Difference between revisions of "Team:ZJU-China/Project/tp"

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                               <li><a href="https://2017.igem.org/Team:ZJU-China/Parts">All Parts</a></li>
 
                               <li><a href="https://2017.igem.org/Team:ZJU-China/Parts">All Parts</a></li>
 
                               <li><a href="https://2017.igem.org/Team:ZJU-China/Parts/Basic">Basic Parts</a></li>
 
                               <li><a href="https://2017.igem.org/Team:ZJU-China/Parts/Basic">Basic Parts</a></li>
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                               <li><a href="https://2017.igem.org/Team:ZJU-China/Composite_Part">Composite Parts</a></li>
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                               <li><a href="https://2017.igem.org/Team:ZJU-China/Parts/Composite">Composite Parts</a></li>
 
                               <li><a href="https://2017.igem.org/Team:ZJU-China/Parts/Collection">Parts Collection</a></li>
 
                               <li><a href="https://2017.igem.org/Team:ZJU-China/Parts/Collection">Parts Collection</a></li>
 
                           </ul>
 
                           </ul>
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          <h1 id="trichodermaproof" class="page-header ArticleHead GreenAH">Trichoderma proof<hr></h1>
+
  <h1 id="introduction" class="page-header ArticleHead GreenAH">Introduction</h1>
 +
  <p class="PP">The InterLab study is developed by the Measurement Committee. Since synthetic biology, also been called engineering biology, is based on engineering thinking and methods, it values the reliability and repeatability of measurements. The InterLab study, involving teams and labs around the world，takes green fluorescent protein (gfp) as an example after one strict, detailed protocol to ensure the common, comparable units for measuring, which hopes to test the robustness by comparing the results from different labs.</p>
 +
  <p class="PP">This is the Fourth Measurement InterLab and our team participates in it. The InterLab this year tries to establish a GFP measurement protocol based on engineering principles, find out the similarity between fluorescence measured around the world and make gene expression more precise and reliable through tests of some RBS devices (BCDs). We are provided with materials for standard curves together with positive control, negative control and devices with different promoters and RBSs. Strictly following the requirements and protocol, we have obtained the relevant datas.</p>
  
−
          <h2 id="highlights" class="H2Head">Highlights:</h2>
+
<h1 id="methods" class="GreenAH ArticleHead ">METHODS & MATERIALS<hr></h1>
−
           <ul class="Retract textli">
+
<h2 id="transformation" class="H2Head">Transformation</h2>
−
              <li>Confront culture experiment proved <em>T.atroviride</em> biocontrol effects.</li>
+
           <p class="PP">Transform <em>Escherichia coli</em> DH5α with these following plasmids:</p>
−
              <li>Successfully finding the specific promoters related to mycoparasitism.</li>
+
          <p class="PP">
−
              <li>Designing and Building the Homologous Recombination Kit in T.artovireide.</li>
+
<ul class="Retract textli">
−
          </ul>
+
    <li>Positive control</li>
 +
    <li>Negative control</li>
 +
    <li>Test Device 1: J23101+I13504</li>
 +
    <li>Test Device 2: J23106+I13504</li>
 +
    <li>Test Device 3: J23117+I13504</li>
 +
    <li>Test Device 4: J23101.BCD2.E0040.B0015</li>
 +
    <li>Test Device 5: J23106.BCD2.E0040.B0015</li>
 +
    <li>Test Device 6: J23117.BCD2.E0040.B0015</li>
 +
</ul></p>
 +
<p class="PP"><br>Resuspend DNA in selected wells in the Distribution Kit with 10µL ddH20. Thaw competent cells on ice. Pipette 25µL of competent cells into 1.5mL tube per transformation and add 2µL of resuspended DNA into it. Incubate on ice for 30min. Heat shock tubes at 42°C for 90 sec. Then incubate on ice for 5min.</p>
 +
<p class="PP">Add 200µL SOC media with Chloramphenicol(1000×) to each transformation. Incubate at 37°C for 1 hours, shaking at 200-300rpm.</p>
 +
<p class="PP">Pipette 100µL of each transformation onto LB plates(Chloramphenicol, 1000×). Spread with sterilized spreader. Incubate transformations overnight (14-18hr) at 37°C.</p>
  
−
          <h1 id="biocontroleffects" class="GreenAH ArticleHead ">Biocontrol effects of <em>T.atroviride</em></h1>
+
<h2 id="colonies" class="H2Head">Colonies Selection</h2>
−
              <h2 id="introduction" class="H2Head">Introduction</h2>
+
<p class="PP">Pick 2 single colonies from each of plate and inoculate it on 5-10 mL LB medium with Chloramphenicol(1000×). Grow the cells overnight (16-18 hours) at 37°C and 220 rpm. Check them under the fluorescence microscope before further experiments.</p>
−
                <p class="PP">As described above(Link to Project Description), <em>T.atroviride</em> is a common biocontrol fungus, usually inhibiting the phytopathogens growth by multiple ways.Therefore,we firstly tested the inhibition function of our strain from two layers.</p>
+
  
