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

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            <h1 id="modeling" class="page-header ArticleHead GreenAH">Notebook</h1>
  
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            <h2 id="2" class="H2Head">2.25</h2>
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    <div class="col-md-3"></div>
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            <p class="PP">Concluded the winter project and 14 members were picked out. ZJU-China 2017 team was set up. Our story began.</p>
−
    <div class="col-md-9" role="main">
+
            <p class="PP">First meetup. A nice meal with the previous iGEMer.</p>
−
        <p class="bs-docs-section">
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/2/29/ZJU_China_Notebook_1.jpg' alt=''/></div>
  
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+
             <h2 class="H2Head">2.26-3.6</h2>
 +
            <p class="PP">Everyone mined their winter project and attempted to extend the project and find problems.</p>
  
 +
            <h2 class="H2Head">2.28</h2>
 +
            <p class="PP"><strong>1st group meeting in Spring term</strong></p>
 +
            <p class="PP">We have some games to break the ice. Several rules and principles were built up as well as the duty table.</p>
  
−
        <h1 id="mwt" class="page-header ArticleHead GreenAH">Medium Wave Transduction</h1>
+
            <h2  id="3" class="H2Head">3.4</h2>
 +
            <p class="PP"><strong>2nd group meeting in Spring term</strong></p>
 +
            <p class="PP">Everyone shared their understand about the principles on the igem website, especially the medal requests. And a delicious chicken soup was cooked.</p>
  
 +
            <h2 class="H2Head">3.7</h2>
 +
            <p class="PP"><strong>3rd group meeting in Spring term</strong></p>
 +
            <p class="PP">Specially, today is a festival for all girls and the day for group meeting. So we pictured the blackboard and had a group photo. Additionally, we talked about the result of the winter project after discussion with related professors.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/0/00/ZJU_China_Notebook_2.jpg' alt=''/></div>
  
−
        <h2 id="background" class="H2Head">Background</h2>
 
  
−
        <h3 id="intr" class="H3Head">Introduction</h3>
+
            <h2 class="H2Head">3.8-3.11</h2>
−
        <p class="PP">Although we have proved the concept of the chemical signal transduction and some of the result shows that it works well, we still want to try something  interesting and innovative——to transduct the signal to the <em>Trichoderma</em> atroviride directly by electromagnetic wave, such as medium wave.</p>
+
            <p class="PP">We detailed the experimental protocol of the winter project and find out the difficulties.</p>
−
        <p class="PP">Chemical signal transduction depends on the diffusion of DAPG, which will limit its application in some degree. Firstly, to transduct the signal, we have to continually supplement DAPG into our device. Although we have achieved the bio-synthesis of DAPG to cut down the cost, it's still inconvenient to supplement it. Combined with the lack of high volatility, the range of effective functioning is quite limited, which means we have to set more chemical releasing device. It can not be more tiring and it's inconsistent to our idea of automatic agriculture. What's more, the efficiency of DAPG's penetrating into the cortex of the root where our <em>Trichoderma</em> atroviride lives，is also a question we have to concern about.</p>
+
−
        <p class="PP">To avoid these problems，we turned our focus into creating a direct contact between electronic signal and biologic response，which we name it Electrobiologic Interface. We select medium wave as our electronic signal. This kind of interface is independent on chemical compound and our signal can directly spread in space without considering the weather or other factors. And by adjusting the strength of transmission, we can control different range of our <em>Trichoderma</em> atroviride to generate a specific strength of response. Furthermore, it's really a fantastic idea to combine the electronic machine and biologic response with medium wave. We desperately tried to construct such a interface.</p>
+
  
−
        <h3 id="tf" class="H3Head">TRPV-Ferritin</h3>
+
            <h2 class="H2Head">3.11</h2>
−
        <p class="PP"><strong>TRPV</strong></p>
+
            <p class="PP"><strong>4th group meeting in Spring term</strong></p>
−
        <p class="PP">TRPV1, the transient receptor potential cation channel subfamily V member 1, is a highly selective calcium channel，which can response to heat shock as well as capsaicin and finally results in calcium influx. And the calcium influx will result in the expression of downstream genes controlled by calmodulin and its associated protein Calcineurin. Calcineurin can activate relative nuclear transcription factors to open the target gene. <sup>[1]</sup></p>
+
            <p class="PP">The whole team divided into three groups. Two deep mined the winter project and one came up with new ideas.The Brainstorm started. Someone jumped out the winter project. And some interesting ideas appeared. For example rhodopsin as photochemistry signal and resveratrol to attack the injured cells.</p>
−
        <p class="PP"><strong>ferritin</strong></p>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/1/18/ZJU_China_Notebook_3.jpg' alt=''/></div>
−
        <p class="PP">Ferritin, a kind of protein can be found in almost all organisms, can store and release iron to balance the content in cell. The ferritin we use is a fusion protein of ferritin light chain，a flexible linker peptide and ferritin heavy chain. This proteinbinding with iron can form nanoparticles which can be heated when exposed to medium radio frequencies(RF). <sup>[2][3]</sup></p>
+
            <h2 class="H2Head">3.14</h2>
−
        <p class="PP"><strong>TRPV-Ferritin system</strong></p>
+
            <p class="PP"><strong>5th group meeting in Spring term</strong></p>
−
        <p class="PP">When TRPV1 and Ferritin are co-expressed in the same cell, only using medium radio frequencies， Ferritin will be heated and then activate TRPV1. Once been activated, TRPV1 will result in calcium influx and finally facilitate  the expression of downstream genes.<sup>[4][5]</sup></p>
+
            <p class="PP">The previous iGEMer came to our group meeting and shared their experience. Then we talked about the project and received their suggestions. At that time 7 different ideas coexisted. We dug previous ideas and considered their safety and feasibility.</p>
−
        <div class="imgdiv col-md-6 col-sm-6"><img class="textimg" src="https://static.igem.org/mediawiki/2017/8/8d/ZJU_China_MWF_xpt5.png"></div>
+
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        <div class="imgdiv col-md-6 col-sm-6"><img class="textimg" src="https://static.igem.org/mediawiki/2017/3/37/ZJU_China_MWF_xpt2.jpg"></div>
+
−
        <p class="capture col-md-6 col-sm-6">Fig.1 TRPV1-Ferritin System</p>
+
−
        <p class="capture col-md-6 col-sm-6">Fig.2 Combination of TRPV1-Ferrtin and CDRE-reporter</p>
+
  
