Difference between revisions of "Team:China Tongji/Notebook"

 
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<!-- content start -->
 
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    <div class="bugDiv"><img class="bugImg" src="https://static.igem.org/mediawiki/2015/2/25/China-Tongji-projectBug.png"></div>
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<div class="bugDiv"><img class="bugImg" src="https://static.igem.org/mediawiki/2015/0/06/China-Tongji-notebookBug.png"></div>
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<p align="left">Project</p>
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<p align="left">Notebook</p>
 
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<!-- myContent start -->
 
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<div class="myContent">
 
<div class="myContent">
 
<div id="contentList">
 
<div id="contentList">
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         <li class="listOne"><p id="listOverview">1. Overview</p>
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         <li class="listOne"><p id="listRecord">1. Record</p>
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         <ul class="listOneDrop" id="listDropOverview">
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         <ul class="listOneDrop" id="listDropRecord">
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                 <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('first1').offsetTop)">1.1 Introduction</li>
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                 <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('first1').offsetTop)">1.1 Plasmid Part</li>
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                 <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('first2').offsetTop)">1.2 Molecular cloning and micro-injection</li>
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                 <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('first2').offsetTop)">1.2 Worm Part</li>
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                 <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('first3').offsetTop)">1.3 Worms testing and tracks recording</li>
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                 <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('first3').offsetTop)">1.3 Efficiency Part</li>
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                 <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('first4').offsetTop)">1.4 Tracks analysis and video edit</li>
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                 <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('first4').offsetTop)">1.4 Equipment Part</li>
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            </ul>
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        </li>
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        <li class="listOne"><p id="listBackground">2. Background</p>
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        <ul class="listOneDrop" id="listDropBackground">
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('second1').offsetTop)">2.1 What is optogenetics?</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('second2').offsetTop)">2.2 Why we use the C.elegans?</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('second3').offsetTop)">2.3 What proteins do we use?</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('second4').offsetTop)">2.4 Reference</li>
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−
            </ul>
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        </li>
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        <li class="listOne"><p id="listProjectDesign">3. Project Design</p>
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        <ul class="listOneDrop" id="listDropProjectDesign">
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('third1').offsetTop)">3.1 Introduction</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('third2').offsetTop)">3.2 General Design</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('third3').offsetTop)">3.3 Plasmid Design</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('third4').offsetTop)">3.4 Equipment Design</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('third5').offsetTop)">3.5 Test Design</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('third6').offsetTop)">3.6 Reference</li>
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−
            </ul>
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        </li>
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−
        <li class="listOne"><p id="listProtocol">4. Protocol</p>
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        <ul class="listOneDrop" id="listDropProtocol">
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth1').offsetTop)">4.1 Introduction</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth2').offsetTop)">4.2 Taq PCR</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth3').offsetTop)">4.3 Pfu PCR</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth4').offsetTop)">4.4 AGE(agarose gel electrophoresis)</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth5').offsetTop)">4.5 Gel extraction</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth6').offsetTop)">4.6 Digestion & ligation</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth7').offsetTop)">4.7 Seamless cloning</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth8').offsetTop)">4.8 Transformation</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth9').offsetTop)">4.9 Plasmid Extraction</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fourth10').offsetTop)">4.10 Microinjection</li>
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−
            </ul>
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−
        </li>
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−
        <li class="listOne"><p id="listSummaryResult">5. Summary and Result</p>
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−
        <ul class="listOneDrop" id="listDropSummaryResult">
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fifth1').offsetTop)">5.1 Plasmids Construction</li>
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                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('fifth2').offsetTop)">5.2 Test Result</li>
+
−
            </ul>
+
−
        </li>
+
−
        <li class="listOne"><p id="listDesign">6. Design</p>
+
−
        <ul class="listOneDrop" id="listDropDesign">
+
−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('sixth1').offsetTop)">6.1 Introduction</li>
+
−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('sixth2').offsetTop)">6.2 Medical treatment potential</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('sixth3').offsetTop)">6.3 Useful tool in scientific research</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('sixth4').offsetTop)">6.4 Conclusion</li>
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−
                <li class="listTwo" onClick="window.scrollTo(0,document.getElementById('sixth5').offsetTop)">6.5 Reference</li>
+
 
             </ul>
 
             </ul>
 
         </li>
 
         </li>
 +
        <li class="listOne"><p id="listTimeline">2. Timeline</p></li>
 
     </div>
 
     </div>
 
     <div id=mainContent>
 
     <div id=mainContent>
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     <!-- maincontent start -->
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     <!-- maincontent start here -->
 
         <div class="fivePx"></div><div class="fivePx"></div>
 
         <div class="fivePx"></div><div class="fivePx"></div>
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         <!-- 1. Overview -->
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         <p class="titleOne" id="Record">1. Record</p>  
−
         <p class="titleOne" id="Overview">1. Overview</p>  
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         <p class="partTwo" id="first1">1.1 Plasmid Part</p>
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         <div class="fivePx"></div>
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         <div class="slidePanel slidePanel1">
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        <p class="titleTwo" id="first1">1.1 Introduction </p>
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            <h3 class="slideTitle" id="slideTitle1dot1">1.1.1 JUNE -- Week 1~4 -- June 6~25<span class="expand">[Expand]</span></h3>
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        <p class="contentP">In our project, we will use theoptogenetic technology and the lights of differentspecific wavelength produced by the light source assembled by ourselves to control the movement of C.elegans and finally construct a movement controlling system.</p>
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            <div class="slideBlock" id="slideBlock1dot1">
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        <div class="fivePx"></div>
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                <h4>1.1.1.1 Week1 -- June 6</h4>
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        <p class="titleTwo" id="first2">1.2 Molecular cloning and micro-injection</p>
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                <h5>June 6</h5>
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        <p class="contentP">We construct the plasmidswhich are inserted our specific promotors and targeted light-sensitive ion channels genes .The specific promotors such as: AIY, pmyo2, pmyo3 and the opsin such as: ChR2,iC1C2, chETA, Blink are all founded on different papers and websites of worms. And then, we insert the plasmids into C.elegans by using themicro-injection technolagy.By doing that, we may can control thebehaviours of C.elegans such as moving forwards or twisting more effectively.</p>
+
                <p>1, Design the PCR primers of chR2-YFP.</p>
−
        <p class="contentP">What's more,we will express GFP,YFP,mcherry in E.coli. By combining the color of microorgasims and C.elegans, we want to construct some interesting scenes to form a C.elegans' fancy world.</p>
+
               
 +
                <h4>1.1.1.2 Week2 -- June 8~14</h4>
 +
                <h5>June 8</h5>
 +
                <p>1, The amplification of CHR2-YFP (use taq PCR protocol)</p>
 +
                <p>2, AGE ( agarose gel electrophoresis )</p>
 +
                <p>3, Gel extraction of chR2-YFP.</p>
 +
                <p>4, Transformation of GFP, YFP, mcherry in E•coli DH5α.</p>
 +
<h5>June 9</h5>
 +
                <p>1, Select a single clone of each plate. (GFP, YFP, mcherry) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.</p>
 +
                <p>2, Transformation of vector with pmyo-3(ppd95.77)</p>
 +
                <h5>June 10</h5>
 +
                <p>1, Select a single clone of plate. (pmyo-3,ppd95.77) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.</p>
 +
                <p>2, Plasmid extraction ofE.coli DH5αwith GFP, YFP and mcherry in it.</p>              
 +
                <h5>June 11</h5>
 +
                <p>1, Plasmid extraction of pmyo-3(ppd95.77)</p>
 +
                <p>2, Digestion of ppd95.77 with pmyo-3 in it and chR2-YFP using BamHI and EcoRI.(digestion protocol)</p>              
 +
                <h5>June 12</h5>
 +
                <p>1, AGE ( agarose gel electrophoresis ) of digested vector---ppd95.77 with pmyo-3.</p>
 +
                <p>2, Gel extraction of pmyo-3.</p>
 +
                <p>3, purification of chR2-YFP witch has been digested with BamHI and EcoRI.</p>               
 +
                <h5>June 14</h5>
 +
                <p>1, Transform of GFP, YFP, mcherryin E•coli BL21.</p>
 +
               
 +
                <h4>1.1.1.3 Week3 -- June 15~21</h4>
 +
                <h5>June 15</h5>
 +
                <p>1, Ligation of pmyo-2 and chR2-YFP. (ligation protocol)</p>
 +
                <p>2, Transformation of ligation product: pmyo3-chR2-YFP (in ppd95.77).</p>
 +
                <h5>June 17</h5>
 +
                <p>1, Select a single clone of plate. (pmyo-3-chR2-YFP, ppd95.77) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.</p>
 +
                <p>2, Plasmid extraction of pmyo3-chR2-YFP.</p>
 +
                <h5>June 19</h5>
 +
                <p>1, Digestion of new plasmid: pmyo3-chR2-YFP using HindIII and EcoRI to check if the ligation step work or not. (it worked! Our first part is done!)</p>
 +
                <h5>June 20</h5>
 +
                <p>1, Transformation of pmyo3-chR2-YFP in order to get more plasmid.</p>
 +
                <p>2, Transformation of pmec3-chR2-YFP, pmec3-dsred and pmec4-chR2-YFP which are offered by professor Li’s lab.</p>
 +
                <p>3, Save the E.coli strain with glycerinum.</p>
 +
                <h5>June 21</h5>
 +
                <p>1, Theamplification of dsred and pmyo-2(use taq PCR protocol---to test the best temperature for the PCR).</p>
 +
                <p>2, the amplification of dsred and pmyo-2(use pfu PCR protocol).</p>
 +
                <p>3, AGE ( agarose gel electrophoresis ) of pmyo-2 and dsred.</p>
 +
                <p>4, Gel extraction of pmyo-2 and dsred.</p>
 +
 
 +
                <h4>1.1.1.4 Week4 -- June 22~25</h4>   
 +
                <h5>June 22</h5>
 +
                <p>1, digestion of pmyo3-chR2-YFP(using HindIII and BamHI)</p>
 +
                <p>2, AGE ( agarose gel electrophoresis ) of digested vector---ppd95.77 with chR2-YFP.</p>
 +
                <p>3, Gel extraction of pmyo-3(PPD95.77).</p>
 +
                <p>4, Digest of pmyo2 with HindIII and BamHI. Digest of dsred with BamHI and EcoRI. </p>
 +
                <p>5, gene purification for pmyo2 and dsred.</p>
 +
                <h5>June 24</h5>
 +
                <p>1, Ligation of pmyo2 with chR2-YFP (in ppd95.77).</p>
 +
                <p>2, Ligation of dsred with pmyo3 and pmyo2(in ppd95.77)</p>
 +
                <h5>June 25</h5>
 +
                <p>1, Digest of pmyo2-chR2-YFP and pmyo2-dsred and pmyo3-dsred.(usingHindIII and EcoRI) To check if we had ligated it successfully.</p>
 +
                <p>2, AGE ( agarose gel electrophoresis ) of digested products. Analyze the result.</p>                           
 +
            </div>
 +
        </div>  
 
          
 
          
 
         <div class="fivePx"></div>
 
         <div class="fivePx"></div>
−
         <p class="titleTwo" id="first3">1.3 Worms testing and tracks recording</p>
+
         <div class="slidePanel slidePanel1">
−
        <p class="contentP">We test the C.elegans with the fluorescence microscope . In the testing, we can select the C.elegans in which our target gene has expressed stably.Then, we observe the movement of worms under specific lengh of wave.Select out the worms which performas expected and recording their tracks in video.</p>
+
            <h3 class="slideTitle" id="slideTitle1dot2">1.1.2 JULY -- Week 2~5 -- July 11~30<span class="expand">[Expand]</span></h3>
−
        <p class="contentP">We next change the duration, the wave length and the intensityof the light we use so that we can grope how different conditions influence the movement of C.elegans in the form of table.</p>
+
            <div class="slideBlock" id="slideBlock1dot2">
−
        <div class="fivePx"></div>
+
                <h4>1.1.2.1 Week2 -- July 11~14</h4>
−
        <p class="titleTwo" id="first4">1.4 Tracks analysis and video edit</p>
+
                <h5>July 11</h5>
−
        <p class="contentP">We analyse the video according to the frame and draw the track lines of each movement.Then we draw the curve graph based on the different conditions and the response of worms.Then, we perfect our video and label the casting part on the worm. </p>
+
                <p>1, Design the PCR primers of Blink gene.</p>
−
        <p class="contentP">This technology will be helpful in the research on neuron's function and interaction. In the future, this technology may also be used in mechanical controlling system and the theraphy of movement defect.</p>
+
                <p>2, Design the PCR primers of pttx-3.</p>  
 +
                <p>3, Design the PCR primers of ptrp4.</p>  
 +
                <p>4, Prepare for competent cells</p>  
 +
                <h5>July 14</h5>
 +
                <p>1, Transformation of the Blink plasmid which was kind offered by Anna’s lab.</p>  
 +
                <p>2, Transformation of pmyo2-chR2-YFP, pmyo2-dsred and pmyo3-dsred in order to get more plasmids.</p>  
 +
                <p>3, GFP, YFP, mcherry transform OP50 and PA14. (OP50 and PA14 are the food of C.elegans)</p>  
  
−
        <p></p><div class="divider"></div>
+
                <h4>1.1.2.2 Week3 -- July 15~21</h4>
−
<!-- 2. Background -->
+
                <h5>July 15</h5>
−
<p class="titleOne" id="Background">2. Background</p>  
+
                <p>1,Select single clones of plate. (pmyo-2-chR2-YFP, pmyo2-dsred and pmyo3-dsred ppd95.77) . Put the E.coli in 4ml LB buffer and cultivate for one night at 37℃.</p>
 +
               
 +
                <h5>July 16</h5>
 +
                <p>1, Plasmid extraction of pmyo-2-chR2-YFP, pmyo2-dsred and pmyo3-dsred.</p>
 +
               
 +
                <h5>July 17</h5>
 +
                <p>1, Recovery of </p>
 +
                <p><b>pNP260( Pnmr-1::flox::ChR2::mCherry.)</b></p>
 +
                <p><b>pCoS2(pnhr-79::Cre):</b></p>
 +
                <p><b>pCoS13(posm-10::loxP::LacZ::STOP::loxP:: ChR2::mCherry::SL2::GFP)</b></p>
 +
                <p><b>pSH116 (pdes-2::Cre)</b></p>
 +
                <p>which are kind offered by Alexander Gottschalk’ lab. </p>
 +
                <p>2, Transformation of pNP260, pCoS2, pCoS13 and pSH116.</p>
 +
                <p>3, Design of the PCR primers of pttx-HindIII-F and pttx3-Xbal-R.</p>
 +
               
 +
                <h5>July 18</h5>
 +
                <p>1, Select single clones of plate.(pNP260, pCoS2, pCoS13 and pSH116). Put the E.coli in 4ml LB buffer and cultivate for 12h at 37℃.</p>
 +
                <p>2, Plasmid extraction.</p>
 +
                <p>3, Make genome DNA from worms.</p>
 +
                <p>4, Make LB plate and LB liquid.</p>
 +
                <p>5, Repeat the experiment: Prepare for competent cells: GFP, YFP, mcherry transform OP50 and PA14.</p>
 +
                <p>6, The amplification of Blink.</p>
 +
                <p>7, AGE ( agarose gel electrophoresis ) of blink PCR products. Analyze the result.</p>
 +
                <p>8, Gel extraction. (all at 300ng/ul)</p>
 +
               
 +
                <h5>July 19</h5>
 +
                <p>1, Use the C.elegans genomic DNA as template and got pttx-3 with the help of Pttx3-HindIII-F and pttx-3-XbaI-R.</p>
 +
                <p>2, AGE ( agarose gel electrophoresis ) of PCR products. Analyze the result.</p>
 
                  
 
                  
−
        <p class="titleTwo" id="second1">2.1 What is optogenetics?</p>
+
                <h5>July 20</h5>
−
        <p class="contentP">Optogenetics involves the use of light to control cells in living tissue, typically <b>neurons</b>, that have been genetically modified to express <b>light-sensitive ion channels</b>. It is a neuromodulation method employed in neuroscience that uses a combination of techniques from optics and genetics to control and monitor the activities of individual neurons in living tissue—even within freely-moving animals—and to precisely measure the effects of those manipulations in real-time.<sup>[1]</sup> The rapid progression of such interdisciplinary "optogenetic" approaches has expanded capabilities for optical imaging and genetic targeting of specific cell types.</p>
+
                <p>1, Transformation of P Blue plasmid.(used to make the mixture of microinjection liquid)</p>
−
        <p class="contentP">The key reagents used in optogenetics are <b>light-sensitive proteins</b>. Spatially-precise neuronal control is achieved using optogenetic actuators like channelrhodopsin, halorhodopsin, and archaerhodopsin, while temporally-precise recordings can be made with the help of optogenetic sensors for calcium (Aequorin, Cameleon, GCaMP), chloride (Clomeleon) or membrane voltage (Mermaid).<sup>[2]</sup></p>
+
                <p>2, Select single clone on the culture plate.Put the E.coli in 4ml LB buffer and cultivate for 12h at 37℃.</p>
−
        <div class="fivePx"></div>
+
                <p>3, Select a single clone of each plate and put each one in a tube that contain the ampicillin and 1ml LB culture media. (OP50 and PA14)</p>
−
        <p class="titleTwo" id="second2">2.2 Why we use the C.elegans?</p>
+
                <p>4, At 37℃ we culture them for 12h.</p>
−
        <p class="contentP">C. elegans(Caenorhabditis elegans) is a small individual,which only has a few cells(959 in the adult hermaphrodite; 1031 in the adult male) and <b>302 neurons</b>. Because of that,C.elegans become one of the <b>simplest organisms</b> with a nervous system. Besides, the body of C.elegans is  transparent and easy to <b>observe</b>. Based on the above, C. elegans is a convenient and effective animal model applied in the optogenetics.</p>
+
               
−
        <p class="contentP">Based on the characteristic of C. elegans,we choose it as our experimental objective.On the one hand, we can easily controll it by casting different waves of light on it .On the other hand, we can also clearly observe it and recort it’s track under the fluorescence microscope.</p>
+
                <h5>July 21</h5>
−
        <div class="fivePx"></div>
+
                <p>1, Add the mixture to a new 15ml tube and add in 4ml LB culture media.</p>
−
        <p class="titleTwo" id="second3">2.3 What proteins do we use?</p>
+
                <p>2, Culture them for 3h until the OD number near 0.8.</p>
−
        <p class="contentP">Each opsin protein requires the incorporation of retinal, a vitaminA-related organic photon-absorbing cofactor, to enable lightsensitivity; this opsin-retinal complex is referred to as rhodopsin.The retinal molecule is covalently fixed in the binding pocketwithin the 7-TM helices and forms a protonated retinal Schiffbase (RSBH+; Figure 1) with a conserved lysine residue locatedon TM helix seven (TM7). The ionic environment of the RSBH+,heavily influenced by the residues lining the binding pocket, dictates the spectral characteristics of each individual protein; upon absorption of a photon, the retinal chromophore isomerizes and triggers a series of structural changes leading to iontransport, channel opening, or interaction with signaling transducer proteins.</p>
+
                <p>3, Add 1mg IPTG in the liquid.</p>
−
        <div class="fivePx"></div>
+
                <p>4, Culture them for 3h in order to gain the protein.</p>
−
        <center><img class="contentImg" src="https://static.igem.org/mediawiki/2015/3/35/China-Tongji-Project-figure2-1.jpg"></center>
+
                <p>5, Plasmid extraction of P blue.</p>
−
        <p class="imgIntroduction"><b>Figure 2-1:</b> Light-mediated isomerization of the retinal Schiff base (RSB). Top: retinal in the all-transstate, as found in the dark-adapted state of microbial rhodopsins andin the light-activated forms of type II rhodopsins of higher eukaryotes. The absorption of a photon converts the retinal from the all-transto the 11-cisconfiguration. Bottom: 11-cisretinal is found only in type II rhodopsins, where it binds to the opsin in the dark state before isomerizing to the all-trans position after photonabsorption.</p>
+
                <p>6, Digestion: Use EcoR I and BamH I digest the pmyo2 and pmyo3 plasmid and the Blink. (use digestion protocol)</p>
 +
                <p>7, Ligase reaction. (use ligation protocol)( pmyo2-blink,pmyo3-blink)</p>
 +
 
 +
                <h4>1.1.2.3 Week4 -- July 22~27</h4>
 +
                <h5>July 22</h5>
 +
                <p>1, Design the PCR plasmid of chETA and ic1c2 which we bought from addgene.</p>
 +
                <p>2, Cultivation of plasmid chETA and ic1c2 from Addgene.Pick a loop of bacteria from the sample, streaking on Amp+ plates, cultivate in 37℃ for 12h.</p>
 +
                <p>3, Transform DH5α(pmyo2-blink,pmyo3-blink). Label the plate and put it in the incubator about one night, 37℃.</p>
 +
               
 +
                <h5>July 23</h5>
 +
                <p>1, Amplify of plasmid chETA and ic1c2 from Addgene.Pick 5 single clone from each plate, add in 4ml LB medium separately, and shaking in 37℃ for 14h.</p>
 +
                <p>2, Make LB plate.</p>
 +
                <p>3, Select a single clone and culture for 12h of each template. (pmyo2-blink,pmyo3-blink)</p>
 +
               
 +
                <h5>July 24</h5>
 +
                <p>1, Plasmid extraction of plasmid chETA and ic1c2. (the results are all around 100ng/ul)</p>
 +
                <p>2, Taq PCR of chETA and ic1c2. (to test the best reaction tempareture)</p>
 +
                <p>3, AGE ( agarose gel electrophoresis ) of PCR products. Analyze the result. We found that 68℃ is the best temperature.</p>
 +
                <p>4, Plasmid Extraction. (pmyo2-blink,pmyo3-blink) the results are all at 200-300ng/ul.</p>
 +
                <p>5, Ligase reaction (again): pmyo2-blink, pmyo3-blink.</p>
 +
               
 +
                <h5>July 25</h5>
 +
                <p>1, Pfu PCR of chETA and ic1c2.</p>
 +
                <p>2, AGE ( agarose gel electrophoresis ) of pfu PCR products.</p>
 +
                <p>3, Gel extraction and recycle the chETA and ic1c2.</p>
 +
                <p>4, Used the C.elegans genomic DNA as template and got pttx-3 with the help of Pttx3-HindIII-F and pttx-3-XbaI-R.</p>
 +
                <p>5, Algarose gel electrophoresis to test PCR result.</p>
 +
                <p>6, Gel extraction and recycle the pttx-3.</p>
 +
                <p>7, Transform DH5α(pmyo2-blink, pmyo3-blink)</p>
 +
                <p>8, Select single clone from culture plate. And culture for 12h.</p>
 +
               
 +
                <h5>July 26</h5>
 +
                <p>1, Plasmid Extraction (pmyo2-blink, pmyo3-blink)</p>
 +
               
 +
                <h5>July 27</h5>
 +
                <p>1, Repeat the experiment: used the C.elegans genomic DNA as template and got pttx-3 with the help of Pttx3-HindIII-F and pttx-3-XbaI-R.</p>
 +
                <p>2, Ran the Algarose gel electrophoresis to test my PCR result.</p>
 +
                <p>3, After confirming the accuracy of my PCR result, we did an gel extraction and recycle the pttx-3.</p>
 +
                <p>4, Transformation of pmyo2, pmyo3 plasmids to get more for the latter experiment.Culture at 37℃ for 16h.</p>
 +
                <p>5, Pfu PCR of chETA and ic1c2.</p>
 +
                <p>6, AGE ( agarose gel electrophoresis ) of pfu PCR products.(chETA and ic1c2)</p>
 +
                <p>7, Gel extraction and recycle the chETA and ic1c2.</p>
 +
                <p>8, Select single clone of AMP LB plate.And culture for 12h.</p>
 +
 
