Team:Peking/JudgingCriteria

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        <li><a href="#010killing">Introduction</a></li>
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<li><a href="#judgingcriteria01">Parts</a></li>
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<li><a href="#judgingcriteria02">Team facilitating</a></li>
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            <ul>
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<li><a href="#judgingcriteria03">Beyond Bench</a></li>
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                <li><a href="#0101killing">1.Hen egg lysozyme</a></li>
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<li><a href="#judgingcriteria04">Medal Criteria</a></li>
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                <li><a href="#0102killing">2.Secretion</a></li>
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                <li><a href="#0103killing">3.Immunity System</a></li>
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        <li><a href="#020killing">Design</a></li>
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        <li><a href="#030killing">Result</a></li>
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  <h2 id="010killing">Introduction</h2>
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  <p> Algal blooms seriously threat the ecological integrity and sustainability of aquatic ecosystems. They can deplete oxygen causing harmful effects to the phytoplankton, and also produce a variety of toxic secondary metabolites such as microcystin. Among many kinds of algae potentially causing water bloom, <i>Microcystis Aeruginosa</i> accounts for a significant proportion [1]. We developed a new approach to control the population of <i>Microcystis Aeruginosa</i> in the water which can overcome the weakness of other methods. Our genetically engineered <i>E. coli</i>, which can express and secrete hen egg lysozyme and kill <i>Microcystis Aeruginosa</i> efficiently, safely, and controllably, with the help of α- hemolysin type I secretion system in <i>E. coli</i>. Moreover, an immunity system is introduced into the <i>E. coli</i> in case that secreted lysozyme could potentially be harmful to out genetically engineered <i>E. coli</i>.
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    <p><h2>Judging Criteria</h2></p>
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    </p>
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  <h2 id="020killing">Design</h2>
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    <p>Through five months hard work, we have witnessed great development of our project, and we have prepared for the criteria for the Giant Jamboree. We successfully submitted 12 newly designed iGEM Registry Standard part while using existing iGEM parts as well. We have also investigated the algal bloom in Taihu Lake. Moreover, we have helped several teams including BIT, BNU, and OUC-China, and we have received assistance from the ZJU-China and OUC-China. We also popularized iGEM to undergraduates in Peking University campus. In conclusion, we have met all the requirements for an iGEM team, so we believe that we deserve Gold Medal.</p>
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      <h3 id="0101killing">1.Hen egg lysozyme</h3>
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    <p>Judging form: https://igem.org/Team_Judging</p>
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<p><i>Microcystis Aeruginosa</i> is a species of freshwater cyanobacteria which can form harmful algal blooms (HABs) [1]. It almost has the same cell wall components with gram negative bacteria, such as outer membrane, peptidoglycan and inner membrane. Peptidoglycan, as an important structural component of bacterial cell wall, can provide resistance against turgor pressure [2]. Peptidoglycan can be cleaved by bacterial cell-wall hydrolases (BCWHs), causing the lysis of bacteria. So we put our attention to lysozymes, the well-known and best-studied group of BCWHs. </p>
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<p>Among the various kinds of lysozymes, we choose to work with hen egg lysozyme. Hen egg lysozyme, also known as lysozyme C (chicken-type), is one of the most widely used lysozyme, which has high antibacterial effect and easily available. Hen egg lysozyme is a kind of 1,4 -β-N- acetylmuramidase which causes the cleaving of the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the bacterial peptidoglycan, further causing the lysis of bacteria [3]. The hen egg lysozyme gene was de novo synthesized from commercial company (Genscript, Nanjing, China), and we cloned this gene into the plasmid pET-21a to test the efficiency of lysozyme being expressed from engineered <i>E. coli</i>. This plasmid was transformed into <i>E. coli</i> BL21(DE3) and defined as strain A.</p>
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    <p><h3 id="judgingcriteria01">1.iGEM Registry of Standard Biological Parts</h3></p>
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    <p>We have designed and constructed 12 brand new BioBrick Parts, and these parts were well documented on the Part Registry pages. The parts are shown as follows:</p>
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<img src="https://static.igem.org/mediawiki/2014/e/ee/Peking2014ylq_1.png" />