−
              <h2 id="experimentaldesign" class="H2Head">Experimental Design</h2>
+
<h2 id="calibration" class="H2Head">Calibration</h2>
−
                <p class="PP">Considering the complicated and disturbed environment condition, we planned to keep asepsis during inoculating the fungi on the plants. Howerver, it was not easy for us to do plant tissue culture in MS medium because of the limited time and instruments. Luckily, UESTC-China connected with us after CCiC(Conference of China iGEMer Community), they used tobacco cells to do transgenesis operation this year.So we had built deep mutual collaboration,they assisted us in cultivating a set of tobacco seedlings in sterilized culture medium.After the seedlings have root and leaf differentiation,inculating <em>T.atroviride</em>, <em>P.nicotianae</em> and both of these two strains on the root of the seedings.(Here we selected Phytophthora nicotianae as our antagonizing phytopathogen for its great damage in tobacco culturing in a very short time.) Culturing these seedlings for a month, taking photos everyday and observe the differences. /n On the other side,we did confront culture experiments to see the inhibition ability of <em>T.atroviride</em>. T.artoviride and <em>P.nicotianae</em> were inoculated on the either side of a PDA medium and then incubated on 25°C for two weeks.Hypha growth condition on this medium can reflect the interaction between these two strains,we also model the growth process based on the photos we took during our confront culture.  This model helped us predict the growth of two strains and evaluate the inhibition intensity of T.artoviride.</p>
+
<p class="PP">We used the plate reader Synergy Neo2 for all the measurements and we used black 96 well plates with flat, transparent bottom.</p>
  
−
              <h2 id="results" class="H2Head">Result</h2>
+
<h3 class="H3Head">OD600 Reference Point</h3>
−
                <p class="PP">The differences between treatment and control groups were very obvious according to our tests in vivo.We selected four points of time of tobacco photos for comparison here.</p>
+
−
                <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/0/0e/ZJU_China_Project_TP_P1.png"></div>
+
  
−
        <p class="PP capture">P1:Photos for four tobacco groups in four different moments. The inoculation date was set to the Day 0 (BI:Before Inoculating)</p>
+
<p class="PP">Add 100 µl LUDOX into wells A1, B1, C1, D1 and 100 µl of H2O into wells A2, B2, C2, D2. Measure absorbance 600nm of all samples in all standard measurement modes in instrument, pathlength correction was turned off. The temperature setting was 24℃. Record the data.</p>
  
−
                <p class="PP">The intruding of <em>P.nicotianae</em> was very harmful to tobacco growth.Within three days after <em>P.nicotianae</em> infected, the leaves of tobaccos began to wilt and the stem lodged in six days.We also counted the time point of stem lodging for each groups.<br><br></p>
+
<h3 class="H3Head">Fluorescein Fluorescence Standard Curve</h3>
 +
<p class="PP">Spin down fluorescein stock tube. Prepare 2x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 µM.</p>
 +
<p class="PP">Prepare the serial dilutions of fluorescein as shown below. Set 4 copies.</p>
  
−
          <!-- Table -->
 
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−
                  <th class="yellowTable"> </th>
 
−
                  <th class="yellowTable">Control</th>
 
−
                  <th class="yellowTable"><em>P.nicotianae</em></th>
 
−
                  <th class="yellowTable"><em>T.atroviride</em></th>
 
−
                  <th class="yellowTable">Coculture</th>
 
−
              </tr>
 
  
−
              <tr>
+
<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/7/71/ZJUChina_interlab_screenshot1.png"></div>
−
                  <th class="greyTable">Leaf wilting</th>
+
<p class="capture">Fig.1.dilution</p>
−
                  <td>/</td>
+
<p class="PP">Measure the plate in plate reader, the excitation filter was set to 485nm/5nm and the emission filter was set to 530nm/30nm. Pathlength correction was turned off. The gain setting was 70. Fluorescence was from the top. The temperature setting was 24℃. Record the data.</p>
−
                  <td>less than 3 DAI</td>
+
−
                  <td>/</td>
+
−
                  <td>4 DAI</td>
+
−
              </tr>
+
  
−
              <tr>
+
<h2 id="cellmeasurement" class="H2Head">Cell Measurement</h2>
−
                  <th class="greyTable">Stem lodging</th>
+
<p class="PP">Measure OD<sub>600</sub> of the overnight cultures prepared after colony selection, then dilute the culture to a target OD600 of 0.02 in LB medium + Chloramphenicol with the help of Excel(Dilution Calculation) sheet. Incubate the cultures at 37°C and 220 rpm. Take 500 µL samples of the cultures from each of the 8 devices, two colonies per device, at 0, 2, 4, and 6 hours of incubation and add them into 96 well plates as shown below. Place samples on ice before measurements</p>
−
                  <td>/</td>
+
<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/a/a1/ZJUChina_interlab_screenshot2.png"></div>
−
                  <td>6 DAI</td>
+
           <p class="capture">Fig.2.loading samples</p>
−
                  <td>/</td>
+
           <p class="PP">Measure the samples (OD and Fl measurement). The cell measurement was under the same condition with fluorescein fluorescence standard curve and used the same plate.</p>
−
                  <td>10 DAI</td>
+
<h1 id="results" class="ArticleHead GreenAH">Results<hr></h1>
−
              </tr>
+
−
           </table>
+
−
          </div>
+
−
           <p class="PP capture">T1:Blank shank symptoms appearing time,DAI(Days after inoculating). "/" means no related symptoms appeared.</p>
+
  