 +
                <h4>1.1.2.4 Week5 -- July 28~31</h4>
 +
                <h5>July 28</h5>
 +
                <p>1, Plasmid Extraction(pmyo2, pmyo3---ppd95.77).</p>
 +
               
 +
                <h5>July 29</h5>
 +
                <p>1, Make Backbone, transformation of backbone.Culture at 37℃ for 16h. (according by protocol offered by iGEM)</p>
 +
                <p>2, DigestchETA and ic1c2 genes, pmyo2 and pmyo3 vectors with BamHI and EcoRI.(digestion protocol)</p>
 +
                <p>3, Ligation of pmyo2-chETA, pmyo2-ic1c2, pmyo3-chETA and pmyo3-ic1c2. (ligation protocol)</p>
 +
               
 +
                <h5>July 30</h5>
 +
                <p>1, Transformation of pmyo2-chETA, pmyo2-ic1c2, pmyo3-chETA and pmyo3-ic1c2.Culture at 37℃ for 16h.</p>
 +
                <p>2, Select single clone from culture plate (pmyo2-chETA, pmyo2-ic1c2).And culture for 12h.(there is no clone of pmyo3-chETA and pmyo3-ic1c2 )</p>
 +
                <p>3, Select single clone from culture plate.(Backbone)</p>
 +
               
 +
                <h5>July 31</h5>
 +
                <p>1, Plasmid Extraction (pmyo2-chETA, pmyo2-ic1c2).</p>
 +
                <p>2, Plasmid Extraction (Backbone).</p>
 +
                <p>3, Digest of pmyo2-chETA and pmyo2-ic1c2 using BamHI and EcoRI. Make sure that the gene had been successfully ligated into the plasmids.</p>
 +
                <p>4, Digest of backbone with PstI and EcoRI. Make sure our backbone is made in the right way.</p>
 +
             
 +
            </div>
 +
        </div>  
 
         <div class="fivePx"></div>
 
         <div class="fivePx"></div>
−
          
+
         <div class="slidePanel slidePanel1">
−
        <p class="contentP">Opsin genes are divided into two distinct superfamilies: microbial opsins (type I) and animal opsins (type II). Bucause we study C.elegans, we only introduction type II here. Type II opsin genes are present only in highereukaryotes and are mainly responsible for vision (Sakmar, 2002). A small fraction of type II opsins also play roles in circadian rhythm and pigment regulation (Sakmar, 2002; Shichidaand Yamashita, 2003). Type II opsins primarily function as Gprotein-coupled receptors (GPCRs) and appear to all use the11-cisisomer of retinal (or derivatives) for photon absorption(Figure 1, bottom)</p>
+
            <h3 class="slideTitle" id="slideTitle1dot3">1.1.3 AUGUST -- Week 1~5 -- August 1~31<span class="expand">[Expand]</span></h3>
−
       
+
            <div class="slideBlock" id="slideBlock1dot3">
−
        <h3>2.3.1 ChRs(ChR2)</h3>
+
                <h4>1.1.3.1 Week1 -- August 1~7</h4>
−
        <p class="contentP">The first known and described ChR, channelrhodopsin-1(ChR1), was identified as a light-gated ion channel inChlamydomonas reinhardtii, a green unicellular alga from temperate freshwater environments (Nagel et al., 2002). ChR1 has broad cation conductance, includingfor Na+,K+, and even Ca2+ions (Lin et al., 2009; Tsunoda andHegemann, 2009). Channelrhodopsin-2 (ChR2),was later characterized from the same organism.Similar to ChR1, ChR2 also conducts cations (Nagel et al.,2003; Tsunoda and Hegemann, 2009), and both ChRs exhibitfast on and off kinetics. When introduced into neurons, ChRscan insert into the plasma membrane and mediate membranepotential changes in response to blue light (Boyden et al.,2005; Ishizuka et al., 2006; Li et al., 2005). </p>
+
                <h5>August 1</h5>
−
        <p class="contentP">Indeed, the photocycle of ChR2 (Figure 2 and Figure 3(Yizhar et al.,2011b))has different spectral characteristics .In ChR2, adark-adapted state absorbing at 470 nm (D470) converts rapidlyupon illumination to the conducting state P520, via the shortlived photointermediates P500 and P390. Illumination of theopen channel at this step with green light terminates the photocurrent (Bamann et al., 2008; Berndt et al., 2009) by photochemically shifting the channel back into a closed state, which may bethe dark-adapted state D470 or the light-adapted state P480(Stehfest and Hegemann, 2010), effectively resetting the photocycle. This photocycle-shortcut pathway may be relevant only atvery high light intensities with wild-type ChR2.</p>
+
                <p>1, Transformation of pmyo2-chETA and pmyo2-ic1c2 in order to get more plasmids.Culture at 37℃ for 16h.</p>
−
       
+
                <p>2, Select single clone from culture plate (pmyo2-chETA and pmyo2-ic1c2.).And culture for 12h.</p>
−
        <div class="fivePx"></div>
+
                <p>3, Try to ligate pmyo3-chETA and pmyo3-ic1c2 again as last time we failed. Digestion and ligation.</p>
−
        <center><img class="contentImg" width="600" src="https://static.igem.org/mediawiki/2015/5/5d/China-Tongji-Project-figure2-2.jpg"></center>
+
                <p>4, Transformation of pmyo3-chETA and pmyo3-ic1c2, Culture at 37℃ for 16h.</p>
−
        <p class="imgName" align="center"><b>Figure 2-2:</b> The working principle of ChR2.</p>
+
               
−
        <div class="fivePx"></div>
+
                <h5>August 2</h5>
−
       
+
                <p>1, Select single clone from culture plate (pmyo3-chETA and pmyo3-ic1c2.).And culture for 12h.</p>
−
        <div class="fivePx"></div>
+
                <p>2, Plasmid Extraction (pmyo2-chETA, pmyo2-ic1c2).</p>
−
        <center><img class="contentImg" src="https://static.igem.org/mediawiki/2015/c/c0/China-Tongji-Project-figure2-3.jpg"></center>
+
                <p>3, Plasmid Extraction (pmyo3-chETA, pmyo3-ic1c2).</p>
−
        <p class="imgIntroduction"><b>Figure 2-3:</b> Simplified model for the photocycle of ChRs. The D470 dark state is converted by a light-induced isomerization of retinal via the early intermediate P500 andthe transient P390 intermediate to the conducting-state P520. The recovery of the D470 dark state proceeds either thermally via the nonconducting P480intermediate or photochemically via possible short-lived intermediates (green arrow). The late or desensitized P480 state can also be activated (blue arrow) toyield the early intermediate P500. Additional parallel cycles may be present (Yizhar et al., 2011b)</p>
+
               
−
        <div class="fivePx"></div>
+
                <h5>August 3</h5>
−
       
+
                <p>1, Digest ofpmyo3-chETA, pmyo3-ic1c2 to check if we had ligated them right. (the result turn out that the pmyo3-chETA is right)</p>
−
        <h3>2.3.2 chETA</h3>
+
                <p>2, Transformation of pmyo3-chETA in order to get more plasmids.</p>
−
        <p class="contentP">Inanother approach addressing both desensitization and deactivation, considering the crystalstructure of BR led to modification of the counterion residue E123 of ChR2 to threonine oralanine; the resulting faster opsin is referred toas ChETA (Gunaydin et al. 2010).</p>
+
                <p>3, Give pmyo2-chR2, pmyo2-chETA, pmyo2-ic1c2, pmyo3-chR2, pmyo3-chRTA and pmec4-dsred to company to test the sequences.</p>
−
        <p class="contentP">This substitution introduced two advantagesover wild-type ChR2. First, it reduced desensitization during light exposure, with the resultthat light pulses late in high-frequency trainsbecame as likely as early light pulses to drivespikes (a very important property referred to astemporal stationarity).</p>
+
               
−
        <p class="contentP">Second, it destabilizedthe active conformation of retinal, speedingspontaneous isomerization to the inactive stateafter light-off and thus closing the channelmuch more quickly after cessation of light thanwild-type or improved ChR2 variants. Theresulting functional consequences of ChETAmutations are temporal stationarity, reducedextra spikes, reduced plateau potentials, andimproved high-frequency spike followingat 200 Hz or more over sustained trains, even within intact mammalian brain tissue(Gunaydin et al. 2010)</p>
+
                <h5>August 4</h5>
−
       
+
                <p>1, Select single clone from culture plate (pmyo3-chETA.).And culture for 12h.</p>
−
        <div class="fivePx"></div>
+
                <p>2, Plasmid Extraction (pmyo3-chETA).</p>
−
        <center><img class="contentImg" src="https://static.igem.org/mediawiki/2015/8/82/China-Tongji-Project-figure2-4.jpg"></center>
+
                <p>3, Pfu PCR of pttx-3 from C.elegans genome.</p>
−
        <p class="imgIntroduction"><b>Figure 2-4:</b> Engineered channelrhodopsin-2 variant with faster deactivation kinetics, resulting in: (1) high-fidelity light-driven spiking over sustained trains at least up to 200 Hz; (2) reduced multiplets and plateau potentials; (3) faster recovery from inactivation, and (4) improved temporal stationarity of performance in sustained trains.</p>
+
                <p>4, AGE ( agarose gel electrophoresis ) of pfu PCR products.(pttx3) However, nothing out.</p>
−
        <div class="fivePx"></div>
+
                <p>5, Trying to ligate pmyo2-ic1c2 once again.Then transformation of it.Cultured in 37℃ for 16h.</p>
−
       
+
               
−
        <h3>2.3.3 iC1C2</h3>
+
                <h5>August 5</h5>
−
        <p class="contentP">Scientists have designed and characterized aclass of channelrhodopsins (originally cation-conducting) converted into chloride-conductinganion channels. These tools enable fast optical inhibition of action potentials and can beengineered to display step-function kinetics for stable inhibition, outlasting light pulses and fororders-of-magnitude-greater light sensitivity of inhibited cells.</p>  
+
                <p>1, Select single clone from culture plate (pmyo2-ic1c2).And culture for 12h.
−
        <p class="contentP">The engineered iC1C2 was designed based on the 2012 crystal structure of C1C2 to conduct chloride ions instead of cations, utilizing physiological chloride gradients to precisely inhibit action potentials in response to blue light. The resulting inhibition is much more light-sensitive than with prior optogenetic inhibitory tools and involves reversible input resistance changes. Light sensitivity of expressing cells is further improved. The channel pore is open and flow of chloride ions across the cell membrane is elevated between the blue and red light pulses, thereby greatly reducing spike probability in expressing neurons without the need for continuous light delivery.</p>
+
                <p>2, Plasmid extraction.( pmyo2-ic1c2)</p>
−
       
+
                <p>3, Give pmyo2-ic1c2 to company, and let it test the sequence.</p>
−
        <div class="fivePx"></div>
+
                <p>4, Pfu PCR of pttx-3 from C.elegans genome again. (use different program and different temperature,) </p>
−
        <center><img class="contentImg" width="400" src="https://static.igem.org/mediawiki/2015/3/34/China-Tongji-Project-figure2-5.jpg"></center>
+
                <p>5, AGE ( agarose gel electrophoresis ) of pfu PCR products.(pttx-3)</p>
−
        <p class="imgName" align="center"><b>Figure 2-5:</b> C1C2 structure, with the nineresidues mutated in C1C2_4x and C1C2_5x in orange.</p>
+
                <p>6, Gel extraction and recycle the pttx-3. (10ng/ul)</p>
−
        <div class="fivePx"></div>
+
               
−
       
+
                <h5>August 6</h5>
−
        <div class="fivePx"></div>
+
                <p>1, Digest of pttx-3 with SalI and BamHI. (using digestion protocol)</p>
−
        <center><img class="contentImg" width="300" src="https://static.igem.org/mediawiki/2015/c/c5/China-Tongji-Project-figure2-6.jpg"></center>
+
                <p>2, Digest of the new ppd95.77 vector with SalI and BamHI. (using digestion protocol)</p>
−
        <p class="imgName" align="center"><b>Figure 2-6:</b> C1C2’s best reaction situation.</p>
+
                <p>3, Ligate of pttx-3 into ppd95.77. (using ligation protocol)</p>
−
        <div class="fivePx"></div>
+
                <p>4, Transformation of pttx-3 in ppd95.77. culture in 37℃ for 16h.</p>
−
       
+
               
−
        <h3>2.3.4 Blink </h3>
+
                <h5>August 7</h5>
−
        <p class="contentP">A blue-light-induced K(+) channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+) selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+) equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities.</p>
+
                <p>1, Yesterday’s transformation has no clone grow.</p>
−
       
+
                <p>2, Pfu PCR of pttx-3 again.</p>
−
        <div class="fivePx"></div>
+
                <p>3, Design the primers of pmec-4. (add HindIII and BamHI)</p>
−
        <p class="titleTwo" id="second4">2.4 References</p>
+
                <p>4, AGE ( agarose gel electrophoresis ) of pfu PCR products.(pttx-3)</p>
−
        <p class="reference">[1] Deisseroth, K.; Feng, G.; Majewska, A. K.; Miesenbock, G.; Ting, A.; Schnitzer, M. J. (2006). "Next-Generation Optical Technologies for Illuminating Genetically Targeted Brain Circuits". Journal of Neuroscience 26 (41): 10380–6. doi:10.1523/JNEUROSCI.3863-06.2006. PMC 2820367. PMID 17035522.</p>
+
                <p>5, Gel extraction and recycle the pttx-3. (13ng/ul)</p>
−
        <p class="reference">[2] Mancuso, J. J.; Kim, J.; Lee, S.; Tsuda, S.; Chow, N. B. H.; Augustine, G. J. (2010). "Optogenetic probing of functional brain circuitry". Experimental Physiology 96 (1): 26–33. doi:10.1113/expphysiol.2010.055731. PMID 21056968.</p>
+
                <p>6, Ligate of pttx-3 into ppd95.77. (using ligation protocol)</p>
−
        <p class="reference">[3] The Microbial Opsin Familyof Optogenetic Tools; Feng Zhang,Johannes Vierock, Ofer Yizhar, Lief E. Fenno, Satoshi Tsunoda, Arash Kianianmomeni, et al.(2011) Cell147,1446-1457.</p>
+
                <p>7, Transformation of pttx-3 in ppd95.77. culture in 37℃ for 16h.</p>
−
        <p class="reference">[4] Lief Fenno,Ofer Yizharand Karl Deisseroth, 2011. The Development andApplication of Optogenetics ; Neurosci34:389–412.</p>
+
−
        <p class="reference">[5] http://web.stanford.edu/group/dlab/optogenetics/sequence_info.html.</p>
+
−
        <p class="reference">[6] Andre Berndt,Soo Yeun Lee,Charu Ramakrishnan, and Karl Deisseroth (2014); Structure-Guided Transformationof Channelrhodopsin into aLight-Activated Chloride Channel; SCIENCE 344,420-423.</p>
+
−
        <p class="reference">[7] http://web.stanford.edu/group/dlab/optogenetics/sequence_info.html.</p>
+
  
 +
                <h4>1.1.3.2 Week2 -- August 8~13</h4>
 +
                <h5>August 8</h5>
 +
                <p>1, Yesterday’s transformation has still no clone grow.</p>
 +
                <p>2, Have a lab meeting about why our ligation has so many problems. We decided to try a new method---seamless cloning.</p>
 +
               
 +
                <h5>August 9</h5>
 +
                <p>1, As we are going to do seamless cloning, we have to design new primers of pttx-3, chR2, chETA, dsRed and ic1c2, blink. </p>
 +
               
 +
                <h5>August 10</h5>
 +
                <p>1, Make LB liquid.Make LB AMP plates.</p>
 +
                <p>2, Taq PCR of pttx-3, chR2, chETA, dsRed and ic1c2, blink to test the best temperature of PCR reaction.</p>
 +
                <p>3, AGE ( agarose gel electrophoresis ) of taq PCR products. (pttx-3, chR2, chETA, dsRed and ic1c2)</p>
 +
                <p>4, Pfu PCR of pttx-3.</p>
 +
               
 +
                <h5>August 11</h5>
 +
                <p>1, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx-3)</p>
 +
                <p>2, Gel extraction and recycle the pttx-3. (17ng/ul)</p>
 +
                <p>3, Digest of ppd95.77 with SalI and BamHI.</p>
 +
                <p>4, Seamless clone of pttx-3 into nppd95.77. (use seamless clone protocol)</p>
 +
                <p>5, Transformation of pttx-3 ppd95.77. culture for 16h on AMP LB plate in 37℃.</p>
 +
               
 +
                <h5>August 12</h5>
 +
                <p>1, Select single clone on AMP LB plate.(pttx-3 ppd95.77) culture in 37℃ for 12h.</p>
 +
                <p>2, Plasmid extraction of pttx-3 ppd95.77.</p>
 +
                <p>3, Digest of pttx-3 with SalI and BamHI to test the ligation result. (turns out to be right!)</p>
 +
                <p>4, Transformation of pttx-3 ppd95.77 in order to get more right plasmids.</p>
 +
                <p>5, Pfu PCR of chR2, chETA, dsRed and ic1c2, blink.</p>
 +
                <p>6, AGE ( agarose gel electrophoresis ) of pfu PCR products. (chR2, chETA, dsRed and ic1c2, blink)</p>
 +
                <p>7, Gel extraction and recycle the chR2, chETA, dsRed and ic1c2, blink. (around 80ng/ul)</p>
 +
                <p>8, Digest 1.5 ul of pttx-3 ppd95.77.</p>
 +
                <p>9, Seamless clone of pttx-3 with chR2, chETA, dsRed, ic1c2 and blink. (seamless clone protocol)</p>
 +
                <p>10, Transformation of pttx-3-chR2, PTTX-3-chETA, PTTX-3-dsRed, pttx-3-ic1c2 and pttx-3-blink.Cultured in 37℃ for 16h.</p>
 +
               
 +
                <h5>August 13</h5>
 +
                <p>1, Select single clone of pttx-3-chR2, pttx-3-chETA, pttx-3-dsRed and pttx-3-ic1c2 (pttx-3-blink has no clone.). Cultured at 37℃ for 12h.</p>
 +
                <p>2, Plasmid extraction. (pttx-3-chR2, pttx-3-chETA, pttx-3-dsRed and pttx-3-ic1c2)</p>
 +
                <p>3, Digest of (pttx-3-chR2, pttx-3-chETA, pttx-3-dsRed and pttx-3-ic1c2 with BamHI and EcoRI to test if we had successfullyligate the gene into the vector, which turn out that these are all right.</p>
 +
                <p>4, Send them to company for a sequence test.</p>
  
−
        <p></p><div class="divider"></div>
+
                <h4>1.1.3.3 Week3 -- August 15~21</h4>
−
        <!-- 3. Project Design -->
+
                <h5>August 15</h5>
−
<p class="titleOne" id="ProjectDesign">3. Project Design</p>  
+
                <p>1, Pfu PCR of blink again.</p>
−
        <p class="titleTwo" id="third1">3.1 Introduction</p>
+
                <p>2, AGE ( agarose gel electrophoresis ) of pfu PCR products. (blink)</p>
−
        <p class="contentP">In this part, we will illustrate how we designed our project in a <b>Q&A </b>way.To help you understand our project better, this section will be divided into 3 parts: Plasmid Design, Equipment Design and Test Design.</p>
+
                <p>3, Gel extraction and recycle the blink. (around 50ng/ul)</p>
 +
                <p>4, Digest of pttx-3 ppd95.77 with BamHI and EcoRI.</p>
 +
                <p>5, Seamless clone of pttx-3-blink.</p>
 +
                <p>6, Transformation of pttx3-blink. Culture in 37℃ for 16h.</p>
 +
                <p>7, Taq PCR of ptwk16 to test the best reaction situation.</p>
 +
                <p>8, AGE ( agarose gel electrophoresis ) of taq PCR products. (ptwk16)</p>
 +
                <p>9, Pfu PCR of ptwk16.</p>
 +
               
 +
                <h5>August 16</h5>
 +
                <p>1, Select single clone of pttx-3-blink.Cultured at 37℃ for 12h.</p>
 +
                <p>2, Plasmid extraction of pttx-3-blink.</p>
 +
                <p>3, AGE ( agarose gel electrophoresis ) of pfu PCR products. (ptwk16)</p>
 +
                <p>4, Gel extraction and recycle the blink. (around33ng/ul)</p>
 +
                <p>5, Digest of ptwk16 with HindIII and SalI. Digest the vector ppd95.77 with HindIII and SalI.</p>
 +
                <p>6, Ligation of ptwk16 into ppd95.77 with T4 ligase. (12h)</p>
 +
                <p>7, AGE ( agarose gel electrophoresis ) of pttx-3-blink to test if we got the right result.</p>
 +
               
 +
                <h5>August 17</h5>
 +
                <p>1, Transformation of ptwk16 ppd95.77. culture in 37℃ for 16h.</p>
 +
                <p>2, Transformation of the back bone we made in order to get more to prepare for the later backbone making.Culture in 37℃ for 16h.</p>
 +
               
 +
                <h5>August 18</h5>
 +
                <p>1, Select single clone of ptwk16 ppd95.77. Cultured at 37℃ for 12h.</p>
 +
                <p>2, Select single clone of backbone.Cultured at 37℃ for 12h.</p>
 +
                <p>3, Plasmid extraction of ptwk16 ppd95.77 and backbone.</p>
 +
                <p>4, Digest of ptwk16 ppd95.77 with HindIII and SalI to test if we had ligated it in a right way.</p>
 +
               
 +
                <h5>Augest 19</h5>
 +
                <p>1, Pfu PCR of blink, chR2, dsred, ic1c2 and chETA.</p>
 +
                <p>2, AGE ( agarose gel electrophoresis ) of pfu PCR products. (blink, chR2, dsred, ic1c2 and chETA)</p>
 +
                <p>3, Gel extraction and recycle the blink. (around 50ng/ul)</p>
 +
                <p>4, Digest of ptwk16 ppd95.75 with BamHI and HindIII.</p>
 +
                <p>5, Seamless clone of ptwk16-blink, ptwk16-chR2, ptwk16-dsred, ptwk16-ic1c2 and ptwk16-chETA.</p>
 +
                <p>6, Transformation of ptwk16-blink, ptwk16-chR2, ptwk16-dsred, ptwk16-ic1c2 and ptwk16-chETA.Cultured in 37℃ for 16h.</p>
 +
               
 +
                <h5>August 20</h5>
 +
                <p>1, Select the single clone of ptwk16-blink, ptwk16-ic1c2 and ptwk16-chETA. (ptwk16-chR2, ptwk16-dsred has not been ligated successfully.) culture in 37℃ for 12h</p>
 +
                <p>2, plasmid extraction of ptwk16-blink, ptwk16-ic1c2 and ptwk16-chETA.</p>
 +
                <p>3, Digest of ptwk16-blink, ptwk16-ic1c2 and ptwk16-chETA with BamHI and EcoRI to test if we had ligated them in the right way. (It turns out to be right.)</p>
 +
                <p>4, Ligate of ptwk16-chR2, ptwk16-dsRed again.</p>
 +
                <p>5, Transformation of ptwk16-chR2, ptwk16-dsred.Culture in 37℃ for 16h.</p>
 +
               