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    <h3><b>Table of iGEM Registry of Standard Biological Parts</b></h1>
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    <table border="2" id="iGEM Registry of Standard Biological Parts">
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        <tr><th>Part number</th><th>Characterisation</th><th>Function</th></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378001">BBa_K1378001</a></td><td>MlrA</td><td>An enzyme degradating toxin microystin</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378003">BBa_K1378003</a></td><td>Microvirin(MVN)</td><td>A lectin from <i>Microcystis.aeruginosa</i></td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378005">BBa_K1378005</a></td><td>INPNC-linker2-MVN-linker1-mRFP</td><td>Fusion protein testing INP direction</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378007">BBa_K1378007</a></td><td>INPNC-MVN and constitutively expressing GFP</td><td><i>E.coli</i>-algae binding effect testing</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378009">BBa_K1378009</a></td><td>INPNC-linker(rTEV)-MVN and constitutively expressing GFP</td><td><i>E.coli</i>-algae binding experiments control group</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378022">BBa_K1378022</a></td><td>pelB-MlrA</td><td>MlrA with a signal peptide from Sec pathway</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378023">BBa_K1378023</a></td><td>TorA-MlrA</td><td>MlrA with a signal peptide from TAT pathway</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378024">BBa_K1378024</a></td><td>MlrA-terminator</td><td>Modified MlrA</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378025">BBa_K1378025</a></td><td>pelB-MlrA-terminator</td><td>Modified pelB-MlrA</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378026">BBa_K1378026</a></td><td>TorA-MlrA-terminator</td><td>Modified TorA-MlrA</td></tr>
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              <tr><td><a href="http://parts.igem.org/Part:BBa_K1378031">BBa_K1378031</a></td><td>Holin</td><td>Hole protein in suicide system</td></tr>
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        <tr><td><a href="http://parts.igem.org/Part:BBa_K1378032">BBa_K1378032</a></td><td>Endolysin</td><td>Muralytic enzyme in suicide system</td></tr>
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    </table>
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    <p>We should emphasize that these <i>de novo</i> parts were built from scratch, and the part <a href="http://parts.igem.org/Part:BBa_K1378005">BBa_K1378005</a>, <a href="http://parts.igem.org/Part:BBa_K1378007">BBa_K1378007</a> and <a href="http://parts.igem.org/Part:BBa_K1378025">BBa_K1378025</a> have experimental data available both on our wiki:<a href="https://2014.igem.org/Team:Peking/Parts">https://2014.igem.org/Team:Peking/Parts</a>and on part registry pages.</p>
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    <p>We also used parts<a href="http://parts.igem.org/Part:BBa_K554009">BBa_K554009</a>, <a href="http://parts.igem.org/Part:BBa_K523013">BBa_K523013</a><a href="http://parts.igem.org/Part:BBa_K208006"> BBa_K208006</a>from iGEM Part Distribution and further distribution directly from the Registry. These parts have played important roles in the killing and its improvement parts of our project.</p>
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<figcaption>Figure 2: HlyA is the C-terminal signal sequence; HlyB and HlyD are the membrane proteins involved in type I secretion; TolC is an outer membrane protein that is essential in the type I secretion pathway together with membrane proteins HlyB and HlyD.</figcaption>
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<p><h3 id="judgingcriteria02">2.Team communication</h3></p>
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<p>The significance of team interactions has long been recognized by Peking iGEM and this year our team members and instructors had visited several other teams and was involved in a regional Jamboree-like iGEM Club in Wuhan this summer.
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<a href="https://2014.igem.org/Team:Peking/TeamCom#teamcommunication02">https://2014.igem.org/Team:Peking/TeamCommunication</a>
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<p>Furthermore, we have helped other teams in this summer, including BIT, BNU, and OUC-China.<a href="https://2014.igem.org/Team:Peking/TeamCom#teamcommunication03">https://2014.igem.org/Team:Peking/Mutualfacilitation</a> We also received help from ZJU-China that they helped us to transfer specific genes into E. coli’s genome. We also helped two high school students from Beijing Huiwen Middle School and Beijing No.4 High School.</p>
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<h3 id="0102killing">2.Secretion</h3>
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<p><h3 id="judgingcriteria03">3.Investigation beyond bench</h3></p>
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<p>To achieve our goal of controlling the growth of <i>Microcystis Aeruginosa</i>, our <i>E. coli</i> should have the ability to secrete hen egg lysozyme. Therefore it`s necessary to introduce a secretion system to our <i>E. coli</i>.</p>