 +
            <h2 class="H2Head">3.18</h2>
 +
            <p class="PP"><strong>6th group meeting in Spring term</strong></p>
 +
            <p class="PP">We still discussed about the new and old ideas. Fortunately, we didn’t choose the idea of Metarhitium against mosquito. Or I can’t imagine what our lab would be like. But the idea about degradation of melanin was interesting. However, after duplicate checking in Taobao, that idea was over. In addition, it was the birthday of one of our members. So there was another party.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/4/46/ZJU_China_Notebook_4.jpg' alt=''/></div>
  
−
        <h2 id="design" class="H2Head">Design</h2>
+
            <h2 class="H2Head">3.21</h2>
−
        <h3 id="desint" class="H3Head">Introduction</h3>
+
            <p class="PP">7th group meeting in Spring term.</p>
−
        <p class="PP">We try to use medium wave for <em>Trichoderma</em> atroviride operation. To achieve our goal, we plan to build up the TRPV-Ferritin system, depending on which we can turn the medium waves into heat, inducing Calcium influx and changing the intracellular Ca<sup>2+</sup> concentration. Therefore, we can operate <em>Trichoderma</em> atroviride using autologous promoter regulated by Ca<sup>2+</sup>.</p>
+
            <p class="PP">The main part started to tend to the Trichoderma. And other ideas exposed their limitation.</p>
−
        <p class="PP">However, the experimental period of <em>Trichoderma</em> atroviride is too long, together with the fact that its genetic background is unclear, so we choose another fungi, Saccharomyces cerevisiae for substitution to check the function. Its own Calmodulinsignal transduction pathway is used to activate the downstream gene.</p>
+
            <p class="PP">In those days, members were learning about basic molecular clone skills. And we exchange our experience in the group meeting.</p>
  
−
        <h3 id="expd" class="H3Head">Experimental Design</h3>
+
            <h2 class="H2Head">3.25</h2>
−
        <p class="PP"><strong>Proof of Concept</strong></p>
+
            <p class="PP"><strong>8th group meeting in Spring term</strong></p>
 +
            <p class="PP">The theme of our project was confirmed. And the team divided into groups again to study different directions about Trichoderma.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/f/f9/ZJU_China_Notebook_5.png' alt=''/></div>
  
 +
            <h2 class="H2Head">3.29</h2>
 +
            <p class="PP"><strong>9th group meeting in Spring term</strong></p>
 +
            <p class="PP">Talked about which the specific Trichoderma we could use and what kind of function we wanted to achieve and something about safety and gene circuit.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/d/dd/ZJU_China_Notebook_6.jpg' alt=''/></div>
  
−
        <p class="PP"><strong>fluo-4</strong> —— To test the change in intracellular Ca<sup>2+</sup> concentration, we used Fluo 4-AM to detect the concentration of intracellular calcium ion.</p>
+
            <h2  id="4" class="H2Head">4.4</h2>
−
        <p class="PP">Fluo 4-AM is a kind of acetylmethyl ester derivative of Fluo 4, and able to enter the cells after culture. After that, AM will be hydrolyzed by the intracellular esterase and release Fluo 4, which can bind Ca<sup>2+</sup> and give out fluorescence. So, Fluo 4-AM enables us to test the intracellular Ca<sup>2+</sup> concentration.</p>
+
            <p class="PP"><strong>10th group meeting in Spring term</strong></p>
−
        <div class="imgdiv"><img style="width: 50% !important;" class="textimg" src="https://static.igem.org/mediawiki/2017/9/93/ZJU_China_MWF_xpt3.png"></div>
+
            <p class="PP">Discussed the available regulatory in Trichoderma and its function of immune and hyperparasitism. The experimental training came to the next phase. Everyone tried to construct a complete device from the very single part.</p>
−
        <p class="capture">Fig.3 Construction of CDRE promoter</p>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/1/17/ZJU_China_Notebook_7.jpg' alt=''/></div>
  