 +
                <h5>August 21</h5>
 +
                <p>1, Select the single clone of ptwk16-blink, ptwk16-ic1c2 and ptwk16-chETA. (ptwk16-chR2, ptwk16-dsred) culture in 37℃ for 12h</p>
 +
                <p>2, Plasmid extraction of ptwk16-chR2, ptwk16-dsred.</p>
 +
                <p>3,digest of ptwk16-chR2, ptwk16-dsred with BamHI and EcoRI to test if the result is right.</p>
 +
                <p>4, Start to make backbone which we are going to send to iGEM. Design the seamless clone PCR primers of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA, pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.</p>
 +
                <p>5, Make some chloramphenicol LB plates.</p>
 
          
 
          
−
        <div class="fivePx"></div>
+
                <h4>1.1.3.4 Week4 -- August 22~28</h4>
−
        <p class="titleTwo" id="third2">3.2 General Design</p>
+
                <h5>August 22</h5>
−
        <h3>3.2.1 Q: WHY choose to control the locomotion of C.elegans?</h3>
+
                <p>1, Taq PCR of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA, pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA to test the best reaction situation.<p>
−
        <p class="contentP"><b>A:</b> At first we wanted to make out something FUNCY for people can see, so controlling the locomotion of C.elegans become the first choice. Besides, this work has some potential in treating paralyzed animal, even maybe treat people in the future.Right now, there are already researchers successfully made paralyzed mouse move it leg muscle again. </p>
+
                <p>2, AGE ( agarose gel electrophoresis ) of these taq PCR products.<p>
−
        <p class="contentP">In all kinds of expressions of locomotion, the study of forward and reverselocomotion serves as an entry into understanding theworm’s motor circuit. And we also tried to control the turning of C.elegans. If we can make the worms to go forward, go back and turn left or right, we may work out something like Snakylines, which is fancy and attractive.</p>
+
                <p>3, Pfu PCR of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.<p>
−
       
+
                <p>4, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.)<p>
−
        <h3>3.2.2 Q: HOW to control the locomotion of C.elegans?</h3>
+
                <p>5, Gel extraction and recycle the blink. (around 50ng/ul)<p>
−
        <p class="contentP"><b>A:</b> At first we have to find neurons or muscles which are related to the movement of C.elegans. Next step is trying to activate or restrain these neurons and muscles by expressing channelrhodopsin2 (chR2) or its improved versions. As we have illustrated in project background, chR2 is a channel which is located at the cell membrane. When use appropriate light toirradiate the worm, the particular tissue will be activated or restrained, and then the whole C.elegan will be controlled by the light.</p>
+
                <p>6, Digest of backbone with PstI and EcoRI.<p>
−
       
+
                <p>7, AGE ( agarose gel electrophoresis ) of digestion products. <p>
−
        <div class="fivePx"></div>
+
                <p>8, Gel extraction and recycle the blink. (around 30ng/ul)<p>
−
        <p class="titleTwo" id="third3">3.3 Plasmid Design</p>
+
               
−
        <h3>3.3.1 Q: How to express chR2 at the certainneurons and muscles we want?</h3>
+
                <h5>August 23</h5>
−
        <p class="contentP"><b>A:</b> We use specific promoter to drive the chR2 at the specific tissue. Besides, we also tried to use cre-loxp system for 2 promoters which can overlap at one single neuron. This may be a good way to express at single neuron. (Unfortunately, this experiment failed at last.) After reading papers, we choose 5 promoters at last.</p>
+
                <p>1, Seamless clone of backbone---pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.<p>
−
       
+
                <p>2, Transformation of backbone---pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.Culture on chloramphenicol LB plates in 37℃ for 16h.<p>
−
        <p class="contentP"><b>Pmyo-2:</b> Encodes a muscle-type specific myosin heavy chain isoform. Myo-2 is expressed in pharyngeal muscle. We supposed that we can use pmyo-2 because it expresses specifically in pharyngeal muscle, which may lead worm turning when irradiated by appropriate light.</p>
+
                <p>3, Select the single clone of backbone---pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA. Culture onchloramphenicol LB plates in 37℃ for 12h.<p>
−
        <p class="contentP"><b>Pmyo-3:</b> Encodes MHC A, the minor isoform of MHC (myosin heavy chain) that is essential for thick filament formation, and for viability, movement, and embryonic elongation.Expressed in body muscle, the somatic sheath cell covering the hermaphrodite gonad, and also expressed in enteric muscle, vulval muscles of the hermaphrodite and the diagonal muscles of the male tail. (from Wormatlas)
+
                <p>4, Pfu PCR of pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.<p>
−
        We decided to use pmyo-3 to construct a plasmid which can let our ChR2s express in worm’s body muscle which is directly related with worm movements.In this way, we may achieve our purpose.</p>
+
                <p>5, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.) However, nothing came out.<p>
−
        <p class="contentP"><b>Pttx-3:</b> Encodes a LIM homeodomain protein required for functions of the interneuron AIY. Expressed at AIY neuron only, in this case the targeted illumination system was used to stimulate AIY only when theworm’s head swung in a particular direction. This work provides new functional evidenceof the chemosensory circuit’s complexity and robustness, and is an example of ‘closed-loop’ optogeneticsstimulation based on behavior.</p>
+
               
−
        <p class="contentP"><b>Pmec-3:</b> Encodes a founding member of the LIM (Lin-11, Isl-1, Mec-3) homeodomain family of transcriptional regulators.During C. elegans development, mec-3 activity is required for proper differentiation and maturation of the mechanosensory neurons. Mec-3 is expressed in the mechanosensory neurons(from Wormatlas). We hope that this may make C.elegans move backward when we irradiate the appropriate light.</p>
+
                <h5>August 24</h5>
−
       
+
                <p>1, Plasmid extraction of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.<p>
−
        <h3>3.3.2 Q: WHY choose those rhodopsins?</h3>
+
                <p>2, Digest of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA to check if we had ligate the right parts into plasmid.<p>
−
        <p class="contentP"><b>A: For ChR2: (Excitation)</b></p>
+
                <p>3, Send them to company to test the sequence.<p>
−
        <p class="contentP">It is the most basic one,and at the same time is the easiest one for us to get. So we choose ChR2 to confirm that our experiment can be completed.</p>
+
                <p>4, Pfu PCR of pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA again. (change the reaction temperature.)<p>
−
        <p class="contentP"><b>For iC1C2 : (Inhibition)</b></p>
+
                <p>5, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.) pttx3-chR2, pttx3-dsred and ptwk16-chR2 show on the gel.<p>
−
        <p class="contentP">Activated by brief blue light stimulation at low intensities, remains open in the dark for an extended period of time and gets deactivated by red light. Without the need for continuous light delivery.</p>
+
                <p>6, Gel extraction and recycle the pttx3-chR2, pttx3-dsred and ptwk16-chR2.<p>
−
        <p class="contentP"><b>For ChETA:(Excitation)</b></p>
+
               
−
        <p class="contentP">(1) Faster deactivation kinetics;</p>
+
                <h5>August 25</h5>
−
        <p class="contentP">(2) High-fidelity light-driven spiking over sustained trains at least up to 200 Hz;</p>
+
                <p>1, Digest the backbone with EcoRI and PstI.<p>
−
        <p class="contentP">(3) Reduced multiples and plateau potentials;</p>
+
                <p>2, Seamless clone of backbone--- pttx3-chR2, pttx3-dsred and ptwk16-chR2.<p>
−
        <p class="contentP">(4) Faster recovery from inactivation, improved temporal stationarity of performance in sustained trains;</p>
+
                <p>3, Transformation of backbone---pttx3-chR2, pttx3-dsred and ptwk16-chR2.Cultured on chloramphenicol LB plates in 37℃ for 19h.<p>
−
        <p class="contentP">(5) Destabilized the active conformation of retinal, speeding spontaneous isomerization to the inactive state after light-off and thus closing the channel much more quickly after cessation of light than wild-type or improved ChR2 variants. </p>
+
               
−
       
+
                <h5>August 26</h5>
−
        <div class="fivePx"></div>
+
                <p>1, Onlybackbone-ptwk16-chR2 grown some clones on the plate. Select single clone on the plate. Cultured it on chloramphenicol LB plates in 37℃ for 19h.<p>
−
        <p class="titleTwo" id="third4">3.4 Equipment Design</p>
+
                <p>2, Plasmid extraction of backbone-ptwk16-chR2.<p>
−
        <h3>3.4.1 Q: Why should we choose LED light sources rather than ordinary light sources?</h3>
+
                <p>3, Digest of backbone-ptwk16-chR2 to make sure that we had made the right backbone. <p>
−
        <p class="contentP"><b>A:</b> In this program we use LED light sources instead of using optical filters. </p>
+
               
−
        <p class="contentP">Compared to other light sources, the LED light sources are easier to control. By using C4W cube, we can connect more than two different LEDs in one light path. So it means that we can change the light instantaneously without infecting the observation of our worms. </p>
+
                <h5>August 27</h5>
−
        <p class="contentP">At the same time, compared to the normal light sources, our light sources’ power is larger, which means that we can have a wider field of vision.</p>
+
                <p>1, Design new primers of pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.<p>
−
        <p class="contentP">LED has another advantage that LED is instant available, which means we needn’t to wait if we turn off it by accident. We can realize the flash mode (modulate pulse) due to this feature. </p>
+
                <p>2, As we couldn’t have PstI, EcoRI, SpeI and BamHI in our backbone plasmid, we have to do some point mutation.<p>
−
       
+
                <p>3, The overlap PCR of backbone---pmyo2-chR2, pmyo2-dsred, pmyo3-chR2 to mutate the PstI site in them.<p>
−
        <h3>3.4.2 Q: Why should we refit our LED from 1W to 5W?</h3>
+
               
−
        <p class="contentP"><b>A:</b> The LED which we choose originally is 1W, whose power is larger than ordinary light sources. But we choose chance our LED from 1W to 5W, which means that when we testing the reaction of our C.elegent, we can have a wider field of vision. So that we can observe it for a long time which benefit to our analyzation later. </p>
+
                <h5>August 28</h5>
−
        <p class="contentP">After the refit, we find that the heat dispersion is still very well, which means that it won’t affect the time we use of the LED.</p>
+
                <p>1, The overlap PCR of backbone---pmyo2-chR2, pmyo2-dsred, pmyo3-chR2 to mutate the PstI site in them.<p>
−
       
+
                <p>2, Pfu PCR of pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA using new primers to test the best reaction situation.<p>
−
        <h3>3.4.3 Q: Why should we choose DC2100 as our LED driver instead of normal LED driver?</h3>
+
                <p>3, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA)<p>
−
        <p class="contentP"><b>A:</b> The DC2100 is advanced version of LED driver. It has a current-limiting program to avoid the LED from being damaged. </p>
+
                <p>4, Pfu PCR of pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA using new primers.</p>
−
        <p class="contentP">Compared to ordinary LED drivers, the DC2100 can control the current more accurately, which means that we can test the optimum light intensity to active or repress the worms.</p>
+
−
        <p class="contentP">By using DC2100, we can modulate pulse which other LED drivers couldn’t realize.</p>
+
−
        <div class="fivePx"></div>
+
−
        <center><img class="contentImg" src="https://static.igem.org/mediawiki/2015/4/47/China-Tongji-Project-ProjectDesign-figure1.jpg" ></center>
+
−
        <p class="imgName" align="center"><b>Figure 3-1:</b> DC2100 VS traditional LED Driver</p>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <p class="titleTwo" id="third5">3.5 Test Design</p>
+
−
        <h3>3.5.1 Q: why should we standardize our test method?</h3>
+
−
        <p class="contentP"><b>A:</b> To evaluate the reaction of these gene modified worms, we find some different aspects to observe them which are the trace, the speed and its angle when the C.elegent makes a turn. So standardize the video is very important for us to analyze the speed and the trace.</p>
+
−
        <p class="contentP">So we use 5-10-10 routine to make the video of the worms, so that it can benefit our analyzation later.</p>
+
−
       
+
−
        <h3>3.5.2 Q: What is 5-10-10 routine? Why should we use this style?</h3>
+
−
        <p class="contentP"><b>A:</b> The 5-10-10 routine means that the first 5 seconds leave the worm in white light, after that give it a 10 second of LED light, at last leave it in white light for about 10 seconds or more. The 5-10-10 routine is better for us to analyze the speed of those worms. And the first 5 seconds white light is use to observe the normal behavior of the worms which can make comparison to the following period. The third period is use to observe how long the worm can get right. </p>
+
−
       
+
−
        <h3>3.5.3 Q: Why should we analyze the trace of the C.elegent?</h3>
+
−
        <p class="contentP"><b>A:</b> The trace of the c.elegent is very useful to our project. It can show the movement of these worms visually. We can find the worm keep going or turn left/right or stop even recede during we give the light. After combine the trace with the coordinate, we can change the graphic information into digital information which is easier for us to analyze. </p>
+
−
       
+
−
        <h3>3.5.4 Q: What kind of software do we use to record the reaction of worms? Why?</h3>
+
−
        <p class="contentP"><b>A:</b> To record the behavior of the worms, we use DP7200 camera and software called Biolife DP to make a video. Compare to those ordinary cameras, DP7200 can change the color temperature of the background. It means that no matter what color the background is, we can always change it to a white background relatively. This can make sure that we can have a high quality video to analyze.</p>
+
−
       
+
−
        <h3>3.5.5 Q: Why should we use a red glassine paper to filtering the white light when testing the worms?</h3>
+
−
        <p class="contentP"><b>A:</b> The white light contains all kinds of light qualities include the blue light or green light. Using the red glassine paper is to make sure our worms will not infect by the background lights.</p>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <p class="titleTwo" id="third6">3.6 Reference</p>
+
−
        <p class="reference">[1]Steven J. Husson, Alexander Gottschalkand Andrew M. Leifer；Optogenetic manipulation of neuralactivity inC. elegans:Fromsynapseto circuits and behaviour；Biol. Cell (2013)105, 235–250DOI:10.1111/boc.201200069.</p>
+
−
        <p class="reference">[2]Andre Berndt, Soo Yeun Lee, Charu Ramakrishnan, and Karl Deisseroth (2014); Structure-Guided Transformation of Channelrhodopsin into a Light-Activated Chloride Channel; SCIENCE 344,420-423.</p>
+
−
        <p class="reference">[3]LiefFenno, OferYizhar and Karl Deisseroth, 2011. The Development and Application of Optogenetics ;Neurosci 34: 389–412.</p>
+
  
−
        <p></p><div class="divider"></div>
+
                <h4>1.1.3.5 Week5 -- August 29~31</h4>  
−
<!-- 4. Protocol -->
+
                <h5>August 29</h5>
−
<p class="titleOne" id="Protocol">4. Protocol</p>  
+
                <p>1, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA) we got pttx3-blink and ptwk16-ic1c2.</p>
−
        <p class="titleTwo" id="fourth1">4.1 Introduction</p>
+
                <p>2, Gel extraction and recycle pttx3-blink and ptwk16-ic1c2.</p>
−
        <p class="contentP">Our project aims to control C.elegans’ movement by expressing chR2 in their muscle and neuron. In our plan, we will make over 20 parts of 3 kinds of channelrhodopsins with 5 different promoters.</p>
+
                <p>3, Digest of backbone with PstI and EcoRI.</p>
−
        <p class="contentP">First of all, we need to design the PCR primers with primer 5.Then we run taq PCR or pfu PCR to get our parts out of the C.elegans’ genome or plasmids.After that, we do the digestion of gene parts and vector pPD95.77. Use traditional method to do the ligation and transformation. Besides, we also use seamless cloning to deal with some difficult ligations. The last step in molecular construction is plasmid extraction.</p>
+
                <p>4, Seamless clone of backbone-pttx3-blink and ptwk16-ic1c2.</p>
−
<p class="contentP">Then we come to the C.elegans part, which includes microinjection, making NGM and ATR plates and seed plates. All the details will show below.</p>
+
                <p>5, Transfomation of backbone-pttx3-blink and ptwk16-ic1c2.Culture in 37℃ for 19h.</p>
 +
                <p>6, Point mutation of our backbone products.</p>
 +
               
 +
                <h5>August 30</h5>
 +
                <p>1, Select the single clone of ptwk16-ic1c2 on chloramphenicol LB plates. (another has no clone) cultured in 37℃ for 19h.</p>
 +
                <p>2, Point mutation of our backbone products.</p>
 +
                <p>3, Point mutation of our backbone products.</p>
 +
               
 +
                <h5>August 31</h5>
 +
                <p>1, Plasmid extraction of ptwk16-ic1c2.</p>
 +
                <p>2, Digest ptwk16-ic1c2 with PstIEcoRI to check our result.</p>
 +
                <p>3, Point mutation of our backbone products.</p>
 +
         
 +
            </div>
 +
        </div>  
 
         <div class="fivePx"></div>
 
         <div class="fivePx"></div>
−
        <p class="titleTwo"  id="fourth2">4.2 Taq PCR</p>
+
         <div class="slidePanel slidePanel1">
−
        <p class="contentP">(1) Put dNTP, primers, template, taqbuffer and taq enzyme on ice;</p>
+
            <h3 class="slideTitle" id="slideTitle1dot4">1.1.4 SEPTEMBER -- Week 1~2 -- September 1~10<span class="expand">[Expand]</span></h3>
−
        <p class="contentP">(2) Prepare the mix liquid:</p>
+
             <div class="slideBlock" id="slideBlock1dot4">
−
         <div class="fivePx"></div>
+
                <h4>1.1.4.1 Week1 -- September 1~7</h4>
−
        <table class="myTable" align="center" border="3">
+
                <h5>September 1</h5>
−
        <tr><th>Experimental Material</th><th>Dose</th></tr>
+
                <p>1, Pfu PCR of pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA for the last time.</p>
−
        <tr><td>Template</td><td>1ul</td></tr>
+
                <p>2, Point mutation of our backbone products. (Some of them failed.)</p>
−
            <tr><td>Primer-Front</td><td>1ul</td></tr>
+
               
−
            <tr><td>Primer-Reverse</td><td>1ul</td></tr>
+
                <h5>September 2</h5>
−
            <tr><td>dNTPs</td><td>4ul</td></tr>
+
                <p>1, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA) we got ptwk16-blink and ptwk16-chETA.</p>
−
            <tr><td>Taq PCR buffer</td><td>5ul</td></tr>
+
                <p>2, Gel extraction and recycleptwk16-blink and ptwk16-chETA.</p>
−
            <tr><td>taq enzyme</td><td>0.25ul</td></tr>
+
                <p>3, Send all the backbone we have now to company to test the sequence.</p>
−
            <tr><td>ddH2O</td><td>37.75ul</td></tr>
+
               
−
            <tr><td>Total volume</td><td>50ul</td></tr>
+
                <h5>September 3</h5>
−
        </table>
+
                <p>1, Digest of backbone with PstI and EcoRI.</p>
−
        <div class="fivePx"></div>
+
                <p>2, Seamless cloning of backbone-ptwk16-blink and ptwk16-chETA.</p>
−
        <p class="contentP">(3) Mix solution well;</p>
+
                <p>3, Transformation into chloramphenicol LB plates.Cultured in 37℃ for 19h. </p>
−
        <p class="contentP">(4) Use the PCR machine and amplification the gene:</p>
+
                <p>4, Make more LB AMP plates.</p>
−
        <div class="fivePx"></div>
+
               
−
        <table class="myTable" align="center" border="3">
+
                <h5>September 4</h5>
−
        <tr><th>Method</th><th colspan="2">Time</th></tr>
+
                <p>1, Select single clone on the plate. Culture in 37℃ for 13h.</p>
−
        <tr><td>95℃ pre-denaturation</td><td colspan="2">10min</td></tr>
+
                <p>2, Plasmid extraction of backbone-ptwk16-blink and ptwk16-chETA.</p>
−
            <tr><td>95℃ denaturation</td><td>30s</td><td rowspan="3">35cycles</td></tr>
+
                <p>3, Digest of backbone-ptwk16-blink and ptwk16-chETA to test. The result is strange which means that we may failed in this ligation.</p>
−
            <tr><td>60℃ anneal</td><td>30s</td></tr>
+
               
−
            <tr><td>72℃ extend</td><td>1min</td></tr>
+
                <h5>September 5</h5>
−
            <tr><td>4℃ save</td><td colspan="2">end</td></tr>
+
                <p>1, Try a new method to make the failed backbones.Digest the backbone-pmyo2-blink and pmyo2-chETA with BamHI and SpelI.Digest the ptwk16 out of the plasmid with HindIII and SalI.</p>
−
        </table>
+
                <p>2, Pfu PCR of ptwk16.</p>
−
        <div class="fivePx"></div>
+
                <p>3, AGE ( agarose gel electrophoresis ) of pfu PCR products. (ptwk16)</p>
−
        <div class="fivePx"></div>
+
                <p>4, Gel extraction and recycleptwk16.</p>
−
        <p class="titleTwo"  id="fourth3">4.3 Pfu PCR</p>
+
               
−
        <p class="contentP">(1) Put dNTP, primers, template,pfubuffer andpfuenzyme on ice;</p>
+
                <h5>September 6</h5>
−
        <p class="contentP">(2) Prepare the mix liquid:</p>
+
                <p>1, Traditional ligation of backbone-ptwk16-blink and ptwk16-chETA.</p>
−
        <div class="fivePx"></div>
+
                <p>2, Transformation of backbone-ptwk16-blink and ptwk16-chETA.Culture in 37℃ for 13h</p>
−
        <table class="myTable" align="center" border="3">
+
               