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<p>Holding the belief that responsive research lies beyond lab bench, we visited Taihu Lake in July to investigate the algal bloom there. We interviewed the researchers in NIGLAS(Nanjing Institute of Geography and Limnology of the Chinese Academy of Sciences)who monitoring the algal bloom there and we sampled the water in the Taihu Lake and brought these samples back to our lab.
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<p>To date, five kinds of translocation pathways have been identified in <i>E. coli</i>. These pathways can either deliver proteins from the cytosol to the medium through only one-step process, or via a periplasmic intermediate which need two steps. In order to prevent the contact between lysozyme and peptidoglycan, we utilized a one-step process system, type I secretion system, to deliver the lysozyme to the medium directly.</p>
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<a href="https://2014.igem.org/Team:Peking/FieldInvestigations#hptaihua">https://2014.igem.org/Team:Peking/FieldInvestigation</a>
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<p>Type I secretion system, which is also known as ABC transporter, works in a continuous secretion process across both the inner and the outer membrane of gram-negative bacteria. The proteins involved in Type I secretion system form a channel that exports proteins from the cytoplasm to the extracellular environment. </p>
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<p>All of the Type I secretion systems, α-hemolysin(HlyA) secretion system is the best characterized and studied which has been widely used. Therefore we chose to work with this system to achieve the secretion of hen egg lysozyme.</p>
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<p>α-hemolysin(HlyA) secretion system contains 4 parts: They are HlyA, HlyB, HlyD and TolC respectively. HlyA is the C-terminal signal sequence of α-hemolysin, which can be recognized by HlyB. HlyB is an ATP-binding cassette. HlyD is a membrane fusion protein, which can be links between the outer and the inner membrane components of the system. And TolC is a specific outer membrane protein, which forms a long channel throughout the outer membrane and the periplasm, largely open towards the extracellular medium.</p>
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<p>We got the following genes, HlyB(Genscript, Nanjing, China), HlyD (Genscript, Nanjing, China) and TolC (BBa_K554009) from iGEM part. We firstly cloned these three genes with RBSes Bba_B0034 before ATG and inserted them at site after the constitutive promoter, Bba_J23105. Then Gibson Assembling was used to assemble these 3 promoter followed by RBS and genes together. At the same time, we use pET-21a as a backbone to constructed another plasmid, which contains the hen egg lysozyme gene, a Glu-Ser linker, and a HlyA signal sequence at the C-terminal of hen egg lysozyme-GS linker. We did the co-transformation, and put these two plasmid that mentioned above in the same <i>E. coli</i> BL21(DH3), which was defined as Stain A <B>(Fig. 3)</B>.</p>
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<p>In order to improve the efficiency of the hen egg lysozyme secretion, we also constructed a plasmid that contains both the lysozyme-linker-hlyA signal sequence and these 3 components. We transformed this plasmid, whose backbone is pET-21a, into the the <i>E. coli</i> BL21(DH3). This strain was defined as Stain B (Fig. 3B). We could further induce both Stain A and Stain B with IPTG and then tested the effect of the lysozyme secretion as well as the killing effect of the secreted lysozyme. </p>
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<h3 id="0103killing">3. Immunity System</h3>
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<p>The function of lysozyme is to provide hydrolysis of peptidoglycan by lysing bacterial cell-wall. Under the critical threat of lysozymes, bacteria in turn evolved mechanisms to avoid bacteriolysis, such as highly specific and potent lysozyme inhibitors production [4].
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There are several inhibitors that are specific for the hen egg lysozyme. In our project, we introduced the protein ykfE to protect our <i>E. coli</i> effectively against lysozyme while killing <i>Microcystis Aeruginosa</i> with lysozyme. YkfE is the product of the ORFan gene, which is one of the inhibitor of various kinds of lysozyme. The ykfE`s inhibition of lysozyme occurs via a key-lock type of interaction <B>(Fig. 4)</B>, without the conformational changes in the lysozyme inhibitor and lysozyme molecules [5].
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</p>
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<p>There are several inhibitors that are specific for the hen egg lysozyme. In our project, we introduced the protein ykfE to protect our <i>E. coli</i> effectively against lysozyme while killing <i>Microcystis Aeruginosa</i> with lysozyme. YkfE is the product of the ORFan gene, which is one of the inhibitor of various kinds of lysozyme. The ykfE`s inhibition of lysozyme occurs via a key-lock type of interaction <B>(Fig. 4)</B>, without the conformational changes in the lysozyme inhibitor and lysozyme molecules [5]. </p>
 