−
          <p class="PP">We further got the fresh weight and dry weight data on 12th day.</p>
+
<h2 id="OD600" class="H2Head">OD600 Reference point</h2>
−
                <p class="PP">In view of this results, We concluded from this in vivo tests:<br></p>
+
  
−
                  <p class="PP textli Retract" style="line-height: 20px !important;"><br>P.nicotianane was a serious phytopathogen and the infected symptoms appeared very quickly.</p>
+
  <!-- Table -->
−
                  <p class="PP textli Retract" style="line-height: 20px !important;"><em>T.atroviride</em> helped the tobaccos resist the <em>P.nicotianae</em> and delayed the symptoms appearing.</p>
+
  <table class="table">
−
                  <p class="PP textli Retract" style="line-height: 20px !important;"><em>T.atroviride</em> seemed to improve the weights of the plants.</p>
+
  <tr>
 +
<th class="yellowTable"> </th>
 +
<th class="yellowTable">LUDOX-HS40</th>
 +
<th class="yellowTable">H<sub>2</sub>O</th>
 +
</tr>
  
−
                <p class="PP"><br>The Confront culture experiments showed the inhibitory effect of <em>T.atroviride</em> hyphae on <em>P.nicotianae</em> growth, this time-lapse video showed the process clearly and visually.</p>
+
<tr>
−
                <p class="PP">The T.atrovoride grew more fast than <em>P.nicotianae</em> on PDA and inhibited its growth after hyphae hit.Finally the whole medium was colonized by <em>T.atroviride</em> and the hyphae of <em>P.nicotianae</em> were totally engulfed. </p>
+
<th class="grayTable">Replicate 1</th>
 +
<td>0.05</td>
 +
<td>0.035</td>
 +
</tr>
  
 +
<tr>
 +
<th class="grayTable">Replicate 2</th>
 +
<td>0.049</td>
 +
<td>0.037</td>
 +
</tr>
  
−
          <h2 id="conclusions" class="H2Head">Conclusions</h2>
+
<tr>
−
                <p class="PP">In this part, we chose <em>P.nicotianae</em> as the object, proving the inhibitory effect of <em>T.atroviride</em> both in the culture medium and plants. On the other hand, we also paid attention to the differences between in vivo tests and confront culture, although <em>T.atroviride</em> completely inhibited Phytophthora in PDA medium, however, in plants, Trichoderma only delayed the diseases outbreak, but could not eliminate the harm of Phytophthora, this may showed that the further improvement of <em>T.atroviride</em>,like combining with synthetic biology technology, is necessary and meaningful.So the next step was collecting DNA parts which can be used in filamentous fungus and proving that we can express foreign genes in <em>T.atroviride</em>.</p>
+
<th class="grayTable">Replicate 3</th>
 +
<td>0.049</td>
 +
<td>0.039</td>
 +
</tr>
  
−
          <h1 id="gmit" class="GreenAH ArticleHead ">Gene manipulation in <em>T.atroviride</em></h1>
+
<tr>
 +
<th class="grayTable">Replicate 4</th>
 +
<td>0.049</td>
 +
<td>0.039</td>
 +
</tr>
  
−
              <h2 id="overview" class="H2Head">Overview</h2>
+
<tr>
−
                <p class="PP">In order to introduce the <em>T.atroviride</em> as a new kind of Chassis, we accomplished
+
<th class="grayTable">Arith. Mean</th>
−
                    the random insertion manipulation in <em>T.atroviride</em>. According to the report gene(GFP) under the fluorescence microscope, we can prove that we are able to manipulate the <em>T.atroviride</em> at the level of genes and express the genes of interests.  However, to the extented application, especially to enable the <em>T.atroviride</em> to realize their biocontrol  function in the automatic agriculture, we need our <em>T.atroviride</em> to finish more tasks. For instance, we need to establish more complicated pathway in the <em>T.atroviride</em> and to integrate more transcription units to the genome of the <em>T.atroviride</em> at the same time. Therefore, we refer to the research in the saccharomyces cerevisiae of the modularized two-step (M2S) chromosome integration technique, established a <em>T.atroviride</em> homologous recombination kit.
+
<td>0.04925</td>
−
                </p>
+
<td>0.0375</td>
−
              <h2 id="experimentaldesign2" class="H2Head">Experimental Design</h2>
+
</tr>
  