−
        <tr><th>Experimental Material</th><th>Dose</th></tr>
+
                <h5>September 7</h5>
−
        <tr><td>Template</td><td>1ul</td></tr>
+
                <p>1, Select single clone on the plate of backbone-ptwk16-blink and ptwk16-chETA. Culture in 37℃ for 13h.</p>
−
            <tr><td>Primer-Front</td><td>2.5ul</td></tr>
+
                <p>2, Plasmid extraction of backbone-ptwk16-blink and ptwk16-chETA.</p>
−
            <tr><td>Primer-Reverse</td><td>2.5ul</td></tr>
+
                <p>3, Digest test.</p>
−
            <tr><td>dNTPs</td><td>5ul</td></tr>
+
−
            <tr><td>5*Loading Buffer</td><td>10ul</td></tr>
+
−
            <tr><td>Pfu DNA polymerase</td><td>1ul</td></tr>
+
−
            <tr><td>ddH2O</td><td>27ul</td></tr>
+
−
            <tr><td>Total volume</td><td>50ul</td></tr>
+
−
        </table>
+
−
        <div class="fivePx"></div>
+
−
        <p class="contentP">(3) Mix solution well;</p>
+
−
        <p class="contentP">(4) Use the PCR machine and amplification the gene:</p>
+
−
        <div class="fivePx"></div>
+
−
        <table class="myTable" align="center" border="3">
+
−
        <tr><th>Method</th><th colspan="2">Time</th></tr>
+
−
        <tr><td>95℃ pre-denaturation</td><td colspan="2">10min</td></tr>
+
−
             <tr><td>95℃ denaturation</td><td>30s</td><td rowspan="3">35cycles</td></tr>
+
−
            <tr><td>60℃ anneal</td><td>30s</td></tr>
+
−
            <tr><td>72℃ extend</td><td>1min</td></tr>
+
−
            <tr><td>4℃ save</td><td colspan="2">end</td></tr>
+
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        </table> 
+
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        <div class="fivePx"></div>   
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        <div class="fivePx"></div>
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−
        <p class="titleTwo" id="fourth4">4.4 AGE ( agarose gel electrophoresis )</p>
+
−
        <p class="contentP">(1) Make of gel with 0.5g agarose and 50ml 10X TAE, add 2 drops of EB to dye the gel;</p>
+
−
        <p class="contentP">(2) Mix the PCR sample with 10x loading buffer; </p>
+
−
        <p class="contentP">(3) 220V 30min;</p>
+
−
        <p class="contentP">(4) Use UV light to view the result.</p>
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<div class="fivePx"></div>
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        <p class="titleTwo" id="fourth5">4.5 Gel extraction</p>
+
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        <p class="contentP"></p>
+
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        <p class="contentP">(1) Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. In this application, the weight of gel is regarded as equivalent to the volume. For example, 100 mg of gel is equivalent to a 100 μl volume. Transfer the gel slice into a 1.5 ml microfuge tube. </p>
+
−
        <p class="contentP">(2) Add a 3x sample volume of Buffer DE-A. </p>
+
−
        <p class="contentP">(3) Resuspend the gel in Buffer DE-A by vortexing. Heat at 75°C until the gel is completely dissolved (typically, 6-8 minutes) Heat at 40°C if low-melt agarose gel is used. Intermittent vortexing (every 2-3 minutes) will accelerate gel solubilization. </p>
+
−
        <p class="contentP">(4) Add 0.5x Buffer DE-A volume of Buffer DE-B, mix. If the DNA fragment is less than 400 bp, supplement further with a 1x sample volume of isopropanol. </p>
+
−
        <p class="contentP">Example: For a 1% gel slice equivalent to 100 μl, add the following: </p>
+
−
        <p class="contentP">• 300 μl Buffer DE-A </p>
+
−
        <p class="contentP">• 150 μl Buffer DE-B </p>
+
−
        <p class="contentP">If the DNA fragment is < 400 bp, you would also add: </p>
+
−
        <p class="contentP">• 100 μl of isopropanol. </p>
+
−
        <p class="contentP">(5) Place a Miniprep column into a 2 ml microfuge tube (provided) Transfer the solubilized agarose from Step 4 into the column. Centrifuge at 12,000xg for 1 minute. </p>
+
−
        <p class="contentP">(6) Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μl of Buffer W1. Centrifuge at 12,000xg for 30 seconds.</p>  
+
−
        <p class="contentP">(7) Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μl of Buffer W2. Centrifuge at 12,000xg for 30 seconds. </p>
+
−
        <p class="contentP">(8) Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μl aliquot of Buffer W2 and centrifuge at 12,000xg for 1 minute. </p>
+
−
        <p class="contentP">(9) Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000xg for 1 minute. </p>
+
−
        <p class="contentP">(10) Transfer the Miniprep column into a clean 1.5 ml microfuge tube (provided) To elute the DNA, add 25-30 μl of Eluent or deionized water to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12,000xg for 1 minute.</p>
+
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<div class="fivePx"></div>
+
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        <p class="titleTwo" id="fourth6">4.6 Digestion & ligation</p>
+
−
        <h3>4.6.1 Digestion</h3>
+
−
        <p class="contentP">(1) Add enzyme A 1ul and enzyme B 1ul;</p>
+
−
        <p class="contentP">(2) Add plasmid 4ul or gene 10ul;</p>
+
−
        <p class="contentP">(3) Add buffer 2ul;</p>
+
−
        <p class="contentP">(4) Add enough water;</p>
+
−
        <p class="contentP">(5) 37℃ 2h;</p>
+
−
        <p class="contentP">(6) Do agarose gel electrophoresis;</p>
+
−
        <p class="contentP">(7) Gel extraction.</p>
+
−
        <h3>4.6.2 Ligase reaction</h3>
+
−
        <p class="contentP">(1) Add 1ul ligase;</p>
+
−
        <p class="contentP">(2) Add 2ul ligase buffer;</p>
+
−
        <p class="contentP">(3) Add 10ul gene that have digested;</p>
+
−
        <p class="contentP">(4) Add 3ul digested plasmid;</p>
+
−
        <p class="contentP">(5) Add water;</p>
+
−
        <p class="contentP">(6) 12℃ 8h.</p>
+
−
<div class="fivePx"></div>
+
−
        <p class="titleTwo" id="fourth7">4.7 Seamless cloning</p>
+
−
        <p class="contentP">The design of primers of PCR amplification for cloning of your sequence of interest is based on the same principles as the design of PCR primers for any sequence. The only difference is that simply add the 14-18 bases of vector sequence to the 5’end of your sequence-specific PCR primers when designing primers. After PCR clean up, the resulting PCR- amplified insert is ready for Fast Seamless Cloning.</p>
+
−
        <p class="contentP">(1) Digest the vector with two enzymes;</p>
+
−
        <p class="contentP">(2) Set up fast seamless gene cloning reaction: 7.5ul seamless cloning enzyme mix with 1ul linearizedvector and 1.5ul gene;</p>
+
−
        <p class="contentP">(3) 42℃ 30min;</p>
+
−
        <p class="contentP">(4) Transformation.</p>
+
−
<div class="fivePx"></div>
+
−
        <p class="titleTwo" id="fourth8">4.8 Transformation</p>
+
−
        <p class="contentP">(1) Get competence E.coli from -80C fridge;</p>
+
−
        <p class="contentP">(2) Add 15ul plasmid liquid into competence E.coli, put it in ice water for 15-30min. then give it a 42℃ heat shock for 90 sec. finally put it out of the 42℃ water bath as quick as possible. Put it into ice water for 5 min;</p>
+
−
        <p class="contentP">(3) Add 500ul LB into competence E.coli;</p>
+
−
        <p class="contentP">(4) At 37℃ we train them for 1h;</p>
+
−
        <p class="contentP">(5) Add 100ul into a LB plate which has already added ampicillin. Put them in the 37C incubator for 14h-16h.</p>
+
−
        <div class="fivePx"></div>
+
−
        <p class="titleTwo" id="fourth9">4.9 Plasmid Extraction</p>
+
−
        <p class="contentP">(1) Put the bacterium liquid in the EP tube, Centrifuge at 12100rpm for 1 minute at room temperature;</p>
+
−
        <p class="contentP">(2) Pour out the supernatant as clean as possible;</p>
+
−
        <p class="contentP">(3) Add 150 ul P1 to the bacteria sediment, suspend the bacteria in P1 buffer;</p>
+
−
        <p class="contentP">(4) Add 150 ul P2, and shake the tube gentle 6-8 times until the liquid become in clear purple color;</p>
+
−
        <p class="contentP">(5) Add 350 ul P5, and shake it quickly for 12-20 times;</p>
+
−
        <p class="contentP">(6) Centrifuge at 12100rpm for 2 minutes at room temperature;</p>
+
−
        <p class="contentP">(7) Transfer 700 ul of the mixture(from Step 6) into a clean DNA Mini Column assembled in a 2ml collection tube(provided) Centrifuge at 12100rpm for 2 min at room temperature to pass solution through column;</p>
+
−
        <p class="contentP">(8) Pour out the liquid;</p>
+
−
        <p class="contentP">(9) Add 300ul PWT in the column, Centrifuge at 12100rpm for 2 min at room temperature to pass solution through column;</p>
+
−
        <p class="contentP">(10) Pour out the liquid and centrifuge again at 12100rpm for 1 min at room temperature;</p>
+
−
        <p class="contentP">(11) Place column into a new clean 1.5ml micro-centrifuge tube. Add 50ul 50℃ ddH2O directly onto the column matrix and centrifuge at 12100rpm for 2min to elute DNA;</p>
+
−
        <p class="contentP">(12) Exam the OD.</p>
+
−
        <div class="fivePx"></div>
+
−
        <p class="titleTwo" id="fourth10">4.10 Microinjection</p>
+
−
        <h3>4.10.1 Equipment</h3>
+
−
        <p class="contentP">(1) Injection table;</p>
+
−
        <p class="contentP">(2) Inverted DIC microscope;( Zeiss Observe.A1)</p>
+
−
        <p class="contentP">(3) Micromanipulator;(Zeiss)</p>
+
−
        <p class="contentP">(4) Pressurized injection system with needle holder;</p>
+
−
        <p class="contentP">(5) Needle puller. Sutter instruments MODEL P-1000 micropipette pullers.</p>
+
−
        <h3>4.10.2 Materials</h3>
+
−
        <p class="contentP">(1) Microinjection needles;</p>
+
−
        <p class="contentP">(2) Injection pads.(Bring 2% agarose in water to a boil, mix well, and place in a heat block. Using a broken Pasteur pipette or a cut-off P200 tip, place a drop (~100ul) of hot agarose onto a #1, 50X22-mm glass coverslip. Quickly place a second coverslip on the drop and lightly tap it. Alternatively, place several drops on the first coverslip, which should merge and mostly cover the surface after adding the second coverslip;)</p>
+
−
        <p class="contentP">(3) Injection oil. Series 700 Halocarbon oil;</p>  
+
−
        <p class="contentP">(4) Worm pick;</p>
+
−
        <p class="contentP">(5) M9 buffer;</p>
+
−
        <p class="contentP">(6) Worms; (Well-fed, young to middle-aged (≥1day old) gravid hermaphrodites with a full but single row of eggs.)</p>
+
−
        <p class="contentP">(7) Needle-loading pipettes. </p>
+
−
        <h3>4.10.3 Method</h3>
+
−
        <p class="contentP">(1) Fill a needle-loading pipette by capillary action with ≥ 1 ul of DNA injection mix;</p>
+
−
        <p class="contentP">(2) Insert the pipette tip in through the back of the injection needle, and expel injection mix onto the needle's internal filament;</p>
+
−
        <p class="contentP">(3) Place a loaded needle into the needle holder and mount on the manipulator;</p>
+
−
        <p class="contentP">(4) Position the needle so that the tip is in the center of the microscope's field of view using the 5X objective;</p>
+
−
        <p class="contentP">(5) Place a drop of oil on an injection pad and place under a dissecting microscope on top of a small Petri plate cover;</p>
+
−
        <p class="contentP">(6) Scoop one to several worms from a bacteria-free region of an NGM plate with a naked pick and transfer to the oil drop. Avoid contact with the worm's head. Alternatively, first touch the worm pick to the oil, and use the oil droplet to pick up the animals from a bacteria-free region. The idea is to minimize transfer of bacteria to the pad;</p>
+
−
        <p class="contentP">(7) Flame then cool the worm pick and use it to position the worms in the oil drop, and to gently push them down onto the pad. Orient the worms in rows with their ventral sides facing the same direction (opposite the needle direction) If the worms fail to adhere to the pad, move to a new location or rub the bodies with the pick to remove water or bacteria droplets. If adherence is still a problem re-bake the pads or use thicker or higher concentration agarose pads (see above)</p>
+
−
        <p class="contentP">(8) Transfer the slide face-up onto the microscope stage. Center the first worm to be injected and focus using the 5X objective. Move the needle down and in close proximity to the dorsal surface of the first animal. Switch to the 40X objective and focus on the worm;</p>
+
−
        <p class="contentP">(9) First make sure the needle is flowing;</p>
+
−
        <p class="contentP">(10) Insert the needle into the worm;</p>
+
−
        <p class="contentP">(11) Inject the DNA solution;</p>
+
−
        <p class="contentP">(12) Recover the worms: (Return the coverslip to the dissecting scope, and add a drop (~20 ul) of recovery buffer on the worms.)</p>
+
−
        <h3>4.10.4 Preparation of NGM plates</h3>
+
−
        <h4>4.10.4.1 Equipment and Reagents</h4>
+
−
        <p class="contentP">• NaCl</p>
+
−
        <p class="contentP">• Agar</p>
+
−
        <p class="contentP">• Peptone</p>
+
−
        <p class="contentP">• 5 mg/ml cholesterol in ethanol (Do not autoclave!)</p>
+
−
        <p class="contentP">• 1 M KPO4 buffer pH 6.0 (108.3 g KH2PO4, 35.6 g K2HPO4, H2O to 1 litre)</p>
+
−
        <p class="contentP">• 1M MgSO4</p>
+
−
        <p class="contentP">• Petri plates</p>
+
−
        <p class="contentP">• Peristaltic pump</p>
+
−
        <h4>4.10.4.2 Methods</h4>
+
−
        <p class="contentP">(1) Mix 3 g NaCl, 17 g agar, and 2.5 g peptone in a 2 litre Erlenmeyer flask. Add 975 ml H2O. Cover mouth of flask with aluminium foil. Autoclave for 50 min;</p>
+
−
        <p class="contentP">(2) Cool flask in 55°C water bath for 15 min;</p>
+
−
        <p class="contentP">(3) Add 1 ml 1 M CaCl2, 1 ml 5 mg/ml cholesterol in ethanol, 1 ml 1 M MgSO4
+
−
        and 25 ml 1 M KPO4 buffer.Swirl to mix well;</p>
+
−
        <p class="contentP">(4) Using sterile procedures, dispense the NGM solution into petri plates using a peristaltic pump. Fill plates 2/3 full of agar;</p>
+
−
        <p class="contentP">(5) Leave plates at room temperature for 2-3 days before use to allow for detection of contaminants, and to allow excess moisture to evaporate. Plates stored in an air-tight container at room temperature will be usable for several weeks.</p>
+
−
        <h4>4.10.4.3 Seeding NGM plates</h4>
+
−
        <p class="contentP">Using sterile technique, apply approximately 0.05 ml of E. coli OP50 liquid culture to small or medium NGM plates or 0.1 ml to large NGM plates using a pipet. If desired, the drop can be spread using the pipet tip or a glass rod. Spreading will create a larger lawn, which can aid in visualizing the worms. Take care not to spread the lawn all the way to the edges of the plate; keep the lawn in the center. The worms tend to spend most of the time in the bacteria. If the lawn extends to the edges of the plate the worms may crawl up the sides of the plate, dry out and die. Allow the E. coli OP50 lawn to grow overnight at room temperature or at 37°C for 8 hours (cool plates to room temperature before adding worms) Seeded plates stored in an air-tight container will remain usable for 2-3 weeks.</p>
+
−
        <h3>4.10.5 Seeding ATR NGM plates</h3>
+
−
        <p class="contentP">Add 1.75ul 5uM ATR into 1 ml E.coli OP50 liquid.1ml liquid can seed 10 small NGM plates.</p>      
+
  
−
        <p></p><div class="divider"></div>       
+
                <h4>1.1.4.2 Week2 -- September 9~10</h4>
−
<!-- 5. Summary and Result -->
+
                 <h5>September 9</h5>
−
        <p class="titleOne" id="SummaryResult">5. Summary and Result</p> 
+
                 <p>1, Make our sending plasmid powder by freeze dryer.</p>
−
        <div class="fivePx"></div>
+
                  
−
        <p class="titleTwo" id="fifth1">5.1 Plasmids Construction</p>
+
                 <h5>September 10</h5>
−
        <p class="chartName">Chart 5-1: plasmids we build in our project</p>
+
                 <p>1, Make our sending plasmid powder by freeze dryer.</p>
−
        <div class="fivePx"></div>
+
−
        <table class="myTable" id="tableParts" align="center" border="3">
+
−
            <tr>
+
−
                <th>Promoter/Protein</th>
+
−
                <th>ChR2-YFP</th>
+
−
                <th>ChETA-EYFP</th>
+
−
                <th>ic1c2-TS-EYFP</th>
+
−
                <th>blink</th>
+
−
                <th>dsRed</th>       
+
−
            </tr>
+
−
            <tr>
+
−
                <th>Pmyo-2</th>
+
−
                <td class="tdTick" id="tableL1R1">    </td>
+
−
                <td class="tdTick" id="tableL1R2">    </td>
+
−
                <td class="tdTick" id="tableL1R3">    </td>
+
−
                <td class="tdTick" id="tableL1R4">    </td>
+
−
                <td class="tdTick" id="tableL1R5">    </td>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>Pmyo-3</th>
+
−
                <td class="tdTick" id="tableL2R1">    </td>
+
−
                <td class="tdTick" id="tableL2R2">    </td>
+
−
                <td class="tdTick" id="tableL2R3">    </td>
+
−
                <td class="tdTick" id="tableL2R4">    </td>
+
−
                <td class="tdTick" id="tableL2R5">    </td>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>Pttx-3</th>
+
−
                 <td class="tdTick" id="tableL3R1">     </td>
+
−
                 <td class="tdTick" id="tableL3R2">     </td>
+
−
                 <td class="tdTick" id="tableL3R3">    </td>
+
−
                 <td class="tdTick" id="tableL3R4">     </td>
+
−
                 <td class="tdTick" id="tableL3R5">    </td>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>Ptwk-16</th>
+
−
                <td class="tdTick" id="tableL4R1">    </td>
+
−
                <td>    </td>
+
−
                <td class="tdTick" id="tableL4R3">    </td>
+
−
                <td>    </td>
+
−
                <td class="tdTick" id="tableL4R5">    </td>
+
−
            </tr>   
+
−
        </table> 
+
−
        <div class="fivePx"></div>
+
−
        <p class="chartName">( When you move your mouse on to the tick part, you may see our plasmids’ maps below. )</p>
+
−
        <div class="fivePx"></div>
+
−
        <div class="equipmentPic">
+
−
        <div class="partsPicDiv" id="partsL1R1">
+
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/a/ab/China-Tongji-Project-parts-pmyo2-chR2-YFP.png" alt="pmyo2-chR2-YFP"/>
+
−
                <p class="imgWords">pmyo2-chR2-YFP</p>
+
 
             </div>
 
             </div>
−
            <div class="partsPicDiv" id="partsL1R2">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/b/b9/China-Tongji-Project-parts-pmyo2-chETA-EYFP.png" alt="pmyo2-chETA-EYFP"/>
 
−
                <p class="imgWords">pmyo2-chETA-EYFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL1R3">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/5/5d/China-Tongji-Project-parts-pmyo2-iC1C2-TS-EYFP.png" alt="pmyo2-iC1C2-TS-EYFP"/>
 
−
                <p class="imgWords">pmyo2-iC1C2-TS-EYFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL1R4">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/8/8b/China-Tongji-Project-parts-pmyo2-blink.png" alt="pmyo2-blink"/>
 
−
                <p class="imgWords">pmyo2-blink</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL1R5">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/7/76/China-Tongji-Project-parts-pmyo2-dsred.png" alt="pmyo2-dsred"/>
 
−
                <p class="imgWords">pmyo2-dsred</p>
 
−
            </div>
 
−
           
 
−
            <div class="partsPicDiv" id="partsL2R1">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/5/52/China-Tongji-Project-parts-pmyo3-chR2-YFP.png" alt="pmyo3-chR2-YFP"/>
 
−
                <p class="imgWords">pmyo3-chR2-YFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL2R2">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/d/d2/China-Tongji-Project-parts-pmyo3-chETA-EYFP.png" alt="pmyo3-chETA-EYFP"/>
 
−
                <p class="imgWords">pmyo3-chETA-EYFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL2R3">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/2/21/China-Tongji-Project-parts-pmyo3-iC1C2-TS-EYFP.png" alt="pmyo3-iC1C2-TS-EYFP"/>
 
−
                <p class="imgWords">pmyo3-iC1C2-TS-EYFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL2R4">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/c/c9/China-Tongji-Project-parts-pmyo3-blink.png" alt="pmyo3-blink"/>
 
−
                <p class="imgWords">pmyo3-blink</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL2R5">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/6/69/China-Tongji-Project-parts-pmyo3-dsred.png" alt="pmyo3-dsred"/>
 
−
                <p class="imgWords">pmyo3-dsred</p>
 
−
            </div>
 
−
           
 
−
            <div class="partsPicDiv" id="partsL3R1">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/8/8d/China-Tongji-Project-parts-pttx3-chR2-YFP.png" alt="pttx3-chR2-YFP"/>
 
−
                <p class="imgWords">pttx3-chR2-YFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL3R2">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/d/d6/China-Tongji-Project-parts-pttx3-chETA-EYFP.png" alt="pttx3-chETA-EYFP"/>
 
−
                <p class="imgWords">pttx3-chETA-EYFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL3R3">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/1/1d/China-Tongji-Project-parts-pttx3-iC1C2-TS-EYFP.png" alt="pttx3-iC1C2-TS-EYFP"/>
 
−
                <p class="imgWords">pttx3-iC1C2-TS-EYFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL3R4">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/c/c3/China-Tongji-Project-parts-pttx3-blink.png" alt="pttx3-blink"/>
 
−
                <p class="imgWords">pttx3-blink</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL3R5">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/c/c4/China-Tongji-Project-parts-pttx3-dsred.png" alt="pttx3-dsred"/>
 
−
                <p class="imgWords">pttx3-dsred</p>
 
−
            </div>
 
−
           
 
−
            <div class="partsPicDiv" id="partsL4R1">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/8/82/China-Tongji-Project-parts-ptwk16-chR2-YFP.png" alt="ptwk16-chR2-YFP"/>
 
−
                <p class="imgWords">ptwk16-chR2-YFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL4R3">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/a/ae/China-Tongji-Project-parts-ptwk16-iC1C2-TS-EYFP.png" alt="ptwk16-iC1C2-TS-EYFP"/>
 
−
                <p class="imgWords">ptwk16-iC1C2-TS-EYFP</p>
 
−
            </div>
 
−
            <div class="partsPicDiv" id="partsL4R5">
 
−
                <img class="equipImg" src="https://static.igem.org/mediawiki/2015/c/cb/China-Tongji-Project-parts-ptwk16-dsred.png" alt="ptwk16-dsred"/>
 
−
                <p class="imgWords">ptwk16-dsred</p>
 
−
            </div>
 
−
           
 
 
         </div>
 
         </div>
−
          
+
         <!-- part2 -->
 
         <div class="fivePx"></div>
 
         <div class="fivePx"></div>
−
         <p class="titleTwo" id="fifth2">5.2 Test Result</p>
+
         <p class="partTwo" id="first2">1.2 Worm Part</p>
−
         <p class="contentP">With meticulously designs and preciseness experiments, we build up the plasmid we want. By using microinjectiontechnology, we get six strains of worms which could stable inheritance. Then we could test their reactions by means of using our refitted microscopeto find out what functions our plasmids have. To improve the quality of the test, we improve our light sources and refit it from 1W to 5W.</p>
+
         <div class="slidePanel slidePanel2">
−
        <p class="contentP">Here we show our results. </p>
+
            <h3 class="slideTitle" id="slideTitle2dot1">1.2.1 JUNE -- June 20~29<span class="expand">[Expand]</span></h3>
−
          
+
            <div class="slideBlock" id="slideBlock2dot1">
 +
                <h4>1.2.1.1 June 20~29</h4>
 +
                <p>Learn how to do the microinjection on C.elegans.</p>     
 +
            </div>
 +
         </div>
 
         <div class="fivePx"></div>
 
         <div class="fivePx"></div>
−
         <h3>5.2.1Expression Pattern</h3>
+
         <div class="slidePanel slidePanel2">
−
        <center>
+
             <h3 class="slideTitle" id="slideTitle2dot2">1.2.2 JULY -- Week 2~5 -- July 9~31<span class="expand">[Expand]</span></h3>
−
            <img width="700" class="resultImg" src="https://static.igem.org/mediawiki/2015/2/2e/China-Tongji-Project-result-Figure1.png"/>
+
            <div class="slideBlock" id="slideBlock2dot2">
−
             <p class="resultImgName">Figure 5-1: The expression pattern of pmyo2</p>
+
                <h4>1.2.2.1 Week2 -- July 9~13</h4>
−
            <p class="resultImgName">The pymo2 always express in the pharyngeal part and head of the worms.</p>
+
                 <h5>July 9</h5>
−
        </center>
+
                 <p>1, Preparation of NGM plates.</p>
−
        <div class="divider"></div>
+
                 <p>2, Seed NGM plates.</p>
−
       
