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| | <div align="center"> | | <div align="center"> |
| | <div id="HOME"> | | <div id="HOME"> |
| − | | + | |
| − | <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Model">Modeling</a></li></ul></div> | + | <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Hardware">Hardware</a></li></ul></div> |
| − | <ul><li><a href="#intro">Introduction</a></li> | + | <ul><li><a href="#part"><font size="-1">part-introduction</font></a></li> |
| − | <li><a href="#method">Method</a></li>
| + | <li><a href="# video"> video</a></li></ul> |
| − | <li><a href="#r">Refinement:</a></li>
| + | |
| − | <li><a href="# document"> Document</a></li> | + | |
| − | <li><a href="#reference">Reference</a></li>
| + | |
| − | </ul>
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| | </div> | | </div> |
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| | <li><a href="https://2018.igem.org/Team:Nanjing-China/Notebook">NOTEBOOK</a></li> | | <li><a href="https://2018.igem.org/Team:Nanjing-China/Notebook">NOTEBOOK</a></li> |
| | </ul> | | </ul> |
| − | </div> | + | </div> <div class="header"><img src="https://static.igem.org/mediawiki/2018/ 4/ 40/T--Nanjing-China--title- 4.png" width="100%" onload="MM_effectAppearFade(this, 1000, 0, 100, false);MM_effectBlind('HOME', 1000, '0%', '100%', true)"> |
| − | <div class="header"><img src="https://static.igem.org/mediawiki/2018/ 2/ 20/T--Nanjing-China--title- MODEL.png " width="100%" width="100%" onload="MM_effectAppearFade(this, 1000, 0, 100, false);MM_effectBlind('HOME', 1000, '0%', '100%', true)" > | + | </div> |
| − | </div> | + | |
| − | | + | <div class="word"> |
| − | <div class="word" id="intro"> | + | <div style="position:absolute; top:-90px; z-index:3; left:-10px;"> |
| − | <p>This year our team created a mathematical model to optimize the arrangement of the nif gene cluster. This model helped we refined our design and provided some new perspectives of our nitrogen-fixation system in transcriptional level.</p>
| + | <img src="https://static.igem.org/mediawiki/2018/4/4c/T--Nanjing-China--PROJECT_h.png" width="45%" /></div> |
| − | <p>We developed this model with two goals in mind:<br />
| + | <div class="word-1" style="height:20px;"></div> |
| − | 1.We want to achieve the putative best stoichiometric proportion of each nif gene, which is nifB:nifH:nifD:nifK:nifE:nifN:nifX:nifV=1:3:4:4:1:1:1:1.<br />
| + | |
| − | 2.We want our system as simple as possible, that means minimizing numbers of promoters and each nif gene.</p>
| + | |
| − | <p>We made the following assumptions:<br />
| + | |
| − | 1.There are two kinds of promoters, both of which can successfully launch the expression of every nitrogen fixation gene involved in our system. <br />
| + | |
| − | 2.One promoter is stronger(called H) while the other is relatively weak(called L). Under promoter H, each gene’s transcription level is double that of under promoter L.<br />
| + | |
| − | 3.The order of genes has little influence on their transcriptional level.</p>
| + | |
| − | <p>We conducted Real-time Quantitative PCR to detect the transcription level of nif gene cluster and the experimental data we received became an important reference for our modeling.</p>
| + | |
| − | <div class="word-note">
| + | |
| − | <table border="1" cellspacing="0" cellpadding="0"> | + | |
| − | <tr>
| + | |
| − | <td valign="top">
| + | |
| − | <p>gene</p></td>
| + | |
| − | <td valign="top"><p>Average value of Cq</p></td>
| + | |
| − | <td valign="top"><p>Relative expression level</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>16S DNA</p></td>
| + | |
| − | <td width="191" valign="top"><p>6.33</p></td>
| + | |
| − | <td width="191" valign="top"><p> </p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>nifB</p></td>
| + | |
| − | <td width="191" valign="top"><p>19.97</p></td>
| + | |
| − | <td width="191" valign="top"><p>7.80E<sup>-05</sup></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>nifH</p></td>
| + | |
| − | <td width="191" valign="top"><p>17.37</p></td>
| + | |
| − | <td width="191" valign="top"><p>4.74E<sup>-04</sup></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>nifD</p></td>
