did you solve those problems?</p>
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          <h1 id="safety" class="page-header ArticleHead GreenAH">Safety</h1>
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          <h2 id="background" class="H2Head">Background</h2>
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              <!--<p class="PP">As the model microorganism of our project, <em>T.atroviride</em> has strong power of biocontrol as well as multiplying. Considering the potential risk of gene transfer due to the development of conidia, we planned to induce some changes in the genome of <em>T.atroviride</em> so that we can limit the spread of it.</p>-->
 +
 
 +
              <p class="PP">According to the grading of ATCC(American Type Culture Collection), <em>T. atroviride</em> is a grade 1 organism, which means that it is safe to use in almost all conditions. Besides, there is no documented report of <em>T.atroviride</em> infection on human species. However, since we made genetic transformation on T.atroviride, there still remains some uncertain safety risks.</p>
 +
              <p class="PP">As most filamentous fungi does, <em>T.atroviride</em> produce conidia, which enables the engineered Trichoderma to spread and get out of control. The conidia producing ability may cause an escape of exogenous genes.Thus, we made effort to interfere the production of conidia, trying to limit such influence to a minimum level.</p>
 +
 
 +
          <p class="PP">Interestingly, during the culture of <em>T.atroviride</em>, we found an exciting phenomenon. As Figure 1 shows, when the <em>T.atroviride</em> grew in the dark condition, the colony is white. After being transferred to the incubator with light on, some green dots appeared around the primitive white colony. Faced with this puzzling fact, we turned to Prof.Zhang for explanation. He told us that the green dots were developed Trichoderma conidia. That is to say, with the light stimulating the <em>T.atroviride</em>, the conidia developed! In other words, if we inhibit the response of <em>T.atroviride</em> towards light, the conidia may fail to come out.</p>
 +
              <div class="imgdiv"><img class="textimg"  style="width: 50% !important;" src=" https://static.igem.org/mediawiki/2017/c/c5/ZJU_China_Safety_1.jpg "></div>
 +
              <p class="capture">Fig.1 The development of conidia in the dark and light</p>
 +
              <p class="PP">Inspired by this surprising phenomenon, we searched for some information about the mechanism of light-response in <em>T.atroviride</em>. Luckily, we found a key gene named hda-2 in the paper, which plays a significant role in the regulatory network. According to the paper, Δhda-2 mutant strain showed slow growth and the absence of conidia when exposed to blue light, which is the main light stimuli causing the development of conidia. Therefore, we chose the hda-2 as our target to knock out.</p>
 +
 
 +
          <h2 id="design" class="H2Head">Design</h2>
 +
              <p class="PP">In this part, we used a site-specific recombination to delete the gene hda-2, which is indispensable for the development of conidia of <em>T.atroviride</em>. To delete this gene, a deletion  element was constructed to assemble into a vector used for transformation into Agrobacterium. As is shown in the Figure 2,  this element consists of fragments which are ~1.5 kb long corresponding to each 5′- and 3′-flanking regions for the hda-2 open reading frame, with the hygromycin-resistant gene(HygR) inserted between them. When the element is absorbed by <em>T.atroviride</em>, the flanking sequence in the element complements to the sequence near hda-2 gene, which is replaced by the HygR.</p>
 +
              <div class="imgdiv"><img class="textimg"  style="width: 50% !important;" src=" https://static.igem.org/mediawiki/2017/6/67/ZJU_China_Safety_2.png "></div>
 +
              <p class="capture">Fig.2 Pko1 with deletion construct</p>
 +
 
 +
          <h2 id="futurework" class="H2Head">Future work</h2>
 +
              <p class="PP">Due to the lack of time, we did not fulfill the construction of the plasmid used for transformation into Agrobacterium. Previously, we successfully ligated the flanking fragments and the hygromycin-resistant gene by overlap PCR. Furthermore, we confirmed the identity of the whole construct. Therefore, the remaining work is to insert the construct into the vector pko1 and transformation of this plasmid. And we will try to optimize the condition of experiment such as enzyme digestion to make sure our system works.</p>
 +
 