−
        <p class="PP"><strong>CDRE</strong> —— CDRE，calcineurin-dependent response element，is a segment of DNA sequence which can be regulated by specific transcription factor Crz I. This TF is activated by calcineurin in the present of calcium ion. To create a promoter can be up-regulated by calcium influx，we replaced the upstream activating sequence(UAS)of CYC1 promoter with 4 CDREs. It can be a hard work to construct such a promoter with these repeating sequences. Luckily, UCAS helped us to contract this fusion promoter and sent it to us. We choose mRFP as report gene to detect its sensibility.<sup>[6][7]</sup></p>
+
            <h2 class="H2Head">4.8</h2>
−
        <p class="PP">We cultured the yeast in calcium-inducing medium and uninduced medium which contains a relatively low concentration of calcium ion (1xYPD medium). Why we don't choose a medium without any calcium? It's impossible that our yeast or <em>Trichoderma atroviride</em> will have to survive and work in the surrounding without any calcium. And a relatively low concentration may be more similar to the real condition of the soil and rhizosphere. It can also represent the basic level of the calcium when the cell has not been stimulated.</p>
+
            <p class="PP"><strong>11th group meeting in Spring term</strong></p>
−
        <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/c/ca/ZJU_China_newfig4.png"></div>
+
            <p class="PP">We started to choose the certain plant and pathogen. Everyone learned about several candidates and considered their feasibility.</p>
−
        <p class="capture">Fig.4 The color difference of two cultrues</p>
+
−
        <p class="PP">Obviously, the transgenic yeast cultured in the calcium-inducing medium(200mM Ca<sup>2+</sup>) is turning red while the control shows no significant changes. Then we detected the fluorescence of this two cultures. To eliminate the influence of the concentration of the yeast, we calculated the value of Fluorescent Intensity/OD600 to evaluate these two groups.</p>
+
  
−
        <div class="imgdiv col-md-6 col-sm-6"><img class="textimg" style="width: 50% !important;" src="https://static.igem.org/mediawiki/2017/1/19/ZJU_China_MWF_Rplot05.jpeg"></div>
+
            <h2 class="H2Head">4.15</h2>
−
        <div class="imgdiv col-md-6 col-sm-6"><img class="textimg" style="width: 54% !important;" src="https://static.igem.org/mediawiki/2017/e/ea/ZJU_China_MWF_Rplot03.jpeg"></div>
+
            <p class="PP"><strong>13th group meeting in Spring term</strong></p>
−
        <p class="capture col-md-6 col-sm-6">Fig.5 Relative fluorescent intensity of two groups</p>
+
            <p class="PP">Ensured the specific plant and pathogen and talked about the T3SS system about immune and GPCR about sense.</p>
−
        <p class="capture col-md-6 col-sm-6">Fig.6 Relative calcium content in two groups</p>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/8/8e/ZJU_China_Notebook_8.jpg' alt=''/></div>
  
 +
            <h2 class="H2Head">4.20-4.30</h2>
 +
            <p class="PP">Some members began to learn the manipulation skill of Trichoderma in Zhang Lab, Agriculture Constitution, Zhejiang University. They sour out the notes and shared the protocol.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/6/6c/ZJU_China_Notebook_9.jpg' alt=''/></div>
  
−
        <p class="PP">The value indicated that the fluorescence intensity of the calcium-induced group has improved about 118%. It's not quite a huge change and the uninduced group has already shown a great intensity of fluorescence. How come?  We suspect that it's the basic calcium level contributed a lot to the high fluorescent intensity of the uninduced group. To verify this hypothesis, we detected the intracellular calcium ion concentration with Fluo 4-AM whose fluorescent intensity can represent the relative concentration of calcium ion.</p>
+
            <h2 class="H2Head">4.29</h2>
−
                <p class="PP">We are all surprised that the difference of the intracellular ion concentration between these two groups are so small that the induced group is only  about 8% higher than the uninduced one and the basic level is truly high as we suspected. But from another perspective, only 8% change of the intracellular ion concentration can contribute to 118% higher expression level of the downstream gene. It seems that our CDRE promoter is much more sensitive than we expected. </p>
+
            <p class="PP">The first dish of Trichoderma came to our lab. We culture it in the illumination incubator.</p>
−
        <p class="PP">But can our selective calcium channel induce enough calcium influx to activate CDRE promoter, at least 8% higher concentration of calcium ion? We need to examine our TRPV1 and Ferritin carefully.</p>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/c/c4/ZJU_China_Notebook_10.jpg' alt=''/></div>
  
−
        <p class="PP"><strong>Ferritin</strong> —— The original sequence of Ferritin is gotten from addgene (plasmid #79649). It is in the same ORF of TRPV1. We inverted it into our GPD-CYC1 expressing system and transferred this plasmid into BY4741. Here follows the result of western blot. The two red-circled bands are exactly Ferritin we expressed.</p>
+
            <h2  id="5" class="H2Head">5.6</h2>
−
        <div class="imgdiv"><img class="textimg" style="width: 30% !important;" src="https://static.igem.org/mediawiki/2017/4/45/ZJU_China_MWF_another.png"></div>
+
            <p class="PP"><strong>1st group meeting in Summer term</strong></p>
−
        <p class="capture">Fig.7 Western blot of Ferritin</p>
+
            <p class="PP">The class about Trichoderma manipulation and gene manipulation mechanism began, which is some posts online. The great idea about VOC device was come up with. The team allocated into groups to extend the direction about immune, hyperparasitism and VOC.</p>
−
        <p class="PP">To further test the function of Ferritin, we have planned to purify it with the flag tag and test the heat effect under medium radio frequencies. However, due to the time limitation, we haven't finished it.</p>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/c/c0/ZJU_China_Notebook_11.jpg' alt=''/></div>
  