−
                    <h3 id="firstphase" class="H3Head">The first phase: random insertion</h3>
+
<tr>
−
                            <p class="PP">We obtain the constitutive promoter(H3 promoter), which is workable in <em>T.atroviride</em>, as well as the terminator used in <em>T.atroviride</em> called RP27. Then we assembled the promoter, terminator, a reporter gene(eGFP) and hph gene (hygromycin) together with the vector PKD1 to construct the random insertion plasmid. PKD1 backbone is a kind of TDNA vector so that it is able to insert into the <em>T.atroviride</em> genome.</p>
+
<th class="grayTable">Corrected Abs600</th>
−
                    <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/c/cd/ZJU_China_Project_TP_1.jpg"></div>
+
<td>0.01175</td>
−
          <p class="PP">This experiment can prove whether the random insertion can be manipulated in <em>T.atroviride</em> and whether it’s convenient to explore various promoters strength for standardization.</p>
+
<td> </td>
−
                            <p class="PP">Through ATMT(Agrobacterium Tumefaciens-mediated Transformation), hph gene and the reporter gene(GFP) can be imported into the genome DNA. The transformants can grow on the medium with hygromycin, and we can see the GFP under the fluorescence microscope.</p>
+
</tr>
  
−
          <h3 id="secondphase" class="H3Head">The second phase: Trichoderma homologous recombination kit</h3>
+
<tr>
 +
<th class="grayTable">Reference OD600</th>
 +
<td>0.0425</td>
 +
<td> </td>
 +
</tr>
  
−
          <p class="PP">A DNA assembly and chromosomal integration method was established by homologous recombination (HR) in <em>T.atroviride</em>.</p>
+
<tr>
−
          <p class="PP">When using our kit, we transform the plasmids embedded gene Of interests together with the</p>
+
<th class="grayTable">OD600/Abs600</th>
−
          <p class="PP">plasm ids embedded left and right homologous arms into the <em>T.atroviride</em>. In this way, we can finish the homologous recombination process in vivo.</p>
+
<td>3.617021277</td>
−
          <p class="PP">As listed in Table, we have constructed a series of promoter vectors and terminator vectors.</p>
+
<td> </td>
 +
</tr>
 +
  </table>
 +
<p class="capture">Table.OD600 reference point</p>
  
−
          <div style="text-align: center; margin-left: 20%">
+
<h2 id="fluorescein" class="H2Head">Fluorescein Fluorescence Standard Curve</h2>
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                  <tr>
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                      <th class="greyTable">BBa_K2207003</th>
+
−
                      <th class="yellowTable">Trichoderma HR System I SOD-Tubulin dobule promoter</th>
+
−
                  </tr>
+
  
−
                  <tr>
+
          <table class="tableTrans">
−
                      <th class="greyTable">BBa_K2207004</th>
+
              <tr><td>
−
                      <th class="yellowTable">Trichoderma HR System II Ech42-H3 double promoter</th>
+
<img  class="textimg_two" src="https://static.igem.org/mediawiki/2017/9/9c/ZJUChina_interlab_diagram1.png">
−
                  </tr>
+
              </td><td>
−
                  <tr>
+
<img  class="textimg_two" src="https://static.igem.org/mediawiki/2017/b/b2/ZJUChina_interlab_diagram2.png">
−
                      <th class="greyTable">BBa_K2207005</th>
+
              </td>
−
                      <th class="yellowTable">Trichoderma HR System III L1-ADH1-RP27-L2</th>
+
              </tr>
−
                  </tr>
+
          </table>
−
                  <tr>
+
          <p class="capture">Fig.3.standard curve 1</p>
−
                      <th class="greyTable">BBa_K2207006</th>
+
          <p class="capture">Fig.4.standard curve 2</p>
−
                      <th class="yellowTable">Trichoderma HR System IV L2-ADH1-RP27-L3</th>
+
−
                  </tr>
+
−
                  <tr>
+
−
                      <th class="greyTable">BBa_K2207007</th>
+
−
                      <th class="yellowTable">Trichoderma HR System V Homologous Binding SiteA</th>
+
−
                  </tr>
+
−
                  <tr>
+
−
                      <th class="greyTable">BBa_K2207008</th>
+
−
                      <th class="yellowTable">Trichoderma HR System VI Homologous Binding SiteB</th>
+
−
                  </tr>
+
−
              </table>
+
−
          </div>
+
  