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<p>Our construct contains the ykfE gene under control of T7 promoter in the pET-21a plasmid was designated Stain C. This pET-21a plasmid was transformed into <i>E. coli</i> BL21, and the resulting strain was designated as ykfE overexpression strain <B>(Fig. 5)</B>.</p>
 
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<h2 id="030killing">Result</h2>
 
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<h3>1. Growth of algae and the killing efficiency of hen egg lysozymes</h3>
 
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<p>Whether the lysozyme could kill the algae is highly significant for the validity of our design. The growth curve of algae should be measured firstly. OD670nm, the absorbance of Chlorophyll a, was measured to illustrate the algal density. The algae were grinded before measuring OD for higher measurement accuracy. Absorbance was monitored every day until the growth of algae reached a stationary phase <B>(Fig. 1A)</B>.</p>
 
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<p>To quantify the killing efficiency of hen egg lysozyme, The killing efficiency was tested by adding different concentration of hen egg lysozymes into algae culture. Since the corpse of dead algae remains float and still have absorbance, we measure the killing effect both by direct observation and absorbance at specific wavelength<B> (Fig. 1B, C)</B>. </p>
 
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<h3>1. Growth of algae and the killing efficiency of hen egg lysozymes</h3>
 
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<p>The result indicates that 200ng/L lysozyme could kill the algae effectively within 72h. So the hen egg lysozyme, if could be properly expressed by our genetically engineered <i>E. coli</i>, would be a valid approach to kill the algae. </p>
 
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<h3>2.The lysozyme immune system</h3>
 
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<p>Considering the working mechanism of lysozyme that cleaving the peptidoglycan of bacterial cell wall can also wound the "manufacturer", our genetically engineered <i>E. coli</i>. Such a detrimental effect to <i>E. coli</i> was firstly measured<B> (Fig. 2A)</B>. To counteract this effect, the best solution to overcome the detrimental effect is to build an immune system for our genetically engineered <i>E. coli</i>. We utilized ykfE, a native inhibitor of lysozyme of <i>E. coli</i>. The circuit for the strain has been constructed <B>(Fig. 2B)</B>, and further experimental is expected coming soon. </p>
 
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<h3>3. The killing efficiency of Lysozyme expressed by genetically engineered <i>E. coli</i></h3>
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<p><h3 id="judgingcriteria04">4. Medal Criteria</h3></p>
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<p>We have constructed plasmids to express the hen egg lysozyme under the inducible promoter on the expression vector pET-21a. The expression of lysozyme in <i>E. coli</i> was verified by PAGE electrophoresis <B>(Fig. 3)</B>. We have verified that the hen egg lysozyme was successfully expressed in our <i>E. coli</i>. Although we have prepared the analysis that to use sonication lysed <i>E. coli</i> or purified protein to test whether the lysozyme is functionally expressed, the difficulty in the experimental protocol and time limit restrict our further trials. More data could probably be shown in our coming oral and poster presentations. </p>
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<h3>4.Perspective experiments</h3>
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<Figure><img src="https://static.igem.org/mediawiki/2014/8/8e/Peking2014Ycy_Judgingform.png"style="height:1200px"/></Figure>
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<p>Further experiments would focus on testing killing efficiency, testing the lysozyme immune system, and developing the lysozyme secretion system. The construction shown in the design part (LINK) should transport the properly expressed lysozyme to the out membrane space of <i>E. coli</i>. Further work would significantly strengthen the proposed while not fully achieved killing system. We believe the full version of our killing system would potentially efficiently kill algae while protect the <i>E. coli</i> to maintain productive state. </p>
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Revision as of 18:38, 17 October 2014