+
                  
−
        <center>
+
                 <h5>July 11</h5>
−
            <img width="700" class="resultImg" src="https://static.igem.org/mediawiki/2015/4/45/China-Tongji-Project-result-Figure2.jpg"/>
+
                 <p>1, Recover the L1 larva of lite-1 worm from -80 fridge.</p>
−
            <p class="resultImgName">Figure 5-2: The expression pattern of pmyo3</p>
+
                  
−
            <p class="resultImgName">The pymo2 always express in the all parts of the worms.</p>
+
                 <h5>July 13</h5>
−
        </center>
+
                 <p>1, Last time the recovery failed. Recover the L1 larva of lite-1 worm again from -80 fridge.</p>
−
        <div class="divider"></div>
+
−
       
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <h3>5.2.2 Phenotype of each genotype</h3>
+
−
        <p class="chartName">Chart 5-2: The phenotype of each genotype</p>
+
−
        <table class="myTable" align="center" border="3">
+
−
            <tr>
+
−
                <th> </th>
+
−
                <th>Pmyo2-ChR2-YFP</th>
+
−
                 <th>Pmyo2-ChETA-EYFP</th>
+
−
                 <th>Pmyo2-ic1c2-EYFP</th>
+
−
                 <th>Pmyo3-ChR2-YFP</th>
+
−
                <th>Pmyo3-ChETA-EYFP</th>
+
−
                <th>Pmyo3-ic1c2-EYFP</th>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>Speed Change</th>
+
−
                <td> </td>
+
−
                <td class="tdTick"> </td>
+
−
                <td> </td>
+
−
                <td class="tdTick"> </td>
+
−
                <td> </td>
+
−
                <td> </td>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>Direction Change</th>
+
−
                <td class="tdTick"> </td>
+
−
                <td> </td>
+
−
                <td class="tdTick"> </td>
+
−
                <td class="tdTick"> </td>
+
−
                <td> </td>
+
−
                <td class="tdTick"> </td>
+
−
            </tr>
+
−
       
+
−
            <tr>
+
−
                <th>Bending Degree</th>
+
−
                <td class="tdTick"> </td>
+
−
                <td class="tdTick"> </td>
+
−
                <td class="tdTick"> </td>
+
−
                <td> </td>
+
−
                <td class="tdTick"> </td>
+
−
                <td> </td>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>Muscle Contraction</th>
+
−
                <td> </td>
+
−
                <td> </td>
+
−
                <td> </td>
+
−
                <td class="tdTick"> </td>
+
−
                <td> </td>
+
−
                <td> </td>
+
−
            </tr>
+
−
        </table>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <h3>5.2.3 Response index measuring </h3>
+
−
        <p class="chartName">Chart 5-3: The response index of each genotype</p>
+
−
        <table class="myTable" align="center" border="3">
+
−
            <tr>
+
−
                 <th> </th>
+
−
                 <th>Pmyo2-ChR2-YFP</th>
+
−
                 <th>Pmyo2-ChETA-EYFP</th>
+
−
                <th>Pmyo2-ic1c2-EYFP</th>
+
−
                <th>Pmyo3-ChR2-YFP</th>
+
−
                <th>Pmyo3-ChETA-EYFP</th>
+
−
                <th>Pmyo3-ic1c2-EYFP</th>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>response</th>
+
−
                <td>67%</td>
+
−
                <td>38%</td>
+
−
                <td>26%</td>
+
−
                <td>92%</td>
+
−
                <td>42%</td>
+
−
                <td>47%</td>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>no response</th>
+
−
                <td>33%</td>
+
−
                <td>62%</td>
+
−
                <td>74%</td>
+
−
                <td>8%</td>
+
−
                <td>58%</td>
+
−
                <td>53%</td>
+
−
            </tr>
+
−
            <tr>
+
−
                <th>relative error</th>
+
−
                <td>0.025</td>
+
−
                 <td>0.02</td>
+
−
                 <td>0.01</td>
+
−
                 <td>0.015</td>
+
−
                <td>0.042</td>
+
−
                <td>0.049</td>
+
−
            </tr>
+
−
        </table>
+
−
        <div class="divider"></div>
+
−
       
+
−
        <center>
+
−
            <img width="600" class="resultImg" src="https://static.igem.org/mediawiki/2015/e/eb/China-Tongji-Project-result-Figure3.png"/>
+
−
            <p class="resultImgName">Figure 5-3: The response index of each genotype</p>
+
−
        </center>
+
−
        <p class="contentP">All these strains are tested with blue LED (470nm, 5W, 1000mA). Comparing to the other strains, the pmyo3-chR2-YFP has the highest expressionefficiency. While, having no response doesn’t mean the plasmids that we inject do not work. Theexpression quantity may be too low, so that the light we use couldn’t stimulate the worms.</p>
+
  
−
<div class="fivePx"></div>
+
                <h4>1.2.2.2 Week3 -- July 15~21</h4>
−
<h3>5.2.4 Behavior changes and track analysis</h3>
+
                <h5>July 15</h5>
−
<div class="fivePx"></div>
+
                <p>1, The recovery still failed. Recover the L1 larva of lite-1 worm once again!(this time we successes!)</p>
 +
               
 +
                <h5>July 21</h5>
 +
                <p>1, Seed plates with OP50 and ATR (keep in dark place). For 10 3cm plateS(1.5ml agar EACH),seed 1000ul OP50 mixed with 1.75ul 100uM ATR. The OP50 E.coli should be shacked for 12 hours in the conical flask at 37℃(150ml LB added 4-5 single colonies. )</p>
 +
                <p>2, Microinjection pmyo-3::dsRed and Pmyo-3::ChR2 co-injection.(40 worms)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmyo3::dsrRed</td> <td>20ng/ul</td> <td>144.7ng/ul</td> <td>1.38ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmyo-3::ChR2</td> <td>80ng/ul</td> <td>174.7ng/ul</td> <td>4.58ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>162.5ng/ul</td> <td>2.04ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <h4>1.2.2.3 Week4 -- July 23~28</h4>
 +
                <h5>July 23</h5>
 +
                <p>1, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred.Put the worm on a new NGM plate.</p>
 +
                <p>2, Preparation of some new NGM plates.</p>
 +
                <p>3, Seed NGM plates.</p>
 +
               
 +
                <h5>July 24</h5>
 +
                <p>1, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred.Put the worm on a new NGM plate.</p>
 +
               
 +
                <h5>July 25</h5>
 +
                <p>1, Subculture of C.elegans which had been injection of pmyo-3::dsRed and Pmyo-3::ChR2.</p>
 +
                <p>2, Subculture of lite1C.elegans.</p>
 +
               
 +
                <h5>July 26</h5>
 +
                <p>1, Make ATR solution. (help chR2 work in C.elegan)</p>
 +
                <p>2, Subculture of C.elegans which had been injection of pmyo-3::dsRed and Pmyo-3::ChR2.</p>
 +
                <p>3, Subculture of lite1C.elegans.</p>
 +
               
 +
                <h5>July 27</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Put some L4 pmyo3-dsred and pmyo3-chR2 C.elegans on ATR plate.</p>
 +
               
 +
                <h5>July 28</h5>
 +
                <p>1, Microinjection.(pmyo-2::dsRed and Pmyo-2::ChR2 co-injection) for 40 worms.</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmyo2::dsrRed</td> <td>20ng/ul</td> <td>209.7ng/ul</td> <td>0.95ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmyo2::ChR2</td> <td>50ng/ul</td> <td>549ng/ul</td> <td>0.91ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>331.2ng/ul</td> <td>3.62ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>2.52ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
  
−
<div class="Group" id="ControlGroup">
+
                <h4>1.2.2.4 Week5 -- July 29~31</h4>
−
<div class="groupNameDiv">
+
                <h5>July 29</h5>
−
    <span class="groupName">Control group</span>
+
                <p>1, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo2 will express at the worm neck) Put the worm on a new NGM plate.</p>
−
    </div>
+
                <p>2, Subculture of all the C.elegans we have.</p>
−
    <div class="fivePx"></div>
+
               
−
    <h4>5.2.4.1 Lite1 worm without any modified</h4>
+
                <h5>July 30</h5>
−
    <p class="contentP">(1) Video: Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)</p>
+
                <p>1, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo2 will express at the worm neck) Put the worm on a new NGM plate.</p>
−
    <div class="fivePx"></div>
+
                <p>2, Subculture of all the C.elegans we have.</p>
−
    <center>
+
               
−
    <video controls width="550">
+
                <h5>July 31</h5>
−
        <source src="https://static.igem.org/mediawiki/2015/c/c6/China-Tongji-Project-video1-lite1-control.mp4" type="video/mp4"/>
+
                <p>1, Microinjection.( PNP260and PSH116 co-injection)</p>
−
    </video>
+
                <p><b>Mixture:</b></p>
−
    </center>
+
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>PNP260</td> <td>50ng/ul</td> <td>471ng/ul</td> <td>1.06ul</td> </tr>
 +
                <tr class="evenTr"> <td>PSH116</td> <td>50ng/ul</td> <td>516ng/ul</td> <td>0.96ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>90ng/ul</td> <td>271ng/ul</td> <td>4.42ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>1.56ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
                <p>2, subculture of all the C.elegans we have.</p>
 +
                <p>3, put some L4 pmyo-2::dsRed and Pmyo-2::ChR2 C.elegans on ATR plate.</p>
  
−
    <p class="contentP">(2) Track:</p>
+
            </div>
−
    <center>
+
        </div>
−
        <img width="350" class="resultImg" src="https://static.igem.org/mediawiki/2015/e/ea/China-Tongji-Project-result-Figure4.png"/>
+
        <div class="fivePx"></div>
−
        <p class="resultImgName">Figure 5-4: The track of lite1 worm given blue light (470nm, 5W, 1000mA)</p>
+
        <div class="slidePanel slidePanel2">
−
        <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
            <h3 class="slideTitle" id="slideTitle2dot3">1.2.3 AUGUST -- Week 1~5 -- August 1~31<span class="expand">[Expand]</span></h3>
−
        <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
            <div class="slideBlock" id="slideBlock2dot3">
−
    </center>
+
                <h4>1.2.3.1 Week1 -- August 1~6</h4>
−
   
+
                <h5>August 1</h5>
−
    <p class="contentP">(3) This group is lite1 worm which is not sensitive to the lights, while other types of worms make have response to the light. So we choose lite1 worm to be our experimental subject to avoid unnecessaryfactors.</p>
+
                <p>1, Microinjection.( pmyo-3::dsRed and Pmyo-3::ChR2 co-injection AGAIN! Last time we failed)</p>
−
   
+
                <p><b>Mixture:</b></p>
−
    <div class="fivePx"></div>
+
                <div class="divider"></div>
−
    <h4>5.2.4.2 Pmyo2-ChR2-YFP worm without ATR</h4>
+
                <table class="tableQuestion">
−
    <p class="contentP">(1) Video: Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)</p>
+
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
−
    <div class="fivePx"></div>
+
                <tr> <td>Pmyo2-dsred</td> <td>20ng/ul</td> <td>144.7ng/ul</td> <td>1.38ul</td> </tr>
−
    <center>
+
                <tr class="evenTr"> <td>Pmyo2-chR2</td> <td>50ng/ul</td> <td>120.8ng/ul</td> <td>4.41ul</td> </tr>
−
    <video controls width="550">
+
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>271ng/ul</td> <td>2.5ul</td> </tr>
−
        <source src="https://static.igem.org/mediawiki/2015/2/20/China-Tongji-Project-video9.mp4" type="video/mp4"/>
+
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
−
    </video>
+
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
−
    </center>
+
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>2, Subculture of all the C.elegans we have.</p>
 +
                <p>3, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle) Put the right worm on a new NGM plate.</p>
 +
               
 +
                <h5>August 2</h5>
 +
                <p>1, Microinjection.( pmec-3::dsRed and Pmec-3::ChR2 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmec3-dsred </td> <td>20ng/ul</td> <td>159ng/ul</td> <td>1.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmec3-chR2</td> <td>50ng/ul</td> <td>190ng/ul</td> <td>2.6ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.6ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>2, Microinjection.(Pcos13 and Pcos2 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>PCoS13</td> <td>50ng/ul</td> <td>191.5ng/ul</td> <td>2.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>PCoS2</td> <td>50ng/ul</td> <td>327.2ng/ul</td> <td>1.5ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>90ng/ul</td> <td>271ng/ul</td> <td>3.3ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.6ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>3, Subculture of all the C.elegans we have.</p>
 +
                <p>4, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle) Put the right worm on a new NGM plate.</p>
 +
                <p>5, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmec3 will express at the worm’s touch neuron) Put the right worm on a new NGM plate.</p>
 +
               
 +
                <h5>August 3</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle, (pmec3 will express at the worm’s touch neuron, PCoS13 overlap PCos2 will express at AVA neuron, PNP260 overlap PSH116 will express at PVC neuron).Put the right worm on a new NGM plate.</p>
 +
                <p>3, Preparation of some new NGM plates.</p>
 +
                <p>4, Seed NGM plates.</p>
 +
                <p>5, Seed new ATR plates.</p>
 +
               
 +
                <h5>August 4</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle, (pmec3 will express at the worm’s touch neuron, PCoS13 overlap PCos2 will express at AVA neuron, PNP260 overlap PSH116 will express at PVC neuron). Put the right worm on a new NGM plate.</p>
 +
               
 +
                <h5>August 5</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle, (pmec3 will express at the worm’s touch neuron, PCoS13 overlap PCos2 will express at AVA neuron, PNP260 overlap PSH116 will express at PVC neuron). Put the right worm on a new NGM plate.</p>
 +
               
 +
                <h5>August 6</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
  
−
    <p class="contentP">(2) Track:</p>
+
                <h4>1.2.3.2 Week2 -- August 8~14</h4>
−
    <center>
+
                <h5>August 8</h5>
−
        <img width="350" class="resultImg" src="https://static.igem.org/mediawiki/2015/6/6d/China-Tongji-Project-result-Figure5.png"/>
+
                <p>1, Subculture of all the C.elegans we have.</p>
−
        <p class="resultImgName">Figure 5-5: the track of pmyo2-ChR2-YFP with blue light (470nm, 5W, 1000mA, fostered without ATR)</p>
+
               
−
        <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
                <h5>August 9</h5>
−
        <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
                <p>1, Subculture of all the C.elegans we have.</p>
−
    </center>
+
                <p>2, Since the some of C.elegans we injected has not been passaged stalely, so we had to reinject some of them.</p>
−
   
+
                <p>Microinjection.( pmec-3::dsRed and Pmec-3::ChR2 co-injection)</p>
−
    <p class="contentP">(3) This group we use the mediums without ATR to foster thepmyo2-ChR2-YFP worms. We use this group to find out the effect of ATR. The pmyo2As a result, we find out that the ATR is necessary to our project. Only being fostered in the mediums which have ATR do the worms have response to the lights we give.In our project, we also set up other control groups to each strain, and they all have the same phenomenon. We choose pmyo2-ChR2-YFP worms as an example for this kind of worm have the highest efficiency.</p>
+
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmec3-dsred</td> <td>20ng/ul</td> <td>159ng/ul</td> <td>1.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmec3-chR2</td> <td>50ng/ul</td> <td>190ng/ul</td> <td>2.6ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.6ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>Microinjection.( pmyo-2::dsRed and Pmyo-2::ChR2 co-injection) for 40 worms.</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmyo2::dsrRed</td> <td>20ng/ul</td> <td>209.7ng/ul</td> <td>0.95ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmyo2::ChR2</td> <td>50ng/ul</td> <td>549ng/ul</td> <td>0.91ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>331.2ng/ul</td> <td>3.62ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>2.52ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <h5>August 10</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 11</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 12</h5>
 +
                <p>1, Microinjection.( pmyo-3::dsRed and Pmyo-3::ic1c2 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmyo3-dsred</td> <td>20ng/ul</td> <td>159ng/ul</td> <td>1.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmyo3-ic1c2</td> <td>50ng/ul</td> <td>187ng/ul</td> <td>2.6ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.6ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>2, Microinjection.( pmyo-3::dsRed and Pmyo-3::chETA co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmyo3-dsred</td> <td>20ng/ul</td> <td>159ng/ul</td> <td>1.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmyo3 chETA</td> <td>50ng/ul</td> <td>106ng/ul</td> <td>2.9ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.3ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>3, Subculture of all the C.elegans we have.</p>
 +
                <p>4, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 13</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 14</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected.</p>                            
  
−
    <div class="fivePx"></div>
+
                <h4>1.2.3.3 Week3 -- August 15~21</h4>
−
    <h4>5.2.4.3 Functional worms with green light</h4>
+
                <h5>August 15</h5>
−
    <p class="contentP">(1) Video: Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)</p>
+
                <p>1, Microinjection.( pmyo-2::dsRed and Pmyo-2::chETA co-injection)</p>
−
    <div class="fivePx"></div>
+
                <p><b>Mixture:</b></p>
−
    <center>
+
                <div class="divider"></div>
−
    <video controls width="550">
+
                <table class="tableQuestion">
−
        <source src="https://static.igem.org/mediawiki/2015/5/54/China-Tongji-Project-video8.mp4" type="video/mp4"/>
+
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
−
    </video>
+
                <tr> <td>Pmyo2-dsred</td> <td>20ng/ul</td> <td>128ng/ul</td> <td>1.25ul</td> </tr>
−
    </center>
+
                <tr class="evenTr"> <td>Pmyo2-chETA</td> <td>50ng/ul</td> <td>106ng/ul</td> <td>2.9ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>120ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.3ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>Microinjection.( pmyo-2::dsRed and Pmyo-2::ic1c2 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmyo2-dsred</td> <td>20ng/ul</td> <td>156ng/ul</td> <td>1.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmyo2-ic1c2</td> <td>90ng/ul</td> <td>178ng/ul</td> <td>2.55ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>70ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.75ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>2, Subculture of all the C.elegans we have.</p>
 +
                <p>3, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 16</h5>
 +
                <p>1, Microinjection.(pttx-3::dsRed and Pttx-3::chR2 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>pttx-3::dsRed</td> <td>20ng/ul</td> <td>128ng/ul</td> <td>1.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pttx-3::chR2</td> <td>80ng/ul</td> <td>200ng/ul</td> <td>2.9ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>90ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.3ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>Microinjection.( pttx-3::dsRed and Pttx-3::BLINK co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>pttx-3::dsRed</td> <td>20ng/ul</td> <td>128ng/ul</td> <td>1.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pttx-3::BLINK</td> <td>80ng/ul</td> <td>213ng/ul</td> <td>2.8ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>90ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.4ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
               
 +
                <p>Microinjection.( pttx-3::dsRed and Pttx-3::ic1c2 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>pttx-3::dsRed</td> <td>20ng/ul</td> <td>128ng/ul</td> <td>1.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pttx-3::ic1c2</td> <td>70ng/ul</td> <td>167ng/ul</td> <td>2.8ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>90ng/ul</td> <td>331ng/ul</td> <td>3.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.4ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>2, Subculture of all the C.elegans we have.</p>
 +
                <p>3, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 18</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 19</h5>
 +
                <p>Microinjection.(Pcos13 and Pcos2 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>PCoS13</td> <td>50ng/ul</td> <td>191.5ng/ul</td> <td>2.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>PCoS2</td> <td>50ng/ul</td> <td>327.2ng/ul</td> <td>1.5ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>90ng/ul</td> <td>271ng/ul</td> <td>3.3ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.6ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>Microinjection.( PNP260and PSH116 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>PNP260</td> <td>50ng/ul</td> <td>471ng/ul</td> <td>1.06ul</td> </tr>
 +
                <tr class="evenTr"> <td>PSH116</td> <td>50ng/ul</td> <td>516ng/ul</td> <td>0.96ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>90ng/ul</td> <td>271ng/ul</td> <td>4.42ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>1.56ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>              
 +
               
 +
                <h5>August 20~21</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected.</p>
  
−
    <p class="contentP">(2) Track:</p>
+
                <h4>1.2.3.4 Week4 -- August 22~27</h4>
−
    <center>
+
                <h5>August 22</h5>
−
        <img width="350" class="resultImg" src="https://static.igem.org/mediawiki/2015/8/83/China-Tongji-Project-result-Figure6.png"/>
+
                <p>1, Microinjection.( ptwk16-dsRed and ptwk16-blink co-injection)</p>
−
        <p class="resultImgName">Figure 5-6: The track of pmyo2-ChR2-YFP worm given green light (490nm, 3W, 1000mA)</p>
+
                <p><b>Mixture:</b></p>
−
        <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
                <div class="divider"></div>
−
        <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
                <table class="tableQuestion">
−
    </center>
+
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
−
   
+
                <tr> <td>ptwk16-dsRed</td> <td>50ng/ul</td> <td>187ng/ul</td> <td>1.06ul</td> </tr>
−
    <p class="contentP">(3) This group we use green light to stimulate the pmyo2-ChR2-YFP worm. By doing this, we try to find out if the worm have response to all kinds of lights. At last, we find out that our worms would only have response to blue light (470nm, 5W, 1000mA). In our project, we also set up other control groups to each strain, and they all have the same phenomenon. We choose pmyo2-ChR2-YFP worms as an example for this kind of worm have the highest efficiency.</p>
+
                <tr class="evenTr"> <td>ptwk16-blink</td> <td>90ng/ul</td> <td>213ng/ul</td> <td>0.96ul</td> </tr>
−
</div>
+
                <tr> <td>PBlue</td> <td>50ng/u</td> <td>271ng/ul</td> <td>4.42ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>1.56ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>Microinjection.( ptwk16-dsRed and ptwk16-chR2 co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>ptwk16-dsRed</td> <td>50ng/ul</td> <td>197ng/ul</td> <td>1.06ul</td> </tr>
 +
                <tr class="evenTr"> <td>ptwk16-chR2</td> <td>90ng/ul</td> <td>254ng/ul</td> <td>0.93ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>50ng/u</td> <td>271ng/ul</td> <td>4.42ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>1.59ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>2, Subculture of all the C.elegans we have.</p>
 +
                <p>3, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 23~24</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected.</p>
 +
               
 +
                <h5>August 25</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                <p>2, Select the F1 generation of the worm we injected.</p>
 +
                <p>3, Microinjection.( pmyo3-dsRed and pmyo3-blink co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>Pmyo3-dsRed</td> <td>50ng/ul</td> <td>197ng/ul</td> <td>1.06ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pmyo3-blink</td> <td>90ng/ul</td> <td>254ng/ul</td> <td>0.93ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>50ng/u</td> <td>271ng/ul</td> <td>4.42ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>1.59ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>
 +
               
 +
                <p>Microinjection.( pttx-3::dsRed and Pttx-3::BLINK co-injection)</p>
 +
                <p><b>Mixture:</b></p>
 +
                <div class="divider"></div>
 +
                <table class="tableQuestion">
 +
                <tr> <th>Material</th> <th>Final concentration</th> <th>Real concentration</th> <th>Real Volume</th> </tr>
 +
                <tr> <td>pttx-3::dsRed</td> <td>20ng/ul</td> <td>128ng/ul</td> <td>1.25ul</td> </tr>
 +
                <tr class="evenTr"> <td>Pttx-3::BLINK</td> <td>80ng/ul</td> <td>213ng/ul</td> <td>2.8ul</td> </tr>
 +
                <tr> <td>PBlue</td> <td>90ng/ul</td> <td>331ng/ul</td> <td>3.6ul</td> </tr>
 +
                <tr class="evenTr"> <td>Rat Genomic DNA</td> <td>10ng/ul</td> <td>100ng/ul</td> <td>1ul</td> </tr>
 +
                <tr> <td>10XTE</td> <td></td> <td></td> <td>1ul</td> </tr>
 +
                <tr class="evenTr"> <td>ddH2O</td> <td></td> <td></td> <td>0.4ul</td> </tr>
 +
                <tr> <td>Total</td> <td></td> <td></td> <td>10ul</td> </tr>
 +
                </table>
 +
                <div class="fivePx"></div>               
 +
               