−
        <p class="PP"><strong>TRPV1</strong> —— The original sequence of TRPV1 is also gotten from addgene(plasmid #79649). We inverted it into our GPD-CYC1 expressing system and transferred this plasmid into BY4741. Here follows the result of western blot.</p>
+
            <h2 class="H2Head">5.13</h2>
−
        <div class="imgdiv"><img class="textimg"  style="width: 30% !important;" src="https://static.igem.org/mediawiki/2017/a/a7/ZJU_China_MWF_another2.png"></div>
+
            <p class="PP"><strong>2nd group meeting in Summer term</strong></p>
−
        <p class="capture">Fig.8 Western blot for TRPV1 </p>
+
            <p class="PP">Communicated and discussed each group’s design schedule. And a delicate sign-in form was printed.</p>
−
        <p class="PP">The band of TRPV1 is quite shallow. But it's OK because we have perform further experiments to test the function of TRPV1.</p>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/d/d0/ZJU_China_Notebook_12.jpg' alt=''/></div>
−
        <p class="PP">We use Fluo 4-AM to indicate the concentration change of calcium when the our yeast suffers a heat shock or be treated with capsaicin. The result shows that our BY4741 transferred with TRPV1 can be sensed a higher calcium concentration  than the control when it has been heated or treated by capsaicin. As Fig.  indicated, when the transgenic BY4741 is heated, the relative intracellular calcium ion concentration increases 71.6% while it increases 61.9% when treated with capsaicin. Compared with 8% enhancement when the yeast is cultured in high calcium concentration medium(200mM Ca2+), they are really huge changes and we're sure these change will induce a high strength of expression of the CDRE promoter.</p>
+
  
−
        <div class="imgdiv"><img class="textimg"  style="width: 30% !important;" src="https://static.igem.org/mediawiki/2017/3/3a/ZJU_China_MWF_fig9.jpeg"></div>
+
            <h2 class="H2Head">5.24</h2>
−
        <p class="capture">Fig.9 Relative calcium content of differet groups<br><br><br></p>
+
            <p class="PP"><strong>3rd group meeting in Summer term</strong></p>
 +
            <p class="PP">Some Human Practice plans were discussed as well as our team uniform. Several plasmid of Trichoderma design were finished.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/4/4f/ZJU_China_Notebook_13.jpg' alt=''/></div>
  
−
        <div style="text-align: center">
+
            <h2 class="H2Head">5.25</h2>
−
             <a class="CuteButton YellowCB" href="https://2017.igem.org/Team:ZJU-China/Project/st">See Chemical Transduction...</a>
+
             <p class="PP">Brought back two plates of T.atroviride”CTCCSJ-A-QT32133” isolated from grape and 600μL Agrobacterium Tumefaciens ”AGL1” in freezing tube.</p>
−
        </div>
+
  
 +
            <h2 class="H2Head">5.26</h2>
 +
            <p class="PP">Got our team flag!</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/1/19/ZJU_China_Notebook_14.jpg' alt=''/></div>
  
−
        <p class="PP"><strong>Combination</strong></p>
+
            <h2 class="H2Head">5.27</h2>
−
        <p class="PP">Due to the lack of time, we haven't finished the combination of TRPV1 and CDRE promoter. What's more, they're still only expressed in Saccharomyces cerevisiae. We are going to express TRPV1-Ferritin in <em>Trichoderma atroviride</em> and build up its own calcium-sensitive promoter.</p>
+
            <p class="PP"><strong>4th group meeting in Summer term</strong></p>
−
        <h2 class="H2Head">Reference</h2>
+
            <p class="PP">Arabidopsis was cultured. The consumable items of yeast were prepared. Experiments of Trichoderma started.</p>
−
        <p class="ref">[1] Szallasi A, Cortright D N, Blum C A, et al. The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept[J]. Nature reviews Drug discovery, 2007, 6(5): 357-372.</p>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/2/24/ZJU_China_Notebook_15.jpg' alt=''/></div>
−
        <p class="ref">[2] Iordanova B, Robison C S, Ahrens E T. Design and characterization of a chimeric ferritin with enhanced iron loading and transverse NMR relaxation rate[J]. JBIC Journal of Biological Inorganic Chemistry, 2010, 15(6): 957-965.</p>
+
            <p class="PP capture">hph from pKD1 and cleavaged tADH1 backbone 2017.5.27 YJB
−
        <p class="ref">[3] Ponka P, Beaumont C, Richardson D R. Function and regulation of transferrin and ferritin[C]//Seminars in hematology. 1998, 35(1): 35-54.</p>
+
−
        <p class="ref">[4] Stanley S A, Gagner J E, Damanpour S, et al. Radio-wave heating of iron oxide nanoparticles can regulate plasma glucose in mice[J]. Science, 2012, 336(6081): 604-608.</p>
+
−
        <p class="ref">[5] Stanley S A, Sauer J, Kane R S, et al. Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles[J]. Nature medicine, 2015, 21(1): 92-98.</p>
+
−
        <p class="ref">[6] Cyert M S. Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress[J]. Biochemical and biophysical research communications, 2003, 311(4): 1143-1150.</p>
+
−
        <p class="ref">[7] Cyert M S. Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae[J]. Annual review of genetics, 2001, 35(1): 647-672.</p>
+
−
        <br><br><br><br>
+
  
 +
            <h2 class="H2Head">5.28</h2>
 +
            <p class="PP">During the first human practice, three igemers made a presentation to three senior high school igem team. They talked about synthetic biology and igem competition. It’s a meaningful time for both the senior high school students and us.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/5/57/ZJU_China_Notebook_16.jpg' alt=''/></div>
  