−
          <p class="PP"><strong>Step1 Characterize the promoters and terminators of Trichoderma as the parts</strong></p>
+
<br><h2 id="discussion" class="H2Head">Discussion</h2>
−
                  <p class="PP">To be able to integrate more genes at the same time, we need to get more promoters and terminators using in the construction of module.</p>
+
<div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/8/83/ZJUChina_interlab_diagram3.png"></div>
−
                  <p class="PP">Firstly, in addition to the existing constitutive H3 promoter, we searched many papers and chose another published constitutive promoter, Phex. Then we replaced PH3 with this Phex in the above random insertion plasmid and transform this new plasmid into <em>T.atroviride</em>, which will be detected under fluorescence microscope.</p>
+
<p class="capture">Fig.5 Abs 600 measurement</p>
−
                  <p class="PP">Secondly, as a kind of filamentous fungus, <em>T.atroviride</em> inhibit the pathogenic fungus through mycoparasitism by receiving the signals such as agglutinin or others. <em>T.atroviride</em> will twine on the pathogenic fungus, persistently upregulate some genes. The ech42 gene is such a kind of upregulated gene during mycoparasitism so that we surmised there is a kind of inducible promoter in front of the ech42 gene. Therefore, we got a fragment (named it Pech42) in front of the ech42 coding region and replaced the PH3 with it as well. This plasmid also transformed into <em>T.atroviride</em> and was detected under fluorescence microscope.</p>
+
<p class="PP">Abs600 of the samples changed over time as shown, in which the data of each sample is the average of 4 replicates. Abs600 of the samples reflected its concentration, which could show the growth of bacteria. As the picture shown, the growth rate of bacteria generally got slower as time went by, this is probably because at the beginning of the measurement, bacteria was in the later period of logarithmic phase and the resources were not that sufficient, inhibiting the growth. Several samples appeared to be different, such as colony 1 of device 4, which seemed to be in the logarithmic phase and colony 2 of device 1, whose concentration went down after 2 hours of incubation, perhaps low activity resulting in massive death.</p>
−
                  <p class="PP">Besides, thanks to assistance of the laboratory in our university, we obtained two more kinds of constitutive promoters, PSOD and Ptubulin to establish our kit and characterize them. This two kinds of constitutive promoters are widely used parts among laboratories, they are all used to be applied in the metabolic pathway of <em>T.atroviride</em>. Also, another terminator got from the part kits named tADH1 was ready.</p>
+
<p class="PP">In addition, growth curves of each colony were different. This may be caused by different initial concentration of samples after dilution, which led the samples be in different growing statuses. We will mention it later.</p>
 +
          <table class="tableTrans">
 +
              <tr><td>
 +
          <img class="textimg_two" src="https://static.igem.org/mediawiki/2017/6/6e/ZJUChina_interlab_diagram4.png">
 +
              </td><td>
 +
          <img class="textimg_two" src="https://static.igem.org/mediawiki/2017/3/3e/ZJUChina_interlab_diagram5.png">
 +
              </td>
 +
              </tr>
 +
          </table>
 +
          <p class="capture">Fig.6.relative fluoresence measurement</p>
 +
          <p class="capture">Fig.7.absolute fluorescence plot</p>
 +
<p class="PP">The fluorescence differed obviously. The absolute fluorescence and relative fluorescence of device 3, device 5 and device 6 were relatively low, for which their promoters and RBSs might account. For two replicates of device 1, their fluorescence differed greatly and the same as growth, making it hard to compare. Either operational error or colony selection would affect.</p><p class="PP">Furthermore, the tendence of absolute fluorescence turned out to rise and fall, mostly going down as time went by. We thought this was because the protein expression varied in different growth phase and growth of bacteria changed over time.</p>
 +
<h2 id="reflection" class="H2Head">Reflection</h2>
 +
<p class="PP">Some of our operations might affect the results, which can be improved.</p>
 +
<p class="PP">Our lab do not have plate reader, so we borrowed one from other lab. We cultured bacteria and loaded samples in our lab and then took them to the other lab for measurement. Since it took some time to go back and forth, our actual measurement took longer than expectation, so the measurement postponed a little. Though samples were laid on ice, the bacteria could still growth, making the real measurement time different from the demand. Similarly, the cultures settled in the process, leading to the deviation of the concentration after dilution. As a result, the comparability between each sample was affected.</p>
 +
<p class="PP">By the way, the first time we did the OD600 Reference Point measurement, we put the 96 well plate containing LUDOX on ice. Then it turned out that the OD600 of LUDOX was lower than water. Maybe LUDOX denatured in the cold environment on ice referred to the official warning. So perhaps it is better not to put LUDOX on ice or anywhere like that.</p>
 +
<h1 id="feedback" class="ArticleHead GreenAH">Feedback<hr></h1>
 +
<p class="PP">After the InterLab Study measurement, we have something to say about the provided protocol, which goes on like this:</p>
  
 +
          <table class="table nonStyleTable tableTrans" style="align-content: center;background-color: transparent;border: none">
 +
              <tr>
 +
                  <th>
 +
                      <h2 id="advantages" class="H3Head">Advantages<hr></h2>
 +
                      <ul class="Retract textli">
 +
                          <li>The protocol is not only detailed but also clear, so it is easy to operate and avoid differences between teams.</li>
 +
                          <li>The provided Excel sheets make it easier to calculate.</li>
 +
                          <li>There are extra materials for mistakes and failure.</li>
 +
                          <li>Some warnings are necessary, such as LUDOX cannot be stored in refrigerator and oversaturated detector will cause low concentrations linear, but high concentrations saturate or fall for Fluorescein standard curve. It helps a lot in finding solutions to problems.</li>
 +
                      </ul>
 +
                  </th>
 +
                  <th>
 +
                      <h2 id="improvements" class="H3Head">Improvements<hr></h2>
 +
                      <ul class="Retract textli">
 +
                          <li>The PDF version of protocol differs from the Excel sheets in some ways, such as the volume of diluted culture. Though it won’t affect the operation and results, it can cause misunderstanding and confusion.</li>
 +
                          <li>Excel sheets can list out the computational process, which hopes to contribute to data verification and data analysis.</li>
 +
                          <li>More samples and replicates can be involved to avoid mistakes and the time span between measurements can be shortened.</li>
 +
                      </ul>
 +
                  </th>
 +
              </tr>
 +
          </table>
  