| + | |
| − | <td width="191" valign="top"><p>18.34</p></td>
| + | |
| − | <td width="191" valign="top"><p>2.42E<sup>-04</sup></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>nifK</p></td>
| + | |
| − | <td width="191" valign="top"><p>20.77</p></td>
| + | |
| − | <td width="191" valign="top"><p>4.48E<sup>-05</sup></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>nifE</p></td>
| + | |
| − | <td width="191" valign="top"><p>22.20</p></td>
| + | |
| − | <td width="191" valign="top"><p>1.66E<sup>-05</sup></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>nifN</p></td>
| + | |
| − | <td width="191" valign="top"><p>22.24</p></td>
| + | |
| − | <td width="191" valign="top"><p>1.62E<sup>-05</sup></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>nifX</p></td>
| + | |
| − | <td width="191" valign="top"><p>22.92</p></td>
| + | |
| − | <td width="191" valign="top"><p>1.01E<sup>-05</sup></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="191" valign="top"><p>nifV</p></td>
| + | |
| − | <td width="191" valign="top"><p>21.25</p></td>
| + | |
| − | <td width="191" valign="top"><p>3.22E<sup>-05</sup></p></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p><font size="-1">Table1 The result of qPCR </font></p>
| + | |
| | </div> | | </div> |
| | + | <div class="word" id="part"> |
| | + | <p>In order to apply our system to practical use, we also designed a device consisting of 4 modules: incubation, illumination, control and condensation. In our design draft, we hide all the tube-like parts to make the main body of the device clearer.</p> |
| | + | <img src="https://static.igem.org/mediawiki/2018/c/c2/T--Nanjing-China--device-whole.jpg" width="100%" /> |
| | + | <p>The incubation module is a shaker inside an incubator which can shake the flask, so that the engineered strain can grow and participate in reaction in agreeable conditions. </p> |
| | + | <img src="https://static.igem.org/mediawiki/2018/e/e3/T--Nanjing-China--device-mul.jpg" width="65%" /> |
| | + | <p>For the illumination module, we choose xenon modulator tube as the light source. However, it requires high voltage to be excited, so we also add a potential transformer to convert 220 voltage to higher voltage. </p> |
| | + | <p>Another problem is that when xenon modulator tube is illuminating, it would heat the air inside incubator, thus making the environment unfavorable for our engineered strain. To solve this problem, we place a reflux condenser to cool the xenon modulator tube, keeping the most suitable temperature. </p> |
| | + | <img src="https://static.igem.org/mediawiki/2018/f/f5/T--Nanjing-China--device-li.jpg" width="58%" /> |
| | + | <p>The control module is the brain of the entire device. It directly controls the temperature inside the incubator and the speed of shaker. The ill module is the brain of the entire device. The illumination intensity can also be regulated by altering the position of the xenon modulator tube.</p> |
| | + | <img src="https://static.igem.org/mediawiki/2018/7/70/T--Nanjing-China--device-cc.jpg" width="60%" /> |
| | + | <p>Our device can not only be applied to nitrogen fixation process, but also serve as a reactor for other similar light-driven reactions, such as hydrogen production and carbon fixation. We believe, after further promotion it will have a fairly promising prospect.</p> |
| | </div> | | </div> |
| − | <div class="word" id=" method"> | + | <div class="word" id=" video"> |
| − | <h3> Method:</h3>
| + | <div class="word- 2" style="width :70% ;"> |
| − | <p>To start with, we put all genes into two groups. One group is under the strong promoter while the other is under the weak one. We constructed two arrays,weak[i] and expected[i].</p>
| + | <div align="center"> |
| − | | + | <object width="600" height="400" id="t-visit"> |
| − | <table border="1" cellspacing="0" cellpadding="0" width="90%">
| + | <param name="AutoStart" value=0 /> |
| − | <tr> | + | <video width="600" height="400" controls="controls"> |
| − | <td width="40%" valign="top"><p>Parameters(i=1,2,3,4,5,6,7,8)</p></td>