 +
            <h2 class="H2Head">5.31</h2>
 +
            <p class="PP">Our first try to amplify fragment of homologous arms from the genome of T.atroviride.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/9/9c/ZJU_China_Notebook_17.jpg' alt=''/></div>
 +
            <p class="PP">1st genomic DNA 1st pcr prb1(failed) ech42(successed)</p>
  
−
    </div>
+
            <h2  id="6" class="H2Head">6.1</h2>
 +
            <p class="PP">Fine. Today is the Children Festival for us ,children. So everyone two sides up!</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/c/c6/ZJU_China_Notebook_18.jpg' alt=''/></div>
  
−
        <!-- 右侧监听开始 -->
+
            <h2 class="H2Head">6.3</h2>
−
        <div class="col-md-3 disappear-on-top" role="complementary">
+
             <p class="PP"><strong>5th group meeting in Summer term</strong></p>
−
             <nav  style="position: fixed; top: 100px ; left:50px;" class="bs-docs-sidebar hidden-print hidden-xs hidden-sm">
+
            <p class="PP">A normal meeting to report progress and make new plan, new human practice to Science and Technology Museum, experimental skills and team uniforms.</p>
−
                <ul class="nav bs-docs-sidenav shorterli">
+
  
−
                    <li>
+
            <h2 class="H2Head">6.4</h2>
−
                        <a href="https://2017.igem.org/Team:ZJU-China/Project/st#ct">Chemical Transduction</a>
+
            <p class="PP">The first AGL1 competences were finished. There were nearly 100 tubes which was enough for our whole year.</p>
−
                        <ul class="nav shorterli">
+
−
                            <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#overview">Overview</a></li>
+
−
                            <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#introduction">Introduction</a></li>
+
−
                            <li>
+
−
                                <a href="https://2017.igem.org/Team:ZJU-China/Project/st#result">Result</a>
+
−
                                <ul class="nav shorterli">
+
−
                                    <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#biosynthesis">Biosynthesis</a></li>
+
−
                                    <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#first">The First Step</a></li>
+
−
                                    <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#second">The Second Step</a></li>
+
−
                                    <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#third">The Third Step</a></li>
+
−
                                </ul>
+
−
                            </li>
+
  
−
                            <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#tr">Transcriptional Regulation</a></li>
+
            <h2 class="H2Head">6.5</h2>
−
                            <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#future">Future Work</a></li>
+
            <p class="PP">The first extended T.atroviride was mature which are just like our sweet children.</p>
−
                            <li><a href="https://2017.igem.org/Team:ZJU-China/Project/st#ref">Reference</a></li>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/1/10/ZJU_China_Notebook_19.jpg' alt=''/></div>
−
                        </ul>
+
−
                    </li>
+
  
−
                    <li>
+
            <h2 class="H2Head">6.10</h2>
−
                        <a href="#mwt">Medium Wave Transduction</a>
+
            <p class="PP"><strong>6th group meeting in Summer term</strong></p>
−
                        <ul class="nav shorterli">
+
            <p class="PP">It's a very big day. AIM medium was finished which consisted of more than 15 different reagents. Some members were still designing primers and training skills. The parts of VOC device were finished.</p>
−
                            <li>
+
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/0/07/ZJU_China_Notebook_20.jpg' alt=''/></div>
−
                                <a href="#background">Background</a>
+
 
−
                                <ul class="nav shorterli">
+
            <h2 class="H2Head">6.13</h2>
−
                                    <li><a href="#intr">Introduction</a></li>
+
            <p class="PP"><strong>7th group meeting in Summer term</strong></p>
−
                                    <li><a href="#tf">TRPV-Ferritin</a></li>
+
            <p class="PP">It's the last group meeting in this Spring-Summer term. So everyone summarized their whole term work and gave a plan about their first two weeks in the summer holiday(workday).</p>
−
                                </ul>
+
            <p class="PP">Up to then, mini apps in Wechat was finished. Gene of DAPG synthesis were amplified. Several plasmid of yeast were being optimized. Several homologous arms of Trichoderma genome were not finished.</p>
−
                            </li>
+
 
−
                            <li>
+
            <h2 class="H2Head">6.11-6.17</h2>
−
                                <a href="#design">Design</a>
+
            <p class="PP">The genomic DNA of T.atroviride were extracted over 5 times. The PCR experiments were tried more than 10 times. The experiments had to pause because of the final exam.</p>
−
                                <ul class="nav">
+
 
−
                                    <li><a href="#desint">Introduction</a></li>
+
            <h2 class="H2Head">6.17-7.3</h2>
−
                                    <li><a href="#expd">Experimental Design</a></li>
+
            <p class="PP">Everyone prepared for their final exams. And before the summer holiday, we had a group meeting.</p>
−
                                </ul>
+
 
−
                            </li>
+
            <h2  id="7" class="H2Head">7.5</h2>
−
                        </ul>
+
            <p class="PP">New experimental tobaccos came to our lab. We were still exploring the suitable protocol of genomic DNA.</p>
−
                    </li>
+
 
 +
            <h2 class="H2Head">7.6</h2>
 +
            <p class="PP">The beautiful cards were printed.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/c/c9/ZJU_China_Notebook_21.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">7.7</h2>
 +
            <p class="PP">The ferritin protein coding DNA was amplified.</p>
 +
 