 +
                <h5>August 26~27</h5>
 +
                <p>1, Subculture of all the C.elegans we have.</p>              
 +
               
 +
                <h4>1.2.3.5 Week5 -- August 29~31</h4> 
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
                         
 +
            </div>
 +
        </div>
 +
        <div class="fivePx"></div>
 +
        <div class="slidePanel slidePanel2">
 +
            <h3 class="slideTitle" id="slideTitle2dot4">1.2.4 SEPTEMBER -- Week 1~2 -- September 1~15<span class="expand">[Expand]</span></h3>
 +
            <div class="slideBlock" id="slideBlock2dot4">
 +
                <p>1, Subculture of all the C.elegans we have.</p>
 +
            </div>
 +
        </div>
 +
        <!-- part3 -->
 +
        <div class="fivePx"></div>
 +
        <p class="partTwo" id="first3">1.3 Efficiency Testpart</p>
 +
        <div class="slidePanel slidePanel3">
 +
            <h3 class="slideTitle" id="slideTitle3dot1">1.3.1 AUGUST -- Week 1~5 -- August 3~31<span class="expand">[Expand]</span></h3>
 +
            <div class="slideBlock" id="slideBlock3dot1">
 +
                <h4>1.3.1.1 Week1 -- August 3~7</h4>
 +
                <h5>August 3</h5>
 +
                <p>1, Initial test of pmyo3-chR2-YFP C.elegans.since we don’t have enough worms yet, we just test for 2 worm. We found their behavior have obvious change when we turn on the blue light. (470nm)</p>
 +
               
 +
                <h5>August 5</h5>
 +
                <p>1, Initial test of pmyo2-chR2-YFP C.elegan. Since the chR2 is expressed at the neck, we didn’t see any big change of movement when we turn on the light.</p>
 +
               
 +
                <h5>August 6</h5>
 +
                <p>1, Intial test of Pcos13 and Pcos2 C.elegan.</p>
 +
               
 +
                <h5>August 7</h5>
 +
                <p>1, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP and Pcos13 overlap with Pcos2 C.elegans again. </p>
  
−
<div class="Group" id="Pmyo2Group">
+
                <h4>1.3.1.2 Week2 -- August 9~14</h4>
−
<div class="groupNameDiv">
+
                <h5>August 9</h5>
−
    <span class="groupName">Pmyo2</span>
+
                <p>1, Trying to take some small video of the pmyo2-chR2-YFP, pmyo3-chR2-YFP and Pcos13 overlap with Pcos2 C.elegans.</p>
−
    </div> 
+
               
−
    <div class="fivePx"></div> 
+
                <h5>August 10</h5>
−
    <h4>5.2.4.4 pmyo2-ChR2-YFP </h4>
+
                <p>1, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP and Pcos13 overlap with Pcos2 C.elegans.</p>
−
    <p class="contentP">(1) Video: Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)</p>
+
               
−
    <div class="fivePx"></div>
+
                <h5>August 13</h5>
−
    <center>
+
                <p>1, Intial test of Pmyo-3-chETAC.elegan.</p>
−
    <video controls width="550">
+
               
−
        <source src="https://static.igem.org/mediawiki/2015/2/2a/China-Tongji-Project-video4-pmyo2-chR2.mp4" type="video/mp4"/>
+
                <h5>August 14</h5>
−
    </video>
+
                <p>1, Intial test of Pmyo-3-ic1c2 C.elegan.</p>
−
    </center>
+
−
<p class="contentP">(2) Track:</p>
+
−
    <center>
+
−
        <img width="350" class="resultImg" src="https://static.igem.org/mediawiki/2015/d/d8/China-Tongji-Project-result-Figure7.png"/>
+
−
        <p class="resultImgName">Figure 5-7: pmyo2-ChR2-YFP worm given blue light (470nm, 5W, 1000mA)</p>
+
−
        <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
−
        <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
−
    </center>   
+
−
    <p class="contentP">(3) Reactions: We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver. When we give light to this kind of worms, we can find some obvious responses. First, after we give light, the worm would change their direction in about 2 seconds in average. Their reactions are always step back. Secondly if we focus on the movement of their heads, we can find the turning angles change a lot during this time. It means the blue light can stimulate the muscle of their heads and as a result the worm will change the direction. But their behavior will turn to normal at the moment we turn off the light instantaneously. The speed of worm doesn’t have some apparent changes.</p>
+
−
   
+
−
    <div class="fivePx"></div>
+
−
    <h4>5.2.4.5 pmyo2-ChETA-EYFP</h4>
+
−
    <p class="contentP">(1) Video: Please pay attention to the top right corner of the video, the appearance of blue point represents the change from white light to 5W LED blue light (470nm)</p>
+
−
    <div class="fivePx"></div>
+
−
    <center>
+
−
    <video controls width="550">
+
−
        <source src="https://static.igem.org/mediawiki/2015/c/c2/China-Tongji-Project-video6-pmyo2-chETA.mp4" type="video/mp4"/>
+
−
    </video>
+
−
    </center>
+
  
−
    <p class="contentP">(2) Track:</p>
+
                <h4>1.3.1.3 Week3 -- August 15~21</h4>
−
    <center>
+
                <h5>August 15</h5>
−
        <img width="350" class="resultImg" src="https://static.igem.org/mediawiki/2015/9/97/China-Tongji-Project-result-Figure8.png"/>
+
                <p>1, Try to take some small video of thePmyo-3-chETA and Pmyo-3-ic1c2 C.elegans.</p>
−
        <p class="resultImgName">Figure 5-8: pmyo2-ChETA-EYFP worm given blue light (470nm, 5W, 1000mA)</p>
+
                <p>2, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pcos13 overlap with Pcos2, Pmyo-3-chETA and Pmyo-3-ic1c2. </p>
−
        <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
               
−
        <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
                <h5>August 16</h5>
−
    </center>  
+
                <p>1, Intial test of Pmyo-2-ic1c2 andPmyo-2-chETA.</p>
−
    <p class="contentP">(3) We test this kind of worms by using 5W LED blue light (470nm) with 1000mA LED driver.When giving the light, this kind of worms has little responses. But you can see the behavior of the head have changed a lot. It means the blue light can still infect the muscle near the head. When the light is on, the behaviors of the worms become stiff compare to the normal worms. At the same time, after the light is given, you can find the speed of the worm obviously slow down. It is very interested that the worm will stop or even recede when the light is turned off. It means it will take some time for the worm to turn to normal.</p>
+
               
−
   
+
                <h5>August 17</h5>
−
</div>
+
                <p>1, Try to take some small video of thePmyo-2-chETA and Pmyo-2-ic1c2 C.elegans.</p>
 +
                <p>2, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pcos13 overlap with Pcos2, Pmyo-3-chETA Pmyo-3-ic1c2, Pmyo-2-ic1c2 and Pmyo-2-chETA.</p>
 +
               
 +
                <h5>August 19</h5>
 +
                <p>1, Intial test of pttx3-chR2.</p>
 +
                <p>2, Try to take some small video of thepttx3-chR2 C.elegans.</p>
 +
               
 +
                <h5>August 20</h5>
 +
                <p>1, Lab meeting about how to take our video in a standard way.We decided to take 40sec each one: 10s for white light, 10s for 470nm light, 10 for white light and 10s for another wavelength light.In this way, we could use some video software to analyze the test result. </p>
 +
               
 +
                <h5>August 21</h5>
 +
                <p>1, Intial test of ptwk16-blink.</p>
 +
                <p>2, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pmyo-3-chETA Pmyo-3-ic1c2, Pmyo-2-ic1c2, Pmyo-2-chETA pttx3-chR2 and ptwk16-blink.</p>
 +
                <p>3, Take video of these genetypeC.elegans.</p>
  
 +
                <h4>1.3.1.4 Week 4~5 -- August 23~30</h4>
 +
                <h5>August 23,25,27,28,30</h5>
 +
                <p>1, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pmyo-3-chETA Pmyo-3-ic1c2, Pmyo-2-ic1c2, Pmyo-2-chETA pttx3-chR2 and ptwk16-blink.</p>
 +
                <p>2, Take video of these genetypeC.elegans.</p>
 +
            </div>
 +
        </div>
 
         <div class="fivePx"></div>
 
         <div class="fivePx"></div>
−
         <h3>5.2.5 Turning angle measuring</h3>
+
         <div class="slidePanel slidePanel3">
−
        <p class="contentP">As we know pmyo2 is express in pharyngeal of C.elegents, so the light will stimulate the head of the worms directly to the head. As a result, observing the movement of their heads is very significative.As we all know, the head of the worm is always shaking, so the turning angle (the angle of each shake) is a very useful data which reflect the response of the head. In this part, we use turning angle of their heads to evaluate the reaction of their head. </p>
+
             <h3 class="slideTitle" id="slideTitle3dot2">1.3.2 SEPTEMBER -- Week 1~2 -- September 1~15<span class="expand">[Expand]</span></h3>
−
        <p class="contentP">We choose pmyo2 worms as our experimental objects in this part. The results are showed below.</p>
+
             <div class="slideBlock" id="slideBlock3dot2">
−
       
+
                <h4>1.3.2.1 Week1 -- September 1~7</h4>
−
        <center>
+
                <p>1, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pmyo-3-chETA Pmyo-3-ic1c2, Pmyo-2-ic1c2, Pmyo-2-chETA pttx3-chR2 and ptwk16-blink.</p>
−
             <img width="600" class="resultImg" src="https://static.igem.org/mediawiki/2015/2/2f/China-Tongji-Project-result-Figure13.png"/>
+
                <p>2, Take video of these genetypeC.elegans.</p>   
−
            <p class="resultImgName">Figure 5-13: The turning angle measuring of pmyo2-ChR2-YFP (using blue light, 470nm, 5W, 1000mA)</p>
+
                 <h4>1.3.2.2 Week2 -- September 8~16</h4>
−
            <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
                <p>1, Analyze the results with related video software.</p>
−
            <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
                 <p>2, Still test of our C.elegans.</p>
−
        </center>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="600" class="resultImg" src="https://static.igem.org/mediawiki/2015/1/1e/China-Tongji-Project-result-Figure14.png"/>
+
−
            <p class="resultImgName">Figure 5-14: The turning angle measuring of pmyo2-ChETA-EYFP (using blue light, 470nm, 5W, 1000mA)</p>
+
−
            <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
−
            <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
−
        </center>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="600" class="resultImg" src="https://static.igem.org/mediawiki/2015/e/ec/China-Tongji-Project-result-Figure15.png"/>
+
−
            <p class="resultImgName">Figure 5-15: The turning angle measuring of pmyo2-iC1C2-EYFP (using blue light, 470nm, 5W, 1000mA)</p>
+
−
            <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
−
            <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
−
        </center>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="600" class="resultImg" src="https://static.igem.org/mediawiki/2015/f/f4/China-Tongji-Project-result-Figure16.png"/>
+
−
            <p class="resultImgName">Figure 5-16: The turning angle measuring of pmyo2-ChR2-EYFP (given no light)</p>
+
−
            <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
−
             <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
−
        </center>
+
−
       
+
−
        <h4>Analysis:</h4>
+
−
        <p class="contentP">From these figures we can get some useful information.</p>
+
−
        <p class="contentP">(1) Thepmyo2-ChR2-YFP worms have an obvious response. When we give the lights, we can see theamplitude of turning angles become larger. The change of the turning angle becomes drastic.</p>
+
−
        <p class="contentP">(2) Compare to the pmyo2-ChR2-YFP worms, the other worm need a long time to be activated. And the time when the worm is activated has become longer.</p>
+
−
        <p class="contentP">(3) We use the pmyo2 worm given no lights as the control group. We test all the strains of pmyo2 worms, and they have the same reactions. We can see the fluctuation of turning angle is mild compare to those experimental groups. </p>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <h3>5.2.6 The relationship between light intensity and response index</h3>
+
−
        <p class="contentP">By using DC2100 we can achieve the aim that we could control the current of LED accurately. For our LEDs, there is a direct proportion relationship between light intensity and the current which move across it. To test which value is the best to stimulate the C.elegents, we design this part to help us. Accord to the limitation of DC2100, the largest current we can use is 1000mA. So we pick some worms of all strains which have obvious reactions as our experimental material (using 1000mA to test the reactions before). Because 0mA is needn’t to be tested, we choose to start from 50mA. Every strain we pick up 10 worms to test it has reactions or not. After 50Ma has been tested, we test the 100mA and then 150mA and so on. Until we finish the test of 1000ma, we calculate the ratio of having reactions. Here is the graph we get due to the records.The results are showed below. </p>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="500" class="resultImg" src="https://static.igem.org/mediawiki/2015/7/72/China-Tongji-Project-result-Figure17.png"/>
+
−
            <p class="resultImgName">Figure 5-17: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo2-ChR2-YFP worm)</p>
+
−
        </center>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="500" class="resultImg" src="https://static.igem.org/mediawiki/2015/2/2f/China-Tongji-Project-result-Figure18.png"/>
+
−
            <p class="resultImgName">Figure 5-18: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo2-ChETA-EYFP worm)</p>
+
−
        </center>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="500" class="resultImg" src="https://static.igem.org/mediawiki/2015/d/dd/China-Tongji-Project-result-Figure19.png"/>
+
−
            <p class="resultImgName">Figure 5-19: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo2-iC1C2-EYFP worm)</p>
+
−
        </center>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="500" class="resultImg" src="https://static.igem.org/mediawiki/2015/b/b3/China-Tongji-Project-result-Figure20.png"/>
+
−
            <p class="resultImgName">Figure 5-20: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo3-ChR2-YFP worm)</p>
+
−
        </center>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="500" class="resultImg" src="https://static.igem.org/mediawiki/2015/3/35/China-Tongji-Project-result-Figure21.png"/>
+
−
            <p class="resultImgName">Figure 5-21: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo3-ChETA-EYFP worm)</p>
+
−
        </center>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
            <img width="500" class="resultImg" src="https://static.igem.org/mediawiki/2015/b/b4/China-Tongji-Project-result-Figure22.png"/>
+
−
            <p class="resultImgName">Figure 5-22: The changing trend of response index with the change of light intensity (blue LED, 470nm, 5W, using pmyo3-iC1C2-EYFP worm)</p>
+
−
        </center>
+
−
       
+
−
        <h4>From these data, we can find out some conclusions.</h4>
+
−
        <p class="contentP">(1) For all these strains, the response index is getting larger with the increase of the current. </p>
+
−
        <p class="contentP">(2) For pmyo2-ChR2-YFP and pmyo3-ChR2-YFP, when the current increase to about 600 we can see the worm can be totally activated. </p>
+
−
        <p class="contentP">(3) For pmyo2-iC1C2-EYFP and pmyo3-iC1C2-EYFP, when the current increase to about 800 we can see the worm can be totally activated.</p>
+
−
        <p class="contentP">(4) Forpmyo2-ChETA-EYFP and pmyo3-ChETA-EYFP, when the current increase to about 900 we can see the worm can be totally activated.</p>
+
−
        <p class="contentP">(5) For all the strains of worms, 1000mA is the most suitable current. So in our project we use 1000mA blue light (470nm, 5W) to test our worms.</p>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <h3>5.2.7 Interval flash mode</h3>
+
−
        <p class="contentP">As chETA has been optimized in active and recover speed, we also tried internally lighting partten to reappear this character on c.elegans. pmyo2-chETA-eYFP has showed impressive phenomenon.</p>   
+
−
       
+
−
        <table border="2" class="tableTwoTd">
+
−
            <tr>
+
−
                 <td>
+
−
                    <h4>5.2.7.1 lite-1</h4>
+
−
                    <p class="contentP">(1) Video:</p>
+
−
                    <div class="fivePx"></div>
+
−
                    <center>
+
−
                    <video controls width="350">
+
−
                        <source src="https://static.igem.org/mediawiki/2015/0/07/China-Tongji-Project-video10.mp4" type="video/mp4" />
+
−
                    </video>
+
−
                    </center>
+
−
       
+
−
                    <p class="contentP">(2) Track:</p>
+
−
                    <center>
+
−
                        <img width="350" class="resultImg" src="https://static.igem.org/mediawiki/2015/7/7a/China-Tongji-Project-result-Figure23.png"/>
+
−
                        <p class="resultImgName">Figure 5-23: lite-1 worm given blue light (470nm, 5W, 1000mA, 10Hz flash)</p>
+
−
                        <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
−
                        <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
−
                    </center>
+
−
       
+
−
                </td>
+
−
                <td>
+
−
                    <h4>5.2.7.2 pmyo2-chETA-eYFP</h4>
+
−
                    <p class="contentP">(1) Video:</p>
+
−
                    <div class="fivePx"></div>
+
−
                    <center>
+
−
                    <video controls width="350">
+
−
                        <source src="https://static.igem.org/mediawiki/2015/b/bf/China-Tongji-Project-video11.mp4" type="video/mp4" />
+
−
                    </video>
+
−
                    </center>
+
−
                   
+
−
                    <p class="contentP">(2) Track:</p>
+
−
                    <center>
+
−
                        <img width="350" class="resultImg" src="https://static.igem.org/mediawiki/2015/3/37/China-Tongji-Project-result-Figure24.png"/>
+
−
                        <p class="resultImgName">Figure 5-24: pmyo2-chETA-eYFP worm given blue light (470nm, 5W, 1000mA, 10Hz flash)</p>
+
−
                        <p class="resultImgName">[ <span style="color:#F00"><b>Red points</b></span> represents the track under white light. ]</p>
+
−
                        <p class="resultImgName">[ <span style="color:#00F"><b>Blue points</b></span> represents the track under blue light (470nm) ]</p>
+
−
                    </center>
+
−
                </td>
+
−
            </tr>
+
−
            <tr>
+
−
                <td colspan="2">
+
−
                    <h4>Analysis:</h4>
+
−
                    <p class="contentP">The test is doing under 5W LED blue light (470nm) with 1000mA LED driverand 10 Hz flash.
+
−
                    Compared to the control group, pmyo2-chETA-eYFP showed a strong body binding tendency under internal light, mainly caused by the increasing head swing angle, which indicats the capability of chETA. To further explore its unique features, we also compared the results to those worms with same genotype, but given continuous stimulation (Figure 8). We found an enhanced influence on locomotion, as the turn is too sharp that it even changes their heading direction. A possible reason for this difference is that the switch between muscle activation state can be complete in this model. When the channelrhodopsin can’t reach the switching effiency, the tissue turned into a state of platform potential, thus subtle effect can be missing. But with chETA, the quick transforming is expressed.</p>
+
−
                 </td>
+
−
            </tr> 
+
−
        </table>
+
−
       
+
−
        <div class="fivePx"></div>
+
−
        <h3>5.2.8 Muscle contraction </h3>
+
−
        <p class="contentP">From the result we get, we find that pmyo3-ChR2-YFP have an obvious response that the muscle of worm contract.So we try to observe the reactions of this kind of worm in high power field, for the light intensity is larger in high power field. As a result, we can find the muscle of worm contract severely, the video is showed below.</p>
+
−
        <div class="fivePx"></div>
+
−
        <center>
+
−
        <video controls width="550">
+
−
            <source src="https://static.igem.org/mediawiki/2015/c/c2/China-Tongji-Project-video12.mp4" type="video/mp4" />
+
−
        </video>
+
−
        </center>
+
  
−
       
+
            </div>
−
        <!-- 6. Design -->
+
         </div>
−
         <p></p><div class="divider"></div>
+
        <!-- part4 -->  
−
<p class="titleOne" id="Design">6. Design</p>  
+
 
         <div class="fivePx"></div>
 
         <div class="fivePx"></div>
−
         <p class="titleTwo" id="sixth1">6.1 Introduction</p>
+
         <p class="partTwo" id="first4">1.4 Equipment Part</p>
−
         <p class="contentP">Our project focuses on using optogenetics technique to control the simple movement of C.elegans. We have successfully expressed different kinds of opsins in different tissues of the worm, and finallycontrolled it under specific wavelength LED lights. Actually, this technique has much more potential use in medical treatment and scientific research.</p>
+
         <div class="slidePanel slidePanel4">
−
          
+
            <h3 class="slideTitle" id="slideTitle4dot1">1.4.1 JULY -- July 1,28<span class="expand">[Expand]</span></h3>
 +
            <div class="slideBlock" id="slideBlock4dot1">
 +
                <h4>1.4.1.1 Week1 -- July 1</h4>
 +
                <p>1, We pick up the parts we need on the internet under the help of our instructor Wei Li. Ordered them on line.</p>
 +
                <h4>1.4.1.2 Week4 -- July 28</h4>       
 +
                <p>1, We learn about how to build our LED light source together. </p>   
 +
            </div>
 +
         </div>
 
         <div class="fivePx"></div>
 
         <div class="fivePx"></div>
−
         <p class="titleTwo" id="sixth2">6.2 Medical treatment potential</p>
+
         <div class="slidePanel slidePanel4">
−
        <h3>6.2.1 Treatment of paralysis</h3>
+
            <h3 class="slideTitle" id="slideTitle4dot2">1.4.2 AUGUST -- Week 1~2 -- August 1~8<span class="expand">[Expand]</span></h3>
−
        <p class="contentP">Right now, we could already make some simple control of C.elegans’ movement, so optogenetics has potential to help the disabled to gain their athletic ability again. After reading papers we found that this work has been done by professor Linda Green Smith, LondonUniversity.They insert opsins into embryonic stem cell of mouse, and they add signal molecule to let the stem cell grow into neuron which can transport signal between spine and other parts of body. Next, they input these neurons into mouse whose ischiadic nerve has been cut. 5 weeks later, they used blue light to stimulate the nerve and they found that the mouse’s muscle on leg has shrink reaction. So as we can see, this technique will finally be used on human being. At the same time, right now the only way to help patient is using electric to stimulate the muscle, however, this method will bring extra pain to the patient. Once the optogenetics used on paralysis treatment, the stimulation will be more gently so that the patient will feel better.</p>
+
            <div class="slideBlock" id="slideBlock4dot2">
−
       
+
                <h4>1.4.2.1 Week1 -- August 1~7</h4>
−
        <h3>6.2.2 Treatment of neurodegenerative diseases</h3>
+
                <h5>August 1</h5>
−
        <p class="contentP">Optogenetics is a new thought to treat neurodegenerative diseases, for instance, parkinson’s disease is an illness which has strong impact on human brain health. Prof. Fan Yang’s group expressed chETA at culturing neuroglia. They found that under the stimulation of blue light, the neuron will differentiate to neuron, at the same time, the neuron cells they got has obvious molecular maker which owns by dopaminergic neuron. So we have great chance to use optogenetics into the treatment of neurodegenerative diseases.</p>
+
                <p>1, All our equipment has been arrived, we started to build it. We met some problems during the process, for instance, some of the parts did not fit in with each other.</p>
−
       