| + | <source src="https://static.igem.org/mediawiki/2018/1/15/T--Nanjing-China--device.mp4" type="video/mp4"></video> |
| − | <td width="60%" valign="top"><p>Meanings</p></td>
| + | </object> |
| − | </tr>
| + | </div> |
| − | <tr>
| + | </div> |
| − | <td valign="top"><p>weak[i]</p></td>
| + | <div class="word-2"> |
| − | <td valign="top"><p>the relative expression level of each <Em>nif</Em> gene under the weak promoter</p></td>
| + | <img src="https://static.igem.org/mediawiki/2018/c/c3/T--Nanjing-China--real-device.png" width="60%" /> |
| − | </tr>
| + | </div> |
| − | <tr>
| + | |
| − | <td valign="top"><p>weak[i]*</p></td>
| + | |
| − | <td valign="top"><p>the relative expression level of each <Em>nif</Em> gene under the weak promoter after normalization</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td valign="top"><p>expected[i]</p></td>
| + | |
| − | <td valign="top"><p>the ideal stoichiometric proportion</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td valign="top"><p>expected[i]*</p></td>
| + | |
| − | <td valign="top"><p>the ideal stoichiometric proportion after normalization</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td valign="top"><p>strong[i]</p></td>
| + | |
| − | <td valign="top"><p>the relative expression level of each <Em>nif</Em> gene under the strong promoter after normalization</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td valign="top"><p>e<sub>i</sub></p></td>
| + | |
| − | <td valign="top"><p>the ideal stoichiometric proportion of the i<sup>th</sup> gene after all preprocessings </p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td valign="top"><p>a<sub>i</sub></p></td>
| + | |
| − | <td valign="top"><p>the relative expression level of the i<sup>th</sup> gene under the weak promoter after all preprocessings</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td valign="top"><p>m<sub>i</sub></p></td>
| + | |
| − | <td valign="top"><p>the number of the i<Sup>th</Sup> gene under the strong promoter</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td valign="top"><p>n<sub>i</sub></p></td>
| + | |
| − | <td valign="top"><p>The number of the i<Sup>th</Sup> gene under the weak promoter</p></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p align="center"><font size="-1">Table 2 The table of parameters in our model</font></p>
| + | |
| | </div> | | </div> |
| − | <p>Then we did some necessary preprocessings. Firstly, we found the smallest data in weak[i] and called it “min”. We normalized all the other data accordingly by doing:</p>
| + | |
| − | <div align="center" style="width:100%;"><img src="https://static.igem.org/mediawiki/2018/4/46/T--Nanjing-China--model-1-1.jpg" height="55px" /></div>
| + | </div> |
| − | <div align="center" style="width:100%;"><img src="https://static.igem.org/mediawiki/2018/e/e4/T--Nanjing-China--model-1-2.jpg" height="55px" /></div>
| + | |
| − | <p>We constructed strong[i]:</p>
| + | |
| − | <div align="center"><em>strong[i]=2×weak[i]*</em> </div> <br />
| + | |
| − | <p>Secondly, to guarantee the existence of a solution, we adjusted expected[i]* by examining whether it is greater than or equal to the corresponding weak[i]*, if not, we did:</p>
| + | |
| − | <div align="center"><em>expected[i]*=weak[i]* </em> </div> <br />
| + | |
| − | <p>
| + | |
| − | After that, we began the organization. In order to minimize the total numbers of genes, we arranged the strong promoter group first, and considered the weak group later. Because each gene can be considered separately, here we only describe the organization of the i<sup>th</sup> gene as an example.<br />
| + | |
| − | For the i<sup>th</sup> gene, we tried adding one copy of it under the strong promoter. If </p>
| + | |
| − | <div align="center"><em>|e<sub>i</sub>-2×a<sub>i</sub>|<e<sub>i</sub>, </em></div>
| + | |
| − | <p>we actually added it. Until we have added (m<sub>i</sub>+1) i<sup>th</sup> genes, and got</p>
| + | |
| − | <div align="center"><em>|e<sub>i</sub>-2(m<sub>i</sub>+1)×a<sub>i</sub>|>=|e<sub>i</sub>-2mi<sub>i</sub>×a<sub>i</sub>| </em> </div>
| + | |