−
          <p class="PP"><strong>Step2 Obtain the homologous arms L1 L2 L3 and the Homologous arms located in the <em>T.atroviride</em> genome </strong></p>
+
<p class="PP"><br><br>We sincerely hope the InterLab Study this year will success and hope the InterLab Study next year will go well.</p>
−
              <p class="PP">We made the contact with the author of the M2S technique and obtained the homologous arms L1 L2 L3 from him. Besides, we chose the upregulated gene when mycoparasitism, ech42, and picked up the upstream and downstream of the coding region as the homologous arms located in the genome of <em>T.atroviride</em>. </p>
+
</div>
−
 
+
−
          <p class="PP"><strong>Step3 assemble the plasmids</strong></p>
+
−
              <p class="PP">Now we can assemble the parts we got into the plasmids’ backbones.</p>
+
−
                    <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/7/74/ZJU_China_Project_TP_2.png"></div>
+
−
                  <p class="PP capture">a: the part of head-to-head promoter module</p>
+
−
                    <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/8/85/ZJU_China_Project_TP_3.jpg"></div>
+
−
                    <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/d/d9/ZJU_China_Project_TP_4.jpg"></div>
+
−
                  <p class="PP capture">b: the part of terminator module together with the homologous arms L1,L2 and L2,L3 for assembly;</p>
+
−
                    <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/c/ca/ZJU_China_Project_TP_5.jpg"></div>
+
−
                  <p class="PP capture">c: the part of L1, resistance gene and the Homologous arms, ech42-LB which is the integration chromosomal locus located in the genome of <em>T.atroviride</em>.</p>
+
−
                    <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/c/c4/ZJU_China_Project_TP_6.jpg"></div>
+
−
                  <p class="PP capture">d: the part of L3 and ech42-RB </p>
+
−
 
+
−
          <p class="PP">Therefore, we can utilize the three-module strategy to finish the  dual-transcription unit assembly.</p>
+
−
              <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/5/56/ZJU_China_Project_TP_add.jpg"></div>
+
−
              <p class="PP">Modularized head-to-head promoter module and a pair of terminators was assembled with two genes. Thus, two transcription units were assembled. Then the two transcription units were transformed into <em>T.atroviride</em> for assembly and integration with modules of selective markers and integration sites.</p>
+
−
              <p class="PP">The process of integrating the several dual-transcription units into the genome of <em>T.atroviride</em> bases on the homologous recombination in vivo. The important point of this process is the dedicated overlap regions L1-L3 so that each fragment can assemble with adjacent fragments by the overlap regions. Plus, these overlap sequences were designed to have least homology with the genome of <em>T.atroviride</em> by sequence analysis.</p>
+
−
 
+
−
          <h2 id="results2" class="H2Head">Result</h2>
+
−
              <p class="PP">The first phase：random insertion</p>
+
−
              <p class="PP">According to the GFP observed under the fluorescence microscope(P2), we can prove that PKD1 plasmid contains both H3 promoter and Hex promoter can work. Meanwhile, the hex promoter is weaker than the H3 promoter.</p>
+
−
            <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/2/20/ZJU_China_Project_TP_P2.png"></div>
+
−
 
+
−
 
+
−
            <p class="PP capture">P2:Fluorescence detection for <em>T.atroviride</em> hyphae.WT:Wide type;PKD1:Hyphae contain PKD1 plasmids within H3 promoter; Hex:Hyphae contain PKD1 plasmids within Hex promoter instead of H3.(The magnification factor was 10X40 for pic A&D,10X10 for pic B&E and 10X20 for pic C&F)</p>
+
−
            <p class="PP">We put the hyphae picked from the <em>T.atroviride</em> which confront cultured with the <em>P.nicotianae</em> on a clean glass slide, next to the hyphae from <em>T.atroviride</em> without <em>P.nicotianae</em>. The different fluorescence strength between them reveals that Pech42 was activated by <em>P.nicotianae</em> (P3,P4).</p>
+
−
            <div class="imgdiv"><img class="textimg" src="https://static.igem.org/mediawiki/2017/c/c7/ZJU_China_Project_TP_P3.png"></div>
+
−
            <p class="PP capture">P4:The relative fluorescent intensity of the hyphae contain the plasmids within Pech42.</p>
+
−
 
+
−
          <h2 id="futurework" class="H2Head">Future work</h2>
+
−
                <p class="PP">In the future, we are going to establish the <em>T.atroviride</em> parts library, including the promoters, terminators and more “L” homologous arm from L1 to Ln which can be more suitable for <em>T.atroviride</em>. Thus, our method can integrate multiple transcription units at the same time.</p>
+
−
 
+
−
    <h1 id="reference" class="GreenAH ArticleHead ">Reference<hr></h1>
+
−
          <p class="PP">[1]Li S, Ding W, Zhang X, et al. Development of a modularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2016, 9(1):232.</p>
+
−
          <p class="PP">[2]…….再补给你</p>
+
−
 