 +
            <h2 class="H2Head">7.9</h2>
 +
            <p class="PP">We came to Zhejiang Science and Technology Museum and prepared several interesting games for children and teenagers in AST Space. During the games, we advertised synthetic biology.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/4/41/ZJU_China_Notebook_23.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">7.14</h2>
 +
            <p class="PP">With the senior high igemer, we came to the Zhejiang Food And Drug Administration to discuss the safety of synthetic biology production and the principles and laws in China.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/b/ba/ZJU_China_Notebook_24.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">7.16</h2>
 +
            <p class="PP">After over a month, we used the 17th and 18th genomic DNA as the templet and amplified all the homologous arms. It is the sentence ”Love for me is not about rice or soup, touch or kiss, is the dream as a hero in my shattered life. ” that helped us to tide over every failure.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/3/33/ZJU_China_Notebook_25.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">7.19</h2>
 +
            <p class="PP">We started the hex1 and ech42 promoter plasmids construction and finished 4 fragments link of two HRs, hph and the vector. The plasmid with TRPV1, Ferritin and PTH340 was transformed into the yeast.</p>
 +
 
 +
            <h2 class="H2Head">7.20</h2>
 +
            <p class="PP">It's a normal day with a little fun.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/0/0e/ZJU_China_Notebook_26.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">7.21</h2>
 +
            <p class="PP">Our PC game finished! And everyone fell love into it.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/1/15/ZJU_China_Notebook_27.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">7.22</h2>
 +
            <p class="PP">The VOC device received several series of data. We tried to sort out the data and adjust our hardware and software.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/d/dd/ZJU_China_Notebook_28.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">7.23</h2>
 +
            <p class="PP">Wiki modules were done. All the data can be saw on the cell phone.</p>
 +
 
 +
            <h2 class="H2Head">7.24-8.6</h2>
 +
            <p class="PP">The one step-clone of Homo Recombination plasmid suffered several failures. However, the manipulation of yeast got better. The transformation experiment was explored out. About the molecular cloning, some backbones and fragments were obtained.</p>
 +
 
 +
            <h2  id="8" class="H2Head">8.7</h2>
 +
            <p class="PP">The hex1 and ech42 promoter fragments were obtained.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/9/99/ZJU_China_Notebook_29.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">8.13</h2>
 +
            <p class="PP">The E.coli with hex1-es-pkD1 plasmid and ech42-ol-pkD1 plasmid were obtained. The PCR checkout result was satisfying.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/f/fb/ZJU_China_Notebook_30.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">8.15</h2>
 +
            <p class="PP">1st group meeting in Summer holiday</p>
 +
            <p class="PP">After military training and internship, members got together again. VOC had detected a lot of data and matched website was built up. Several models were designed and their requests were put forward. Additionally, a new photo was taken for a new start.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/3/3c/ZJU_China_Notebook_31.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">8.16</h2>
 +
            <p class="PP">The sequence result was correct. So we finished the construction of two plasmids for random insertion.</p>
 +
 
 +
            <h2 class="H2Head">8.18</h2>
 +
            <p class="PP">DAPG was extracted and was verified by HPLC</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/d/df/ZJU_China_Notebook_32.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">8.20</h2>
 +
            <p class="PP">Hex1 and ech42 plasmids were transformed into AGL1 and verified by PCR.</p>
 +
 
 +
            <h2 class="H2Head">8.22-8.24</h2>
 +
            <p class="PP">Four members went to Hunan Province to test our device and did human practice with China Tobacco.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/6/64/ZJU_China_Notebook_33.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">8.25-8.28</h2>
 +
            <p class="PP">Four members went to the CCiC meeting to communicate and exchange ideas with other igem.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/9/9e/ZJU_China_Notebook_34.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">8.27-9.4</h2>
 +
            <p class="PP">TRPV-PYES2.1 and phlFcheck-pKD1 were amplified. And phlF protein was extracted.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/6/6f/ZJU_China_Notebook_35.jpg' alt=''/></div>
 +
 
 +
            <h2  id="9" class="H2Head">9.15</h2>
 +
            <p class="PP">A unique Kit for Homologous Recombination was designed.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/6/6f/ZJU_China_Notebook_36.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">9.20</h2>
 +
            <p class="PP"><strong>1st group meeting in Autumn term</strong></p>
 +
            <p class="PP">The safety of our project was emphasized. Some part of our project came to the phase of reaping the harvest. The three model were detailed.</p>
 +
 
 +
            <h2 class="H2Head">9.23</h2>
 +
            <p class="PP"><strong>2nd group meeting in Autumn term</strong></p>
 +
            <p class="PP">The green fluorescence of three different promoters in T.atroviride were detected. And we took photos. The result was obvious.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/1/1d/ZJU_China_Notebook_37.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">9.30</h2>
 +
            <p class="PP"><strong>4th group meeting in Autumn term</strong></p>
 +
            <p class="PP">The duty of wiki were handed out to members. And we discussed job beyond experiments.</p>
 +
 
 +
            <h2  id="10" class="H2Head">10.1</h2>
 +
            <p class="PP">The serial number was handed out. And we started to standardize our parts.</p>
 +
 
 +
            <h2 class="H2Head">10.10</h2>
 +
            <p class="PP"><strong>5th group meeting in Autumn term</strong></p>
 +
            <p class="PP">The part of application and auto agriculture were discussed which was just like the brainstorm. Everyone had great ideas. And several questions left to be considered.</p>
 +
 
 +
            <h2 class="H2Head">10.14</h2>
 +
            <p class="PP"><strong>6th group meeting in Autumn term</strong></p>
 +
            <p class="PP">Some western blot were finished and more proteins were confirmed to be expressed by yeast and E.coli.</p>
 +
 