Judging Criteria

Through five months hard work, we have witnessed great development of our project, and we have prepared for the criteria for the Giant Jamboree. We successfully submitted 12 newly designed iGEM Registry Standard part while using existing iGEM parts as well. We have also investigated the algal bloom in Taihu Lake. Moreover, we have helped several teams including BIT, BNU, and OUC-China, and we have received assistance from the ZJU-China and OUC-China. We also popularized iGEM to undergraduates in Peking University campus. In conclusion, we have met all the requirements for an iGEM team, so we believe that we deserve Gold Medal.

Judging form: https://igem.org/Team_Judging

1.iGEM Registry of Standard Biological Parts

We have designed and constructed 12 brand new BioBrick Parts, and these parts were well documented on the Part Registry pages. The parts are shown as follows:

Table of iGEM Registry of Standard Biological Parts

Part numberCharacterisationFunction
BBa_K1378001MlrAAn enzyme degradating toxin microystin
BBa_K1378003Microvirin(MVN)A lectin from Microcystis.aeruginosa
BBa_K1378005INPNC-linker2-MVN-linker1-mRFPFusion protein testing INP direction
BBa_K1378007INPNC-MVN and constitutively expressing GFPE.coli-algae binding effect testing
BBa_K1378009INPNC-linker(rTEV)-MVN and constitutively expressing GFPE.coli-algae binding experiments control group
BBa_K1378022pelB-MlrAMlrA with a signal peptide from Sec pathway
BBa_K1378023TorA-MlrAMlrA with a signal peptide from TAT pathway
BBa_K1378024MlrA-terminatorModified MlrA
BBa_K1378025pelB-MlrA-terminatorModified pelB-MlrA
BBa_K1378026TorA-MlrA-terminatorModified TorA-MlrA
BBa_K1378031HolinHole protein in suicide system
BBa_K1378032EndolysinMuralytic enzyme in suicide system

We should emphasize that these de novo parts were built from scratch, and the part BBa_K1378005, BBa_K1378007 and BBa_K1378025 have experimental data available both on our wiki:https://2014.igem.org/Team:Peking/Partsand on part registry pages.

We also used partsBBa_K554009, BBa_K523013 BBa_K208006from iGEM Part Distribution and further distribution directly from the Registry. These parts have played important roles in the killing and its improvement parts of our project.

2.Team communication

The significance of team interactions has long been recognized by Peking iGEM and this year our team members and instructors had visited several other teams and was involved in a regional Jamboree-like iGEM Club in Wuhan this summer. https://2014.igem.org/Team:Peking/TeamCommunication

Furthermore, we have helped other teams in this summer, including BIT, BNU, and OUC-China.https://2014.igem.org/Team:Peking/Mutualfacilitation We also received help from ZJU-China that they helped us to transfer specific genes into E. coli’s genome. We also helped two high school students from Beijing Huiwen Middle School and Beijing No.4 High School.

3.Investigation beyond bench

Holding the belief that responsive research lies beyond lab bench, we visited Taihu Lake in July to investigate the algal bloom there. We interviewed the researchers in NIGLAS(Nanjing Institute of Geography and Limnology of the Chinese Academy of Sciences)who monitoring the algal bloom there and we sampled the water in the Taihu Lake and brought these samples back to our lab. https://2014.igem.org/Team:Peking/FieldInvestigation

4. Medal Criteria