+
−
 
+
−
      </div>
+
  
  
Line 675: Line 712:
 
   <nav  style="position: fixed; top: 100px ; left:50px; " class="bs-docs-sidebar hidden-print hidden-xs hidden-sm">
 
   <nav  style="position: fixed; top: 100px ; left:50px; " class="bs-docs-sidebar hidden-print hidden-xs hidden-sm">
 
     <ul class="nav bs-docs-sidenav">
 
     <ul class="nav bs-docs-sidenav">
 +
      <li><a href="#introduction">Introduction</a></li>
 
       <li>
 
       <li>
−
         <a href="#trichodermaproof">Trichoderma proof</a>
+
         <a href="#methods">Methods&Materials</a>
 
           <ul class="nav">
 
           <ul class="nav">
−
             <li><a href="#highlights">Highlights</a></li>
+
             <li><a href="#transformation">Transformation</a></li>
 +
            <li><a href="#colonies">Colonies Selection</a></li>
 +
            <li><a href="#calibration">Calibration</a></li>
 +
            <li><a href="#cellmeasurement">Cell Measurement</a></li>
 
           </ul>
 
           </ul>
 
       </li>
 
       </li>
 
       <li>
 
       <li>
−
         <a href="#biocontroleffects">Biocontrol effects of <em>T.atroviride</em></a>
+
         <a href="#results">Results</a>
 
           <ul class="nav">
 
           <ul class="nav">
−
             <li><a href="#introduction">Introduction</a></li>
+
             <li><a href="#OD600">OD600</a></li>
−
             <li><a href="#experimentaldesign">Experimental Design</a></li>
+
             <li><a href="#fluorescein">Fluorescein</a></li>
−
             <li><a href="#results">Results</a></li>
+
             <li><a href="#discussion">Discussion</a></li>
−
             <li><a href="#conclusions">Conclusions</a></li>
+
             <li><a href="#reflection">Reflection</a></li>
 
           </ul>
 
           </ul>
 
       </li>
 
       </li>
 
       <li>
 
       <li>
−
         <a href="#gmit">Gene manipulation in <em>T.atroviride</em></a>
+
         <a href="#feedback">Feedback</a>
 
         <ul class="nav">
 
         <ul class="nav">
−
           <li><a href="#overview">Overview</a></li>
+
           <li><a href="#advantages">Advantages</a></li>
−
            <li><a href="#experimentaldesign2">Experimental Design</a></li>
+
          <li><a href="#improvements">Improvements</a></li>
−
            <li><a href="#results2">Result</a></li>
+
−
            <li><a href="#futurework">Future Work</a></li>
+
 
         </ul>
 
         </ul>
 
       </li>
 
       </li>
−
        <li><a href="#reference">Reference</a></li>
 
 
     </ul>
 
     </ul>
  

Revision as of 15:06, 24 October 2017

Introduction

The InterLab study is developed by the Measurement Committee. Since synthetic biology, also been called engineering biology, is based on engineering thinking and methods, it values the reliability and repeatability of measurements. The InterLab study, involving teams and labs around the world，takes green fluorescent protein (gfp) as an example after one strict, detailed protocol to ensure the common, comparable units for measuring, which hopes to test the robustness by comparing the results from different labs.

This is the Fourth Measurement InterLab and our team participates in it. The InterLab this year tries to establish a GFP measurement protocol based on engineering principles, find out the similarity between fluorescence measured around the world and make gene expression more precise and reliable through tests of some RBS devices (BCDs). We are provided with materials for standard curves together with positive control, negative control and devices with different promoters and RBSs. Strictly following the requirements and protocol, we have obtained the relevant datas.

METHODS & MATERIALS

Transformation

Transform Escherichia coli DH5α with these following plasmids:

  • Positive control
  • Negative control
  • Test Device 1: J23101+I13504
  • Test Device 2: J23106+I13504
  • Test Device 3: J23117+I13504
  • Test Device 4: J23101.BCD2.E0040.B0015
  • Test Device 5: J23106.BCD2.E0040.B0015
  • Test Device 6: J23117.BCD2.E0040.B0015


Resuspend DNA in selected wells in the Distribution Kit with 10µL ddH20. Thaw competent cells on ice. Pipette 25µL of competent cells into 1.5mL tube per transformation and add 2µL of resuspended DNA into it. Incubate on ice for 30min. Heat shock tubes at 42°C for 90 sec. Then incubate on ice for 5min.

Add 200µL SOC media with Chloramphenicol(1000×) to each transformation. Incubate at 37°C for 1 hours, shaking at 200-300rpm.

Pipette 100µL of each transformation onto LB plates(Chloramphenicol, 1000×). Spread with sterilized spreader. Incubate transformations overnight (14-18hr) at 37°C.

Colonies Selection

Pick 2 single colonies from each of plate and inoculate it on 5-10 mL LB medium with Chloramphenicol(1000×). Grow the cells overnight (16-18 hours) at 37°C and 220 rpm. Check them under the fluorescence microscope before further experiments.