 +
            <h2 class="H2Head">10.17</h2>
 +
            <p class="PP"><strong>7th group meeting in Autumn term</strong></p>
 +
            <p class="PP">We exchanged our wiki and pointed out problems. The phlF and DAPG were verified to work.</p>
 +
 
 +
            <h2 class="H2Head">10.21</h2>
 +
            <p class="PP"><strong>8th group meeting in Autumn term</strong></p>
 +
            <p class="PP">The TRPV1 was verified to work. The following timetable was cleared.</p>
 +
 
 +
            <h2 class="H2Head">10.22</h2>
 +
            <p class="PP">Green fluorescence was detected which verified that the gene of synthesis of DAPG were transformed into T.atroviride.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/4/45/ZJU_China_Notebook_38.jpg' alt=''/></div>
 +
 
 +
            <h2 class="H2Head">10.23-10.30</h2>
 +
            <p class="PP">Wiki, Wiki, Monday Wikiday, Tuesday Wikiday, Wednesday Wikiday, Thursday Wikiday, Weekend Wikiweekend.</p>
 +
            <div class="imgdiv"><img class="textimg" src='https://static.igem.org/mediawiki/2017/d/d4/ZJU_China_Notebook_39.jpg' alt=''/></div>
 +
 
 +
            <div style="text-align: center;">
 +
                <br><br>
 +
                <a class="CuteButton YellowCB" href="https://2017.igem.org/Team:ZJU-China/Protocols">More About Protocols...</a>
 +
            </div>
 +
        </div>
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                    <li><a href="#2">February</a></li>
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                    <li><a href="#3">March</a></li>
 +
                    <li><a href="#4">April</a></li>
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Revision as of 05:37, 30 October 2017

Notebook

2.25

Concluded the winter project and 14 members were picked out. ZJU-China 2017 team was set up. Our story began.

First meetup. A nice meal with the previous iGEMer.

2.26-3.6

Everyone mined their winter project and attempted to extend the project and find problems.

2.28

1st group meeting in Spring term

We have some games to break the ice. Several rules and principles were built up as well as the duty table.

3.4

2nd group meeting in Spring term

Everyone shared their understand about the principles on the igem website, especially the medal requests. And a delicious chicken soup was cooked.

3.7

3rd group meeting in Spring term

Specially, today is a festival for all girls and the day for group meeting. So we pictured the blackboard and had a group photo. Additionally, we talked about the result of the winter project after discussion with related professors.

3.8-3.11

We detailed the experimental protocol of the winter project and find out the difficulties.

3.11

4th group meeting in Spring term

The whole team divided into three groups. Two deep mined the winter project and one came up with new ideas.The Brainstorm started. Someone jumped out the winter project. And some interesting ideas appeared. For example rhodopsin as photochemistry signal and resveratrol to attack the injured cells.

3.14

5th group meeting in Spring term

The previous iGEMer came to our group meeting and shared their experience. Then we talked about the project and received their suggestions. At that time 7 different ideas coexisted. We dug previous ideas and considered their safety and feasibility.

3.18

6th group meeting in Spring term

We still discussed about the new and old ideas. Fortunately, we didn’t choose the idea of Metarhitium against mosquito. Or I can’t imagine what our lab would be like. But the idea about degradation of melanin was interesting. However, after duplicate checking in Taobao, that idea was over. In addition, it was the birthday of one of our members. So there was another party.

3.21

7th group meeting in Spring term.

The main part started to tend to the Trichoderma. And other ideas exposed their limitation.

In those days, members were learning about basic molecular clone skills. And we exchange our experience in the group meeting.

3.25

8th group meeting in Spring term

The theme of our project was confirmed. And the team divided into groups again to study different directions about Trichoderma.

3.29

9th group meeting in Spring term

Talked about which the specific Trichoderma we could use and what kind of function we wanted to achieve and something about safety and gene circuit.

4.4

10th group meeting in Spring term

Discussed the available regulatory in Trichoderma and its function of immune and hyperparasitism. The experimental training came to the next phase. Everyone tried to construct a complete device from the very single part.

4.8

11th group meeting in Spring term

We started to choose the certain plant and pathogen. Everyone learned about several candidates and considered their feasibility.

4.15

13th group meeting in Spring term

Ensured the specific plant and pathogen and talked about the T3SS system about immune and GPCR about sense.

4.20-4.30

Some members began to learn the manipulation skill of Trichoderma in Zhang Lab, Agriculture Constitution, Zhejiang University. They sour out the notes and shared the protocol.

4.29

The first dish of Trichoderma came to our lab. We culture it in the illumination incubator.

5.6

1st group meeting in Summer term

The class about Trichoderma manipulation and gene manipulation mechanism began, which is some posts online. The great idea about VOC device was come up with. The team allocated into groups to extend the direction about immune, hyperparasitism and VOC.

5.13

2nd group meeting in Summer term

Communicated and discussed each group’s design schedule. And a delicate sign-in form was printed.

5.24

3rd group meeting in Summer term

Some Human Practice plans were discussed as well as our team uniform. Several plasmid of Trichoderma design were finished.

5.25

Brought back two plates of T.atroviride”CTCCSJ-A-QT32133” isolated from grape and 600μL Agrobacterium Tumefaciens ”AGL1” in freezing tube.

5.26

Got our team flag!