+
               
−
        <h3>6.2.3 Treatment of depression</h3>
+
                <h5>August 2~3</h5>
−
        <p class="contentP">Last but no least, we know that it’s difficult to cure depression right now. So what if we try to use optogenetics into treatment of depression? One method today to treat depression is using electric stimulation to stimulate neuron in order to make it alive. However, this method may make some damages of brain tissue. With gentle technique like optogenetics, we may find a better way to solute this problem.We can stimulate the dopaminergic neuron and let it secret more dopamine so that the mental condition of patient will be remitted. Besides, there are research shows that when express activated opsins into mice brain, the anxiety behavior will decrease under the irradiation of light. This strongly proved that this technique can be used in treatment of depression one day in the future! </p>
+
                <p>1, Build the LED light source.</p>
−
       
+
               
−
        <div class="fivePx"></div>
+
                <h5>August 4</h5>
−
        <p class="titleTwo" id="sixth3">6.3 Useful tool in scientific research</p>
+
                <p>1, Adjustment of light path.</p>
−
        <h3>6.3.1 Research of neural circuit</h3>
+
                <p>2, Ask for the help about how install and use the LED related software on the computer from microscope engineer.</p>
−
        <p class="contentP">As a quick operate tool, neither will it make extra damage on biological sample like electric stimulation, nor it wouldappear the inaccuratearea like traditional stimulation method.At present, optogenetics can achieve ‘ms’ level optical control on intact mammal neural circuit. This offers wider approaches for research on neuron which has contact with specific cell.</p>
+
               
−
       
+
                <h5>August 5</h5>
−
        <h3>6.3.2 Research of animal behavior</h3>
+
                <p>1, After the first test, we found that our LED power is still not powerful enough. So we bought some new LED source on the internet.</p>
−
        <p class="contentP">Optogenetics can also be used in research of animal behavior. For instance, researchers use optogenetics to make dopamine secret inside drosophila’s body. This will accompany with obvious behavior. So they can figure out the relationship with the dopaminergic neuron’s actions and drosophila’s behavior, which will be very difficult to use traditional method to make position locate.</p>
+
               
−
       
+
                <h5>August 7</h5>
−
        <h3>6.3.3 Research of neuropharmacology experiment</h3>
+
                <p>1, Weld the new 5W LED light source up.</p>
−
        <p class="contentP">Traditional neuropharmacology experiments rely on detection of behaviors, which is slow and inaccurate. Optogenetics let us can operate the behavior easier. Like what we have down in our project, we use this technique finding out the specific function of different muscle or neurons. Besides, traditional method should use couple of days to remove the drug completely, while optogenetics only need very little time.</p>
+
 
−
       
+
                <h4>1.4.2.2 Week2 -- August 8</h4>    
−
        <div class="fivePx"></div>
+
                <p>1, Replace the old LED source with the new one we bought.</p>
−
        <p class="titleTwo" id="sixth4">6.4 Conclusion</p>
+
            </div>
−
        <p class="contentP">We can see from the material above that optogenatics has unlimited potential in real life’s medical care. We still need to work on this topic for a long time!</p>
+
         </div>
−
       
+
−
        <div class="fivePx"></div>
+
−
        <p class="titleTwo" id="sixth5">6.5 Reference</p>
+
−
        <p class="reference">[1] KlingenbegrM, HuangSG. Structure and function of the uncoupling protein from brown adipose issue, Biochem, Biophy, 1999, 271-296.</p>
+
−
        <p class="reference">[2] Study of optogenetics, YiZhang Chen, Frontier.</p>
+
−
         <p class="reference">[3] Optogenetics technique can be used on repairing damaged dopaminergic neuron, Dan Wang, Health Daily.</p>
+
−
        <p class="reference">[4] Optogenetics technique help regaining the function of paralyzed muscle, Dan Chen.Science and Technology Daily.</p>
+
  
−
        <br>
 
 
         <div class="divider"></div>
 
         <div class="divider"></div>
 +
 +
<p class="titleOne" id="Timeline">2. Timeline</p>             
 +
        <div class="divider"></div>
 +
        <center><img class="hoverHand bigImgShow" width="800" src="https://static.igem.org/mediawiki/2015/b/bd/China-Tongji-Notebook-timeline-1600.png" title="800,600" alt="Notebook -- Timeline" ></center>
 +
        <div class="divider"></div>
 +
 +
            <br>
 
</div>
 
</div>
 
</div>
 
</div>
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Latest revision as of 12:55, 18 September 2015

close label

Notebook

  • 1. Record

    • 1.1 Plasmid Part
    • 1.2 Worm Part
    • 1.3 Efficiency Part
    • 1.4 Equipment Part

  • 2. Timeline

    • 1. Record

    1.1 Plasmid Part

    1.1.1 JUNE -- Week 1~4 -- June 6~25[Expand]

    1.1.1.1 Week1 -- June 6

    June 6

    1, Design the PCR primers of chR2-YFP.

    1.1.1.2 Week2 -- June 8~14

    June 8

    1, The amplification of CHR2-YFP (use taq PCR protocol)

    2, AGE ( agarose gel electrophoresis )

    3, Gel extraction of chR2-YFP.

    4, Transformation of GFP, YFP, mcherry in E•coli DH5α.

    June 9

    1, Select a single clone of each plate. (GFP, YFP, mcherry) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.

    2, Transformation of vector with pmyo-3(ppd95.77)

    June 10

    1, Select a single clone of plate. (pmyo-3,ppd95.77) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.

    2, Plasmid extraction ofE.coli DH5αwith GFP, YFP and mcherry in it.

    June 11

    1, Plasmid extraction of pmyo-3(ppd95.77)

    2, Digestion of ppd95.77 with pmyo-3 in it and chR2-YFP using BamHI and EcoRI.(digestion protocol)

    June 12

    1, AGE ( agarose gel electrophoresis ) of digested vector---ppd95.77 with pmyo-3.

    2, Gel extraction of pmyo-3.

    3, purification of chR2-YFP witch has been digested with BamHI and EcoRI.

    June 14

    1, Transform of GFP, YFP, mcherryin E•coli BL21.

    1.1.1.3 Week3 -- June 15~21

    June 15

    1, Ligation of pmyo-2 and chR2-YFP. (ligation protocol)

    2, Transformation of ligation product: pmyo3-chR2-YFP (in ppd95.77).

    June 17

    1, Select a single clone of plate. (pmyo-3-chR2-YFP, ppd95.77) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.

    2, Plasmid extraction of pmyo3-chR2-YFP.

    June 19

    1, Digestion of new plasmid: pmyo3-chR2-YFP using HindIII and EcoRI to check if the ligation step work or not. (it worked! Our first part is done!)

    June 20

    1, Transformation of pmyo3-chR2-YFP in order to get more plasmid.

    2, Transformation of pmec3-chR2-YFP, pmec3-dsred and pmec4-chR2-YFP which are offered by professor Li’s lab.

    3, Save the E.coli strain with glycerinum.

    June 21

    1, Theamplification of dsred and pmyo-2(use taq PCR protocol---to test the best temperature for the PCR).

    2, the amplification of dsred and pmyo-2(use pfu PCR protocol).

    3, AGE ( agarose gel electrophoresis ) of pmyo-2 and dsred.

    4, Gel extraction of pmyo-2 and dsred.

    1.1.1.4 Week4 -- June 22~25

    June 22

    1, digestion of pmyo3-chR2-YFP(using HindIII and BamHI)

    2, AGE ( agarose gel electrophoresis ) of digested vector---ppd95.77 with chR2-YFP.

    3, Gel extraction of pmyo-3(PPD95.77).

    4, Digest of pmyo2 with HindIII and BamHI. Digest of dsred with BamHI and EcoRI.

    5, gene purification for pmyo2 and dsred.

    June 24

    1, Ligation of pmyo2 with chR2-YFP (in ppd95.77).

    2, Ligation of dsred with pmyo3 and pmyo2(in ppd95.77)

    June 25

    1, Digest of pmyo2-chR2-YFP and pmyo2-dsred and pmyo3-dsred.(usingHindIII and EcoRI) To check if we had ligated it successfully.

    2, AGE ( agarose gel electrophoresis ) of digested products. Analyze the result.

    1.1.2 JULY -- Week 2~5 -- July 11~30[Expand]

    1.1.2.1 Week2 -- July 11~14

    July 11

    1, Design the PCR primers of Blink gene.

    2, Design the PCR primers of pttx-3.

    3, Design the PCR primers of ptrp4.

    4, Prepare for competent cells

    July 14

    1, Transformation of the Blink plasmid which was kind offered by Anna’s lab.

    2, Transformation of pmyo2-chR2-YFP, pmyo2-dsred and pmyo3-dsred in order to get more plasmids.

    3, GFP, YFP, mcherry transform OP50 and PA14. (OP50 and PA14 are the food of C.elegans)

    1.1.2.2 Week3 -- July 15~21

    July 15

    1,Select single clones of plate. (pmyo-2-chR2-YFP, pmyo2-dsred and pmyo3-dsred ppd95.77) . Put the E.coli in 4ml LB buffer and cultivate for one night at 37℃.

    July 16

    1, Plasmid extraction of pmyo-2-chR2-YFP, pmyo2-dsred and pmyo3-dsred.

    July 17

    1, Recovery of

    pNP260( Pnmr-1::flox::ChR2::mCherry.)

    pCoS2(pnhr-79::Cre):

    pCoS13(posm-10::loxP::LacZ::STOP::loxP:: ChR2::mCherry::SL2::GFP)

    pSH116 (pdes-2::Cre)

    which are kind offered by Alexander Gottschalk’ lab.

    2, Transformation of pNP260, pCoS2, pCoS13 and pSH116.

    3, Design of the PCR primers of pttx-HindIII-F and pttx3-Xbal-R.

    July 18

    1, Select single clones of plate.(pNP260, pCoS2, pCoS13 and pSH116). Put the E.coli in 4ml LB buffer and cultivate for 12h at 37℃.

    2, Plasmid extraction.

    3, Make genome DNA from worms.

    4, Make LB plate and LB liquid.

    5, Repeat the experiment: Prepare for competent cells: GFP, YFP, mcherry transform OP50 and PA14.

    6, The amplification of Blink.

    7, AGE ( agarose gel electrophoresis ) of blink PCR products. Analyze the result.

    8, Gel extraction. (all at 300ng/ul)

    July 19

    1, Use the C.elegans genomic DNA as template and got pttx-3 with the help of Pttx3-HindIII-F and pttx-3-XbaI-R.

    2, AGE ( agarose gel electrophoresis ) of PCR products. Analyze the result.

    July 20

    1, Transformation of P Blue plasmid.(used to make the mixture of microinjection liquid)

    2, Select single clone on the culture plate.Put the E.coli in 4ml LB buffer and cultivate for 12h at 37℃.

    3, Select a single clone of each plate and put each one in a tube that contain the ampicillin and 1ml LB culture media. (OP50 and PA14)

    4, At 37℃ we culture them for 12h.

    July 21

    1, Add the mixture to a new 15ml tube and add in 4ml LB culture media.

    2, Culture them for 3h until the OD number near 0.8.

    3, Add 1mg IPTG in the liquid.

    4, Culture them for 3h in order to gain the protein.

    5, Plasmid extraction of P blue.

    6, Digestion: Use EcoR I and BamH I digest the pmyo2 and pmyo3 plasmid and the Blink. (use digestion protocol)

    7, Ligase reaction. (use ligation protocol)( pmyo2-blink,pmyo3-blink)

    1.1.2.3 Week4 -- July 22~27

    July 22

    1, Design the PCR plasmid of chETA and ic1c2 which we bought from addgene.

    2, Cultivation of plasmid chETA and ic1c2 from Addgene.Pick a loop of bacteria from the sample, streaking on Amp+ plates, cultivate in 37℃ for 12h.

    3, Transform DH5α(pmyo2-blink,pmyo3-blink). Label the plate and put it in the incubator about one night, 37℃.

    July 23

    1, Amplify of plasmid chETA and ic1c2 from Addgene.Pick 5 single clone from each plate, add in 4ml LB medium separately, and shaking in 37℃ for 14h.

    2, Make LB plate.

    3, Select a single clone and culture for 12h of each template. (pmyo2-blink,pmyo3-blink)

    July 24

    1, Plasmid extraction of plasmid chETA and ic1c2. (the results are all around 100ng/ul)

    2, Taq PCR of chETA and ic1c2. (to test the best reaction tempareture)

    3, AGE ( agarose gel electrophoresis ) of PCR products. Analyze the result. We found that 68℃ is the best temperature.

    4, Plasmid Extraction. (pmyo2-blink,pmyo3-blink) the results are all at 200-300ng/ul.

    5, Ligase reaction (again): pmyo2-blink, pmyo3-blink.

    July 25

    1, Pfu PCR of chETA and ic1c2.

    2, AGE ( agarose gel electrophoresis ) of pfu PCR products.

    3, Gel extraction and recycle the chETA and ic1c2.

    4, Used the C.elegans genomic DNA as template and got pttx-3 with the help of Pttx3-HindIII-F and pttx-3-XbaI-R.

    5, Algarose gel electrophoresis to test PCR result.

    6, Gel extraction and recycle the pttx-3.

    7, Transform DH5α(pmyo2-blink, pmyo3-blink)

    8, Select single clone from culture plate. And culture for 12h.

    July 26

    1, Plasmid Extraction (pmyo2-blink, pmyo3-blink)

    July 27

    1, Repeat the experiment: used the C.elegans genomic DNA as template and got pttx-3 with the help of Pttx3-HindIII-F and pttx-3-XbaI-R.

    2, Ran the Algarose gel electrophoresis to test my PCR result.

    3, After confirming the accuracy of my PCR result, we did an gel extraction and recycle the pttx-3.

    4, Transformation of pmyo2, pmyo3 plasmids to get more for the latter experiment.Culture at 37℃ for 16h.

    5, Pfu PCR of chETA and ic1c2.

    6, AGE ( agarose gel electrophoresis ) of pfu PCR products.(chETA and ic1c2)

    7, Gel extraction and recycle the chETA and ic1c2.

    8, Select single clone of AMP LB plate.And culture for 12h.

    1.1.2.4 Week5 -- July 28~31

    July 28

    1, Plasmid Extraction(pmyo2, pmyo3---ppd95.77).

    July 29

    1, Make Backbone, transformation of backbone.Culture at 37℃ for 16h. (according by protocol offered by iGEM)

    2, DigestchETA and ic1c2 genes, pmyo2 and pmyo3 vectors with BamHI and EcoRI.(digestion protocol)

    3, Ligation of pmyo2-chETA, pmyo2-ic1c2, pmyo3-chETA and pmyo3-ic1c2. (ligation protocol)

    July 30

    1, Transformation of pmyo2-chETA, pmyo2-ic1c2, pmyo3-chETA and pmyo3-ic1c2.Culture at 37℃ for 16h.

    2, Select single clone from culture plate (pmyo2-chETA, pmyo2-ic1c2).And culture for 12h.(there is no clone of pmyo3-chETA and pmyo3-ic1c2 )

    3, Select single clone from culture plate.(Backbone)

    July 31

    1, Plasmid Extraction (pmyo2-chETA, pmyo2-ic1c2).

    2, Plasmid Extraction (Backbone).

    3, Digest of pmyo2-chETA and pmyo2-ic1c2 using BamHI and EcoRI. Make sure that the gene had been successfully ligated into the plasmids.

    4, Digest of backbone with PstI and EcoRI. Make sure our backbone is made in the right way.

    1.1.3 AUGUST -- Week 1~5 -- August 1~31[Expand]

    1.1.3.1 Week1 -- August 1~7

    August 1

    1, Transformation of pmyo2-chETA and pmyo2-ic1c2 in order to get more plasmids.Culture at 37℃ for 16h.

    2, Select single clone from culture plate (pmyo2-chETA and pmyo2-ic1c2.).And culture for 12h.

    3, Try to ligate pmyo3-chETA and pmyo3-ic1c2 again as last time we failed. Digestion and ligation.

    4, Transformation of pmyo3-chETA and pmyo3-ic1c2, Culture at 37℃ for 16h.

    August 2

    1, Select single clone from culture plate (pmyo3-chETA and pmyo3-ic1c2.).And culture for 12h.

    2, Plasmid Extraction (pmyo2-chETA, pmyo2-ic1c2).

    3, Plasmid Extraction (pmyo3-chETA, pmyo3-ic1c2).

    August 3

    1, Digest ofpmyo3-chETA, pmyo3-ic1c2 to check if we had ligated them right. (the result turn out that the pmyo3-chETA is right)

    2, Transformation of pmyo3-chETA in order to get more plasmids.

    3, Give pmyo2-chR2, pmyo2-chETA, pmyo2-ic1c2, pmyo3-chR2, pmyo3-chRTA and pmec4-dsred to company to test the sequences.

    August 4

    1, Select single clone from culture plate (pmyo3-chETA.).And culture for 12h.

    2, Plasmid Extraction (pmyo3-chETA).

    3, Pfu PCR of pttx-3 from C.elegans genome.

    4, AGE ( agarose gel electrophoresis ) of pfu PCR products.(pttx3) However, nothing out.

    5, Trying to ligate pmyo2-ic1c2 once again.Then transformation of it.Cultured in 37℃ for 16h.

    August 5

    1, Select single clone from culture plate (pmyo2-ic1c2).And culture for 12h.

    2, Plasmid extraction.( pmyo2-ic1c2)

    3, Give pmyo2-ic1c2 to company, and let it test the sequence.

    4, Pfu PCR of pttx-3 from C.elegans genome again. (use different program and different temperature,)

    5, AGE ( agarose gel electrophoresis ) of pfu PCR products.(pttx-3)

    6, Gel extraction and recycle the pttx-3. (10ng/ul)

    August 6

    1, Digest of pttx-3 with SalI and BamHI. (using digestion protocol)

    2, Digest of the new ppd95.77 vector with SalI and BamHI. (using digestion protocol)

    3, Ligate of pttx-3 into ppd95.77. (using ligation protocol)

    4, Transformation of pttx-3 in ppd95.77. culture in 37℃ for 16h.

    August 7

    1, Yesterday’s transformation has no clone grow.

    2, Pfu PCR of pttx-3 again.

    3, Design the primers of pmec-4. (add HindIII and BamHI)

    4, AGE ( agarose gel electrophoresis ) of pfu PCR products.(pttx-3)

    5, Gel extraction and recycle the pttx-3. (13ng/ul)

    6, Ligate of pttx-3 into ppd95.77. (using ligation protocol)

    7, Transformation of pttx-3 in ppd95.77. culture in 37℃ for 16h.

    1.1.3.2 Week2 -- August 8~13

    August 8

    1, Yesterday’s transformation has still no clone grow.

    2, Have a lab meeting about why our ligation has so many problems. We decided to try a new method---seamless cloning.

    August 9

    1, As we are going to do seamless cloning, we have to design new primers of pttx-3, chR2, chETA, dsRed and ic1c2, blink.

    August 10

    1, Make LB liquid.Make LB AMP plates.

    2, Taq PCR of pttx-3, chR2, chETA, dsRed and ic1c2, blink to test the best temperature of PCR reaction.

    3, AGE ( agarose gel electrophoresis ) of taq PCR products. (pttx-3, chR2, chETA, dsRed and ic1c2)

    4, Pfu PCR of pttx-3.

    August 11

    1, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx-3)

    2, Gel extraction and recycle the pttx-3. (17ng/ul)

    3, Digest of ppd95.77 with SalI and BamHI.

    4, Seamless clone of pttx-3 into nppd95.77. (use seamless clone protocol)

    5, Transformation of pttx-3 ppd95.77. culture for 16h on AMP LB plate in 37℃.

    August 12

    1, Select single clone on AMP LB plate.(pttx-3 ppd95.77) culture in 37℃ for 12h.

    2, Plasmid extraction of pttx-3 ppd95.77.

    3, Digest of pttx-3 with SalI and BamHI to test the ligation result. (turns out to be right!)

    4, Transformation of pttx-3 ppd95.77 in order to get more right plasmids.

    5, Pfu PCR of chR2, chETA, dsRed and ic1c2, blink.

    6, AGE ( agarose gel electrophoresis ) of pfu PCR products. (chR2, chETA, dsRed and ic1c2, blink)

    7, Gel extraction and recycle the chR2, chETA, dsRed and ic1c2, blink. (around 80ng/ul)

    8, Digest 1.5 ul of pttx-3 ppd95.77.

    9, Seamless clone of pttx-3 with chR2, chETA, dsRed, ic1c2 and blink. (seamless clone protocol)

    10, Transformation of pttx-3-chR2, PTTX-3-chETA, PTTX-3-dsRed, pttx-3-ic1c2 and pttx-3-blink.Cultured in 37℃ for 16h.

    August 13

    1, Select single clone of pttx-3-chR2, pttx-3-chETA, pttx-3-dsRed and pttx-3-ic1c2 (pttx-3-blink has no clone.). Cultured at 37℃ for 12h.

    2, Plasmid extraction. (pttx-3-chR2, pttx-3-chETA, pttx-3-dsRed and pttx-3-ic1c2)

    3, Digest of (pttx-3-chR2, pttx-3-chETA, pttx-3-dsRed and pttx-3-ic1c2 with BamHI and EcoRI to test if we had successfullyligate the gene into the vector, which turn out that these are all right.

    4, Send them to company for a sequence test.

    1.1.3.3 Week3 -- August 15~21

    August 15

    1, Pfu PCR of blink again.

    2, AGE ( agarose gel electrophoresis ) of pfu PCR products. (blink)

    3, Gel extraction and recycle the blink. (around 50ng/ul)

    4, Digest of pttx-3 ppd95.77 with BamHI and EcoRI.

    5, Seamless clone of pttx-3-blink.

    6, Transformation of pttx3-blink. Culture in 37℃ for 16h.

    7, Taq PCR of ptwk16 to test the best reaction situation.

    8, AGE ( agarose gel electrophoresis ) of taq PCR products. (ptwk16)

    9, Pfu PCR of ptwk16.

    August 16

    1, Select single clone of pttx-3-blink.Cultured at 37℃ for 12h.

    2, Plasmid extraction of pttx-3-blink.

    3, AGE ( agarose gel electrophoresis ) of pfu PCR products. (ptwk16)

    4, Gel extraction and recycle the blink. (around33ng/ul)

    5, Digest of ptwk16 with HindIII and SalI. Digest the vector ppd95.77 with HindIII and SalI.

    6, Ligation of ptwk16 into ppd95.77 with T4 ligase. (12h)

    7, AGE ( agarose gel electrophoresis ) of pttx-3-blink to test if we got the right result.

    August 17

    1, Transformation of ptwk16 ppd95.77. culture in 37℃ for 16h.

    2, Transformation of the back bone we made in order to get more to prepare for the later backbone making.Culture in 37℃ for 16h.

    August 18

    1, Select single clone of ptwk16 ppd95.77. Cultured at 37℃ for 12h.

    2, Select single clone of backbone.Cultured at 37℃ for 12h.

    3, Plasmid extraction of ptwk16 ppd95.77 and backbone.

    4, Digest of ptwk16 ppd95.77 with HindIII and SalI to test if we had ligated it in a right way.

    Augest 19

    1, Pfu PCR of blink, chR2, dsred, ic1c2 and chETA.

    2, AGE ( agarose gel electrophoresis ) of pfu PCR products. (blink, chR2, dsred, ic1c2 and chETA)

    3, Gel extraction and recycle the blink. (around 50ng/ul)

    4, Digest of ptwk16 ppd95.75 with BamHI and HindIII.

    5, Seamless clone of ptwk16-blink, ptwk16-chR2, ptwk16-dsred, ptwk16-ic1c2 and ptwk16-chETA.

    6, Transformation of ptwk16-blink, ptwk16-chR2, ptwk16-dsred, ptwk16-ic1c2 and ptwk16-chETA.Cultured in 37℃ for 16h.