Calibration

We used the plate reader Synergy Neo2 for all the measurements and we used black 96 well plates with flat, transparent bottom.

OD600 Reference Point

Add 100 µl LUDOX into wells A1, B1, C1, D1 and 100 µl of H2O into wells A2, B2, C2, D2. Measure absorbance 600nm of all samples in all standard measurement modes in instrument, pathlength correction was turned off. The temperature setting was 24℃. Record the data.

Fluorescein Fluorescence Standard Curve

Spin down fluorescein stock tube. Prepare 2x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 µM.

Prepare the serial dilutions of fluorescein as shown below. Set 4 copies.

Fig.1.dilution

Measure the plate in plate reader, the excitation filter was set to 485nm/5nm and the emission filter was set to 530nm/30nm. Pathlength correction was turned off. The gain setting was 70. Fluorescence was from the top. The temperature setting was 24℃. Record the data.

Cell Measurement

Measure OD600 of the overnight cultures prepared after colony selection, then dilute the culture to a target OD600 of 0.02 in LB medium + Chloramphenicol with the help of Excel(Dilution Calculation) sheet. Incubate the cultures at 37°C and 220 rpm. Take 500 µL samples of the cultures from each of the 8 devices, two colonies per device, at 0, 2, 4, and 6 hours of incubation and add them into 96 well plates as shown below. Place samples on ice before measurements

Fig.2.loading samples

Measure the samples (OD and Fl measurement). The cell measurement was under the same condition with fluorescein fluorescence standard curve and used the same plate.

Results

OD600 Reference point

LUDOX-HS40 H2O
Replicate 1 0.05 0.035
Replicate 2 0.049 0.037
Replicate 3 0.049 0.039
Replicate 4 0.049 0.039
Arith. Mean 0.04925 0.0375
Corrected Abs600 0.01175
Reference OD600 0.0425
OD600/Abs600 3.617021277

Table.OD600 reference point

Fluorescein Fluorescence Standard Curve

Fig.3.standard curve 1

Fig.4.standard curve 2


Discussion

Fig.5 Abs 600 measurement

Abs600 of the samples changed over time as shown, in which the data of each sample is the average of 4 replicates. Abs600 of the samples reflected its concentration, which could show the growth of bacteria. As the picture shown, the growth rate of bacteria generally got slower as time went by, this is probably because at the beginning of the measurement, bacteria was in the later period of logarithmic phase and the resources were not that sufficient, inhibiting the growth. Several samples appeared to be different, such as colony 1 of device 4, which seemed to be in the logarithmic phase and colony 2 of device 1, whose concentration went down after 2 hours of incubation, perhaps low activity resulting in massive death.

In addition, growth curves of each colony were different. This may be caused by different initial concentration of samples after dilution, which led the samples be in different growing statuses. We will mention it later.

Fig.6.relative fluoresence measurement

Fig.7.absolute fluorescence plot

The fluorescence differed obviously. The absolute fluorescence and relative fluorescence of device 3, device 5 and device 6 were relatively low, for which their promoters and RBSs might account. For two replicates of device 1, their fluorescence differed greatly and the same as growth, making it hard to compare. Either operational error or colony selection would affect.

Furthermore, the tendence of absolute fluorescence turned out to rise and fall, mostly going down as time went by. We thought this was because the protein expression varied in different growth phase and growth of bacteria changed over time.

Reflection

Some of our operations might affect the results, which can be improved.

Our lab do not have plate reader, so we borrowed one from other lab. We cultured bacteria and loaded samples in our lab and then took them to the other lab for measurement. Since it took some time to go back and forth, our actual measurement took longer than expectation, so the measurement postponed a little. Though samples were laid on ice, the bacteria could still growth, making the real measurement time different from the demand. Similarly, the cultures settled in the process, leading to the deviation of the concentration after dilution. As a result, the comparability between each sample was affected.

By the way, the first time we did the OD600 Reference Point measurement, we put the 96 well plate containing LUDOX on ice. Then it turned out that the OD600 of LUDOX was lower than water. Maybe LUDOX denatured in the cold environment on ice referred to the official warning. So perhaps it is better not to put LUDOX on ice or anywhere like that.

Feedback

After the InterLab Study measurement, we have something to say about the provided protocol, which goes on like this:

Advantages

  • The protocol is not only detailed but also clear, so it is easy to operate and avoid differences between teams.
  • The provided Excel sheets make it easier to calculate.
  • There are extra materials for mistakes and failure.
  • Some warnings are necessary, such as LUDOX cannot be stored in refrigerator and oversaturated detector will cause low concentrations linear, but high concentrations saturate or fall for Fluorescein standard curve. It helps a lot in finding solutions to problems.

Improvements

  • The PDF version of protocol differs from the Excel sheets in some ways, such as the volume of diluted culture. Though it won’t affect the operation and results, it can cause misunderstanding and confusion.
  • Excel sheets can list out the computational process, which hopes to contribute to data verification and data analysis.
  • More samples and replicates can be involved to avoid mistakes and the time span between measurements can be shortened.



We sincerely hope the InterLab Study this year will success and hope the InterLab Study next year will go well.


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