5.27

4th group meeting in Summer term

Arabidopsis was cultured. The consumable items of yeast were prepared. Experiments of Trichoderma started.

hph from pKD1 and cleavaged tADH1 backbone 2017.5.27 YJB

5.28

During the first human practice, three igemers made a presentation to three senior high school igem team. They talked about synthetic biology and igem competition. It’s a meaningful time for both the senior high school students and us.

5.31

Our first try to amplify fragment of homologous arms from the genome of T.atroviride.

1st genomic DNA 1st pcr prb1(failed) ech42(successed)

6.1

Fine. Today is the Children Festival for us ,children. So everyone two sides up!

6.3

5th group meeting in Summer term

A normal meeting to report progress and make new plan, new human practice to Science and Technology Museum, experimental skills and team uniforms.

6.4

The first AGL1 competences were finished. There were nearly 100 tubes which was enough for our whole year.

6.5

The first extended T.atroviride was mature which are just like our sweet children.

6.10

6th group meeting in Summer term

It's a very big day. AIM medium was finished which consisted of more than 15 different reagents. Some members were still designing primers and training skills. The parts of VOC device were finished.

6.13

7th group meeting in Summer term

It's the last group meeting in this Spring-Summer term. So everyone summarized their whole term work and gave a plan about their first two weeks in the summer holiday(workday).

Up to then, mini apps in Wechat was finished. Gene of DAPG synthesis were amplified. Several plasmid of yeast were being optimized. Several homologous arms of Trichoderma genome were not finished.

6.11-6.17

The genomic DNA of T.atroviride were extracted over 5 times. The PCR experiments were tried more than 10 times. The experiments had to pause because of the final exam.

6.17-7.3

Everyone prepared for their final exams. And before the summer holiday, we had a group meeting.

7.5

New experimental tobaccos came to our lab. We were still exploring the suitable protocol of genomic DNA.

7.6

The beautiful cards were printed.

7.7

The ferritin protein coding DNA was amplified.

7.9

We came to Zhejiang Science and Technology Museum and prepared several interesting games for children and teenagers in AST Space. During the games, we advertised synthetic biology.

7.14

With the senior high igemer, we came to the Zhejiang Food And Drug Administration to discuss the safety of synthetic biology production and the principles and laws in China.

7.16

After over a month, we used the 17th and 18th genomic DNA as the templet and amplified all the homologous arms. It is the sentence ”Love for me is not about rice or soup, touch or kiss, is the dream as a hero in my shattered life. ” that helped us to tide over every failure.

7.19

We started the hex1 and ech42 promoter plasmids construction and finished 4 fragments link of two HRs, hph and the vector. The plasmid with TRPV1, Ferritin and PTH340 was transformed into the yeast.

7.20

It's a normal day with a little fun.

7.21

Our PC game finished! And everyone fell love into it.

7.22

The VOC device received several series of data. We tried to sort out the data and adjust our hardware and software.

7.23

Wiki modules were done. All the data can be saw on the cell phone.

7.24-8.6

The one step-clone of Homo Recombination plasmid suffered several failures. However, the manipulation of yeast got better. The transformation experiment was explored out. About the molecular cloning, some backbones and fragments were obtained.

8.7

The hex1 and ech42 promoter fragments were obtained.

8.13

The E.coli with hex1-es-pkD1 plasmid and ech42-ol-pkD1 plasmid were obtained. The PCR checkout result was satisfying.

8.15

1st group meeting in Summer holiday

After military training and internship, members got together again. VOC had detected a lot of data and matched website was built up. Several models were designed and their requests were put forward. Additionally, a new photo was taken for a new start.

8.16

The sequence result was correct. So we finished the construction of two plasmids for random insertion.

8.18

DAPG was extracted and was verified by HPLC

8.20

Hex1 and ech42 plasmids were transformed into AGL1 and verified by PCR.

8.22-8.24

Four members went to Hunan Province to test our device and did human practice with China Tobacco.

8.25-8.28

Four members went to the CCiC meeting to communicate and exchange ideas with other igem.

8.27-9.4

TRPV-PYES2.1 and phlFcheck-pKD1 were amplified. And phlF protein was extracted.

9.15

A unique Kit for Homologous Recombination was designed.

9.20

1st group meeting in Autumn term

The safety of our project was emphasized. Some part of our project came to the phase of reaping the harvest. The three model were detailed.

9.23

2nd group meeting in Autumn term

The green fluorescence of three different promoters in T.atroviride were detected. And we took photos. The result was obvious.

9.30

4th group meeting in Autumn term

The duty of wiki were handed out to members. And we discussed job beyond experiments.

10.1

The serial number was handed out. And we started to standardize our parts.

10.10

5th group meeting in Autumn term

The part of application and auto agriculture were discussed which was just like the brainstorm. Everyone had great ideas. And several questions left to be considered.

10.14

6th group meeting in Autumn term

Some western blot were finished and more proteins were confirmed to be expressed by yeast and E.coli.

10.17

7th group meeting in Autumn term

We exchanged our wiki and pointed out problems. The phlF and DAPG were verified to work.

10.21

8th group meeting in Autumn term

The TRPV1 was verified to work. The following timetable was cleared.

10.22

Green fluorescence was detected which verified that the gene of synthesis of DAPG were transformed into T.atroviride.

10.23-10.30

Wiki, Wiki, Monday Wikiday, Tuesday Wikiday, Wednesday Wikiday, Thursday Wikiday, Weekend Wikiweekend.




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