    August 20

    1, Select the single clone of ptwk16-blink, ptwk16-ic1c2 and ptwk16-chETA. (ptwk16-chR2, ptwk16-dsred has not been ligated successfully.) culture in 37℃ for 12h

    2, plasmid extraction of ptwk16-blink, ptwk16-ic1c2 and ptwk16-chETA.

    3, Digest of ptwk16-blink, ptwk16-ic1c2 and ptwk16-chETA with BamHI and EcoRI to test if we had ligated them in the right way. (It turns out to be right.)

    4, Ligate of ptwk16-chR2, ptwk16-dsRed again.

    5, Transformation of ptwk16-chR2, ptwk16-dsred.Culture in 37℃ for 16h.

    August 21

    1, Select the single clone of ptwk16-blink, ptwk16-ic1c2 and ptwk16-chETA. (ptwk16-chR2, ptwk16-dsred) culture in 37℃ for 12h

    2, Plasmid extraction of ptwk16-chR2, ptwk16-dsred.

    3,digest of ptwk16-chR2, ptwk16-dsred with BamHI and EcoRI to test if the result is right.

    4, Start to make backbone which we are going to send to iGEM. Design the seamless clone PCR primers of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA, pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.

    5, Make some chloramphenicol LB plates.

    1.1.3.4 Week4 -- August 22~28

    August 22

    1, Taq PCR of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA, pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA to test the best reaction situation.

    2, AGE ( agarose gel electrophoresis ) of these taq PCR products.

    3, Pfu PCR of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.

    4, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.)

    5, Gel extraction and recycle the blink. (around 50ng/ul)

    6, Digest of backbone with PstI and EcoRI.

    7, AGE ( agarose gel electrophoresis ) of digestion products.

    8, Gel extraction and recycle the blink. (around 30ng/ul)

    August 23

    1, Seamless clone of backbone---pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.

    2, Transformation of backbone---pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.Culture on chloramphenicol LB plates in 37℃ for 16h.

    3, Select the single clone of backbone---pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA. Culture onchloramphenicol LB plates in 37℃ for 12h.

    4, Pfu PCR of pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.

    5, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.) However, nothing came out.

    August 24

    1, Plasmid extraction of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA.

    2, Digest of pmyo2-chR2, pmyo2-dsred, pmyo2-blink, pmyo2-ic1c2, pmyo2-chETA, pmyo3-chR2, pmyo3-dsred, pmyo3-blink, pmyo3-ic1c2, pmyo3-chETA to check if we had ligate the right parts into plasmid.

    3, Send them to company to test the sequence.

    4, Pfu PCR of pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA again. (change the reaction temperature.)

    5, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-chR2, pttx3-dsred, pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-chR2, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.) pttx3-chR2, pttx3-dsred and ptwk16-chR2 show on the gel.

    6, Gel extraction and recycle the pttx3-chR2, pttx3-dsred and ptwk16-chR2.

    August 25

    1, Digest the backbone with EcoRI and PstI.

    2, Seamless clone of backbone--- pttx3-chR2, pttx3-dsred and ptwk16-chR2.

    3, Transformation of backbone---pttx3-chR2, pttx3-dsred and ptwk16-chR2.Cultured on chloramphenicol LB plates in 37℃ for 19h.

    August 26

    1, Onlybackbone-ptwk16-chR2 grown some clones on the plate. Select single clone on the plate. Cultured it on chloramphenicol LB plates in 37℃ for 19h.

    2, Plasmid extraction of backbone-ptwk16-chR2.

    3, Digest of backbone-ptwk16-chR2 to make sure that we had made the right backbone.

    August 27

    1, Design new primers of pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA.

    2, As we couldn’t have PstI, EcoRI, SpeI and BamHI in our backbone plasmid, we have to do some point mutation.

    3, The overlap PCR of backbone---pmyo2-chR2, pmyo2-dsred, pmyo3-chR2 to mutate the PstI site in them.

    August 28

    1, The overlap PCR of backbone---pmyo2-chR2, pmyo2-dsred, pmyo3-chR2 to mutate the PstI site in them.

    2, Pfu PCR of pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA using new primers to test the best reaction situation.

    3, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA)

    4, Pfu PCR of pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA using new primers.

    1.1.3.5 Week5 -- August 29~31

    August 29

    1, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA) we got pttx3-blink and ptwk16-ic1c2.

    2, Gel extraction and recycle pttx3-blink and ptwk16-ic1c2.

    3, Digest of backbone with PstI and EcoRI.

    4, Seamless clone of backbone-pttx3-blink and ptwk16-ic1c2.

    5, Transfomation of backbone-pttx3-blink and ptwk16-ic1c2.Culture in 37℃ for 19h.

    6, Point mutation of our backbone products.

    August 30

    1, Select the single clone of ptwk16-ic1c2 on chloramphenicol LB plates. (another has no clone) cultured in 37℃ for 19h.

    2, Point mutation of our backbone products.

    3, Point mutation of our backbone products.

    August 31

    1, Plasmid extraction of ptwk16-ic1c2.

    2, Digest ptwk16-ic1c2 with PstIEcoRI to check our result.

    3, Point mutation of our backbone products.

    1.1.4 SEPTEMBER -- Week 1~2 -- September 1~10[Expand]

    1.1.4.1 Week1 -- September 1~7

    September 1

    1, Pfu PCR of pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA for the last time.

    2, Point mutation of our backbone products. (Some of them failed.)

    September 2

    1, AGE ( agarose gel electrophoresis ) of pfu PCR products. (pttx3-blink, pttx3-ic1c2, pttx3-chETA, ptwk16-dsred, ptwk16-blink, ptwk16-ic1c2, ptwk16-chETA) we got ptwk16-blink and ptwk16-chETA.

    2, Gel extraction and recycleptwk16-blink and ptwk16-chETA.

    3, Send all the backbone we have now to company to test the sequence.

    September 3

    1, Digest of backbone with PstI and EcoRI.

    2, Seamless cloning of backbone-ptwk16-blink and ptwk16-chETA.

    3, Transformation into chloramphenicol LB plates.Cultured in 37℃ for 19h.

    4, Make more LB AMP plates.

    September 4

    1, Select single clone on the plate. Culture in 37℃ for 13h.

    2, Plasmid extraction of backbone-ptwk16-blink and ptwk16-chETA.

    3, Digest of backbone-ptwk16-blink and ptwk16-chETA to test. The result is strange which means that we may failed in this ligation.

    September 5

    1, Try a new method to make the failed backbones.Digest the backbone-pmyo2-blink and pmyo2-chETA with BamHI and SpelI.Digest the ptwk16 out of the plasmid with HindIII and SalI.

    2, Pfu PCR of ptwk16.

    3, AGE ( agarose gel electrophoresis ) of pfu PCR products. (ptwk16)

    4, Gel extraction and recycleptwk16.

    September 6

    1, Traditional ligation of backbone-ptwk16-blink and ptwk16-chETA.

    2, Transformation of backbone-ptwk16-blink and ptwk16-chETA.Culture in 37℃ for 13h

    September 7

    1, Select single clone on the plate of backbone-ptwk16-blink and ptwk16-chETA. Culture in 37℃ for 13h.

    2, Plasmid extraction of backbone-ptwk16-blink and ptwk16-chETA.

    3, Digest test.

    1.1.4.2 Week2 -- September 9~10

    September 9

    1, Make our sending plasmid powder by freeze dryer.

    September 10

    1, Make our sending plasmid powder by freeze dryer.

    1.2 Worm Part

    1.2.1 JUNE -- June 20~29[Expand]

    1.2.1.1 June 20~29

    Learn how to do the microinjection on C.elegans.

    1.2.2 JULY -- Week 2~5 -- July 9~31[Expand]

    1.2.2.1 Week2 -- July 9~13

    July 9

    1, Preparation of NGM plates.

    2, Seed NGM plates.

    July 11

    1, Recover the L1 larva of lite-1 worm from -80 fridge.

    July 13

    1, Last time the recovery failed. Recover the L1 larva of lite-1 worm again from -80 fridge.

    1.2.2.2 Week3 -- July 15~21

    July 15

    1, The recovery still failed. Recover the L1 larva of lite-1 worm once again!(this time we successes!)

    July 21

    1, Seed plates with OP50 and ATR (keep in dark place). For 10 3cm plateS(1.5ml agar EACH),seed 1000ul OP50 mixed with 1.75ul 100uM ATR. The OP50 E.coli should be shacked for 12 hours in the conical flask at 37℃(150ml LB added 4-5 single colonies. )

    2, Microinjection pmyo-3::dsRed and Pmyo-3::ChR2 co-injection.(40 worms)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo3::dsrRed 20ng/ul 144.7ng/ul 1.38ul
    Pmyo-3::ChR2 80ng/ul 174.7ng/ul 4.58ul
    PBlue 120ng/ul 162.5ng/ul 2.04ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0
    Total 10ul

    1.2.2.3 Week4 -- July 23~28

    July 23

    1, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred.Put the worm on a new NGM plate.

    2, Preparation of some new NGM plates.

    3, Seed NGM plates.

    July 24

    1, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred.Put the worm on a new NGM plate.

    July 25

    1, Subculture of C.elegans which had been injection of pmyo-3::dsRed and Pmyo-3::ChR2.

    2, Subculture of lite1C.elegans.

    July 26

    1, Make ATR solution. (help chR2 work in C.elegan)

    2, Subculture of C.elegans which had been injection of pmyo-3::dsRed and Pmyo-3::ChR2.

    3, Subculture of lite1C.elegans.

    July 27

    1, Subculture of all the C.elegans we have.

    2, Put some L4 pmyo3-dsred and pmyo3-chR2 C.elegans on ATR plate.

    July 28

    1, Microinjection.(pmyo-2::dsRed and Pmyo-2::ChR2 co-injection) for 40 worms.

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo2::dsrRed 20ng/ul 209.7ng/ul 0.95ul
    Pmyo2::ChR2 50ng/ul 549ng/ul 0.91ul
    PBlue 120ng/ul 331.2ng/ul 3.62ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 2.52ul
    Total 10ul

    1.2.2.4 Week5 -- July 29~31

    July 29

    1, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo2 will express at the worm neck) Put the worm on a new NGM plate.

    2, Subculture of all the C.elegans we have.

    July 30

    1, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo2 will express at the worm neck) Put the worm on a new NGM plate.

    2, Subculture of all the C.elegans we have.

    July 31

    1, Microinjection.( PNP260and PSH116 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    PNP260 50ng/ul 471ng/ul 1.06ul
    PSH116 50ng/ul 516ng/ul 0.96ul
    PBlue 90ng/ul 271ng/ul 4.42ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 1.56ul
    Total 10ul

    2, subculture of all the C.elegans we have.

    3, put some L4 pmyo-2::dsRed and Pmyo-2::ChR2 C.elegans on ATR plate.

    1.2.3 AUGUST -- Week 1~5 -- August 1~31[Expand]

    1.2.3.1 Week1 -- August 1~6

    August 1

    1, Microinjection.( pmyo-3::dsRed and Pmyo-3::ChR2 co-injection AGAIN! Last time we failed)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo2-dsred 20ng/ul 144.7ng/ul 1.38ul
    Pmyo2-chR2 50ng/ul 120.8ng/ul 4.41ul
    PBlue 120ng/ul 271ng/ul 2.5ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0ul
    Total 10ul

    2, Subculture of all the C.elegans we have.

    3, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle) Put the right worm on a new NGM plate.

    August 2

    1, Microinjection.( pmec-3::dsRed and Pmec-3::ChR2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmec3-dsred 20ng/ul 159ng/ul 1.25ul
    Pmec3-chR2 50ng/ul 190ng/ul 2.6ul
    PBlue 120ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.6ul
    Total 10ul

    2, Microinjection.(Pcos13 and Pcos2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    PCoS13 50ng/ul 191.5ng/ul 2.6ul
    PCoS2 50ng/ul 327.2ng/ul 1.5ul
    PBlue 90ng/ul 271ng/ul 3.3ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.6ul
    Total 10ul

    3, Subculture of all the C.elegans we have.

    4, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle) Put the right worm on a new NGM plate.

    5, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmec3 will express at the worm’s touch neuron) Put the right worm on a new NGM plate.

    August 3

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle, (pmec3 will express at the worm’s touch neuron, PCoS13 overlap PCos2 will express at AVA neuron, PNP260 overlap PSH116 will express at PVC neuron).Put the right worm on a new NGM plate.

    3, Preparation of some new NGM plates.

    4, Seed NGM plates.

    5, Seed new ATR plates.

    August 4

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle, (pmec3 will express at the worm’s touch neuron, PCoS13 overlap PCos2 will express at AVA neuron, PNP260 overlap PSH116 will express at PVC neuron). Put the right worm on a new NGM plate.

    August 5

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected. We are supposed to select the worm with dsred. (pmyo3 will express at the worm’s whole body muscle, (pmec3 will express at the worm’s touch neuron, PCoS13 overlap PCos2 will express at AVA neuron, PNP260 overlap PSH116 will express at PVC neuron). Put the right worm on a new NGM plate.

    August 6

    1, Subculture of all the C.elegans we have.

    1.2.3.2 Week2 -- August 8~14

    August 8

    1, Subculture of all the C.elegans we have.

    August 9

    1, Subculture of all the C.elegans we have.

    2, Since the some of C.elegans we injected has not been passaged stalely, so we had to reinject some of them.

    Microinjection.( pmec-3::dsRed and Pmec-3::ChR2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmec3-dsred 20ng/ul 159ng/ul 1.25ul
    Pmec3-chR2 50ng/ul 190ng/ul 2.6ul
    PBlue 120ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.6ul
    Total 10ul

    Microinjection.( pmyo-2::dsRed and Pmyo-2::ChR2 co-injection) for 40 worms.

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo2::dsrRed 20ng/ul 209.7ng/ul 0.95ul
    Pmyo2::ChR2 50ng/ul 549ng/ul 0.91ul
    PBlue 120ng/ul 331.2ng/ul 3.62ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 2.52ul
    Total 10ul
    August 10

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected.

    August 11

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected.

    August 12

    1, Microinjection.( pmyo-3::dsRed and Pmyo-3::ic1c2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo3-dsred 20ng/ul 159ng/ul 1.25ul
    Pmyo3-ic1c2 50ng/ul 187ng/ul 2.6ul
    PBlue 120ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.6ul
    Total 10ul

    2, Microinjection.( pmyo-3::dsRed and Pmyo-3::chETA co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo3-dsred 20ng/ul 159ng/ul 1.25ul
    Pmyo3 chETA 50ng/ul 106ng/ul 2.9ul
    PBlue 120ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.3ul
    Total 10ul

    3, Subculture of all the C.elegans we have.

    4, Select the F1 generation of the worm we injected.

    August 13

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected.

    August 14

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected.

    1.2.3.3 Week3 -- August 15~21

    August 15

    1, Microinjection.( pmyo-2::dsRed and Pmyo-2::chETA co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo2-dsred 20ng/ul 128ng/ul 1.25ul
    Pmyo2-chETA 50ng/ul 106ng/ul 2.9ul
    PBlue 120ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.3ul
    Total 10ul

    Microinjection.( pmyo-2::dsRed and Pmyo-2::ic1c2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo2-dsred 20ng/ul 156ng/ul 1.25ul
    Pmyo2-ic1c2 90ng/ul 178ng/ul 2.55ul
    PBlue 70ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.75ul
    Total 10ul

    2, Subculture of all the C.elegans we have.

    3, Select the F1 generation of the worm we injected.

    August 16

    1, Microinjection.(pttx-3::dsRed and Pttx-3::chR2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    pttx-3::dsRed 20ng/ul 128ng/ul 1.25ul
    Pttx-3::chR2 80ng/ul 200ng/ul 2.9ul
    PBlue 90ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.3ul
    Total 10ul

    Microinjection.( pttx-3::dsRed and Pttx-3::BLINK co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    pttx-3::dsRed 20ng/ul 128ng/ul 1.25ul
    Pttx-3::BLINK 80ng/ul 213ng/ul 2.8ul
    PBlue 90ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.4ul
    Total 10ul

    Microinjection.( pttx-3::dsRed and Pttx-3::ic1c2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    pttx-3::dsRed 20ng/ul 128ng/ul 1.6ul
    Pttx-3::ic1c2 70ng/ul 167ng/ul 2.8ul
    PBlue 90ng/ul 331ng/ul 3.25ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.4ul
    Total 10ul

    2, Subculture of all the C.elegans we have.

    3, Select the F1 generation of the worm we injected.

    August 18

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected.

    August 19

    Microinjection.(Pcos13 and Pcos2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    PCoS13 50ng/ul 191.5ng/ul 2.6ul
    PCoS2 50ng/ul 327.2ng/ul 1.5ul
    PBlue 90ng/ul 271ng/ul 3.3ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.6ul
    Total 10ul

    Microinjection.( PNP260and PSH116 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    PNP260 50ng/ul 471ng/ul 1.06ul
    PSH116 50ng/ul 516ng/ul 0.96ul
    PBlue 90ng/ul 271ng/ul 4.42ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 1.56ul
    Total 10ul
    August 20~21

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected.

    1.2.3.4 Week4 -- August 22~27

    August 22

    1, Microinjection.( ptwk16-dsRed and ptwk16-blink co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    ptwk16-dsRed 50ng/ul 187ng/ul 1.06ul
    ptwk16-blink 90ng/ul 213ng/ul 0.96ul
    PBlue 50ng/u 271ng/ul 4.42ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 1.56ul
    Total 10ul

    Microinjection.( ptwk16-dsRed and ptwk16-chR2 co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    ptwk16-dsRed 50ng/ul 197ng/ul 1.06ul
    ptwk16-chR2 90ng/ul 254ng/ul 0.93ul
    PBlue 50ng/u 271ng/ul 4.42ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 1.59ul
    Total 10ul

    2, Subculture of all the C.elegans we have.

    3, Select the F1 generation of the worm we injected.

    August 23~24

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected.

    August 25

    1, Subculture of all the C.elegans we have.

    2, Select the F1 generation of the worm we injected.

    3, Microinjection.( pmyo3-dsRed and pmyo3-blink co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    Pmyo3-dsRed 50ng/ul 197ng/ul 1.06ul
    Pmyo3-blink 90ng/ul 254ng/ul 0.93ul
    PBlue 50ng/u 271ng/ul 4.42ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 1.59ul
    Total 10ul

    Microinjection.( pttx-3::dsRed and Pttx-3::BLINK co-injection)

    Mixture:

    Material Final concentration Real concentration Real Volume
    pttx-3::dsRed 20ng/ul 128ng/ul 1.25ul
    Pttx-3::BLINK 80ng/ul 213ng/ul 2.8ul
    PBlue 90ng/ul 331ng/ul 3.6ul
    Rat Genomic DNA 10ng/ul 100ng/ul 1ul
    10XTE 1ul
    ddH2O 0.4ul
    Total 10ul
    August 26~27

    1, Subculture of all the C.elegans we have.

    1.2.3.5 Week5 -- August 29~31

    1, Subculture of all the C.elegans we have.

    1.2.4 SEPTEMBER -- Week 1~2 -- September 1~15[Expand]

    1, Subculture of all the C.elegans we have.

    1.3 Efficiency Testpart

    1.3.1 AUGUST -- Week 1~5 -- August 3~31[Expand]

    1.3.1.1 Week1 -- August 3~7

    August 3

    1, Initial test of pmyo3-chR2-YFP C.elegans.since we don’t have enough worms yet, we just test for 2 worm. We found their behavior have obvious change when we turn on the blue light. (470nm)

    August 5

    1, Initial test of pmyo2-chR2-YFP C.elegan. Since the chR2 is expressed at the neck, we didn’t see any big change of movement when we turn on the light.

    August 6

    1, Intial test of Pcos13 and Pcos2 C.elegan.

    August 7

    1, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP and Pcos13 overlap with Pcos2 C.elegans again.

    1.3.1.2 Week2 -- August 9~14

    August 9

    1, Trying to take some small video of the pmyo2-chR2-YFP, pmyo3-chR2-YFP and Pcos13 overlap with Pcos2 C.elegans.

    August 10

    1, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP and Pcos13 overlap with Pcos2 C.elegans.

    August 13

    1, Intial test of Pmyo-3-chETAC.elegan.

    August 14

    1, Intial test of Pmyo-3-ic1c2 C.elegan.

    1.3.1.3 Week3 -- August 15~21

    August 15

    1, Try to take some small video of thePmyo-3-chETA and Pmyo-3-ic1c2 C.elegans.

    2, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pcos13 overlap with Pcos2, Pmyo-3-chETA and Pmyo-3-ic1c2.

    August 16

    1, Intial test of Pmyo-2-ic1c2 andPmyo-2-chETA.

    August 17

    1, Try to take some small video of thePmyo-2-chETA and Pmyo-2-ic1c2 C.elegans.

    2, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pcos13 overlap with Pcos2, Pmyo-3-chETA Pmyo-3-ic1c2, Pmyo-2-ic1c2 and Pmyo-2-chETA.

    August 19

    1, Intial test of pttx3-chR2.

    2, Try to take some small video of thepttx3-chR2 C.elegans.

    August 20

    1, Lab meeting about how to take our video in a standard way.We decided to take 40sec each one: 10s for white light, 10s for 470nm light, 10 for white light and 10s for another wavelength light.In this way, we could use some video software to analyze the test result.

    August 21

    1, Intial test of ptwk16-blink.

    2, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pmyo-3-chETA Pmyo-3-ic1c2, Pmyo-2-ic1c2, Pmyo-2-chETA pttx3-chR2 and ptwk16-blink.

    3, Take video of these genetypeC.elegans.

    1.3.1.4 Week 4~5 -- August 23~30

    August 23,25,27,28,30

    1, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pmyo-3-chETA Pmyo-3-ic1c2, Pmyo-2-ic1c2, Pmyo-2-chETA pttx3-chR2 and ptwk16-blink.

    2, Take video of these genetypeC.elegans.

    1.3.2 SEPTEMBER -- Week 1~2 -- September 1~15[Expand]

    1.3.2.1 Week1 -- September 1~7

    1, Test of pmyo2-chR2-YFP, pmyo3-chR2-YFP, Pmyo-3-chETA Pmyo-3-ic1c2, Pmyo-2-ic1c2, Pmyo-2-chETA pttx3-chR2 and ptwk16-blink.

    2, Take video of these genetypeC.elegans.

    1.3.2.2 Week2 -- September 8~16

    1, Analyze the results with related video software.

    2, Still test of our C.elegans.

    1.4 Equipment Part

    1.4.1 JULY -- July 1,28[Expand]

    1.4.1.1 Week1 -- July 1

    1, We pick up the parts we need on the internet under the help of our instructor Wei Li. Ordered them on line.

    1.4.1.2 Week4 -- July 28

    1, We learn about how to build our LED light source together.

    1.4.2 AUGUST -- Week 1~2 -- August 1~8[Expand]

    1.4.2.1 Week1 -- August 1~7

    August 1

    1, All our equipment has been arrived, we started to build it. We met some problems during the process, for instance, some of the parts did not fit in with each other.

    August 2~3

    1, Build the LED light source.

    August 4

    1, Adjustment of light path.

    2, Ask for the help about how install and use the LED related software on the computer from microscope engineer.

    August 5

    1, After the first test, we found that our LED power is still not powerful enough. So we bought some new LED source on the internet.

    August 7

    1, Weld the new 5W LED light source up.

    1.4.2.2 Week2 -- August 8

    1, Replace the old LED source with the new one we bought.

    2. Timeline

    Notebook -- Timeline

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