 +
          <h2 id="reference" class="H2Head">References</h2>
 +
              <p class="ref">[1] Osorio-Concepción M, Cristóbal-Mondragón G R, Gutiérrez-Medina B, et al. Histone Deacetylase HDA-2 Regulates Trichoderma atroviride Growth, Conidiation, Blue Light Perception, and Oxidative Stress Responses[J]. Applied and environmental microbiology, 2017, 83(3): e02922-16.</p>
 +
              <p class="ref">[2] Casas-Flores S, Herrera-Estrella A. 2013. The influence of light on the biology of Trichoderma, p 43–66. In Mukherjee PK, Horwitz BA, Singh US, Mukherjee M, Schmoll M. (ed), Trichoderma: biology and applications. CABI, Wallingford, United Kingdom.</p>
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Latest revision as of 16:08, 1 November 2017

Safety

Background

According to the grading of ATCC(American Type Culture Collection), T. atroviride is a grade 1 organism, which means that it is safe to use in almost all conditions. Besides, there is no documented report of T.atroviride infection on human species. However, since we made genetic transformation on T.atroviride, there still remains some uncertain safety risks.

As most filamentous fungi does, T.atroviride produce conidia, which enables the engineered Trichoderma to spread and get out of control. The conidia producing ability may cause an escape of exogenous genes.Thus, we made effort to interfere the production of conidia, trying to limit such influence to a minimum level.

Interestingly, during the culture of T.atroviride, we found an exciting phenomenon. As Figure 1 shows, when the T.atroviride grew in the dark condition, the colony is white. After being transferred to the incubator with light on, some green dots appeared around the primitive white colony. Faced with this puzzling fact, we turned to Prof.Zhang for explanation. He told us that the green dots were developed Trichoderma conidia. That is to say, with the light stimulating the T.atroviride, the conidia developed! In other words, if we inhibit the response of T.atroviride towards light, the conidia may fail to come out.

Fig.1 The development of conidia in the dark and light

Inspired by this surprising phenomenon, we searched for some information about the mechanism of light-response in T.atroviride. Luckily, we found a key gene named hda-2 in the paper, which plays a significant role in the regulatory network. According to the paper, Δhda-2 mutant strain showed slow growth and the absence of conidia when exposed to blue light, which is the main light stimuli causing the development of conidia. Therefore, we chose the hda-2 as our target to knock out.

Design

In this part, we used a site-specific recombination to delete the gene hda-2, which is indispensable for the development of conidia of T.atroviride. To delete this gene, a deletion element was constructed to assemble into a vector used for transformation into Agrobacterium. As is shown in the Figure 2, this element consists of fragments which are ~1.5 kb long corresponding to each 5′- and 3′-flanking regions for the hda-2 open reading frame, with the hygromycin-resistant gene(HygR) inserted between them. When the element is absorbed by T.atroviride, the flanking sequence in the element complements to the sequence near hda-2 gene, which is replaced by the HygR.

Fig.2 Pko1 with deletion construct

Future work

Due to the lack of time, we did not fulfill the construction of the plasmid used for transformation into Agrobacterium. Previously, we successfully ligated the flanking fragments and the hygromycin-resistant gene by overlap PCR. Furthermore, we confirmed the identity of the whole construct. Therefore, the remaining work is to insert the construct into the vector pko1 and transformation of this plasmid. And we will try to optimize the condition of experiment such as enzyme digestion to make sure our system works.

References

[1] Osorio-Concepción M, Cristóbal-Mondragón G R, Gutiérrez-Medina B, et al. Histone Deacetylase HDA-2 Regulates Trichoderma atroviride Growth, Conidiation, Blue Light Perception, and Oxidative Stress Responses[J]. Applied and environmental microbiology, 2017, 83(3): e02922-16.

[2] Casas-Flores S, Herrera-Estrella A. 2013. The influence of light on the biology of Trichoderma, p 43–66. In Mukherjee PK, Horwitz BA, Singh US, Mukherjee M, Schmoll M. (ed), Trichoderma: biology and applications. CABI, Wallingford, United Kingdom.


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