| − | <p>Then we stopped adding it and recorded that we have added m<sub>i</sub> i<sup>th</sup> genes under the strong promoter.<br />
| + | |
| − | For the weak promoter group, we applied a similar method. For the i<sup>th</sup> gene, we tried adding one copy of it under the weak promoter. If</p>
| + | |
| − | <div align="center"> <em>|e<sub>i</sub>-2×m<sub>i</sub>×a<sub>i</sub>-a<sub>i</sub>|<|e<sub>i</sub>-2×m<sub>i</sub>×a<sub>i</sub>|, </em> </div>
| + | |
| − | <p>we actually added it. Until we have added (n<sub>i</sub>+1) i<sup>th</sup> genes, and got </p>
| + | |
| − | <div align="center"><em>|e<sub>i</sub>-2×m<sub>i</sub>×a-(n<sub>i</sub>+1)×a<sub>i</sub>|>=|e<sub>i</sub>-2×mi<sub>i</sub>×a<sub>i</sub>-n<sub>i</sub>×a<sub>i</sub>|</em> </div>
| + | |
| − | <p>Then we stopped adding it and recorded that we have added n<sub>i</sub> i<sup>th</sup> genes under the weak promoter.<br />
| + | |
| − | In that way, we were able to determine numbers of the i<sup>th</sup> gene under the two promoters with which the deviation was the smallest.</p>
| + | |
| − | <div class="word-1" align="center">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/8/8a/T--Nanjing-China--model-1.png" width="100%"/>
| + | |
| − | <p><font size="-1">Fig 1. A flow diagram describing the idea of our modeling process</font></p>
| + | |
| − | </div>
| + | |
| − | <p>According to this flow diagram, we programmed with Python and got the following results:</p>
| + | |
| − | <div class="word-1" align="center">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/e/ed/T--Nanjing-China--model-2.png" width="100%"/>
| + | |
| − | <p><font size="-1">Fig 2. The best arrangement of nif genes according to our calculation</font></p>
| + | |
| − | </div>
| + | |
| − | <p>With this arrangement, the proportion of nifB: nifH: nifD: nifK: nifE: nifN: nifX: nifV = 15.44: 46.93: 71.88: 62.10: 16.44: 16.04: 16.0: 15.94, which is close enough to the ideal proportion among all the solutions.</p>
| + | |
| − | </div>
| + | |
| − | <div class="word" id="r">
| + | |
| − |
| + | |
| − | <h2>Refinement of our model:</h2>
| + | |
| − | <p>We modified the putative best expression level of nifB:nifH:nifD:nifK:nifE:nifN:nifX:nifV to 5:3:4:4:1:1:1:1. We believed in this way, we could better simulate the expression of nitrogenase in our engineered <em>E.coli</em> strains. We made this change because of three reasons.</p>
| + | |
| − | <p>Firstly, nifB is indispensable for assembly nitrogenase no matter in diazotrophs or engineered <em>E.coli</em> strains. Apart from the minimal nitrogen fixation gene cluster, the genomic DNA of wide type <em>Paenibacillus polymyxa </em>includes analogues of nifM, nifU, nifS and other genes which exist in other nitrogen-fixing microorganisms and are essential for the correct folding of nitrogenase iron protein. However, the <em>E.coli </em>genome doesn’t have such analogues. Nevertheless, it has been reported that the excessive expression of nifB could compensate for the absence of nifU and nifS. That is, if nifB is overexpressed in <em>E.coli</em>, these auxiliaries are not necessary. Therefore, the expression level of nifB should be the highest 5.</p>
| + | |
| − | <p>Secondly, compared with nitrogen-fixing microorganisms, <em>E.coli</em> also lacks some genes that provide electron transfer function, such as nifF and niff. So the intracellular reductive power of <em>E.coli</em> is insufficient to accomplish nitrogen fixation. Thus it is necessary to overexpress nifH(nitrogenase reductase) and the value is set to 3 instead of 5 because our semiconductor, the CdS part, can provide additional electrons.</p>
| + | |
| − | <p>Thirdly, we set the expression level of nifD and nifK to be 4 because molybdenum iron protein is an ɑ2β2 allotetramer and is the core of nitrogenase.</p>
| + | |
| − | <p>Based on the new ideal stoichiometric proportion, we adjusted the code and received a more accurate result.</p>
| + | |
| − | <div class="word-1" align="center">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/5/50/T--Nanjing-China--model-3.png" width="100%"/>
| + | |
| − | <p><font size="-1">Fig 3 The best arrangement of nif genes version 2.0.</font></p>
| + | |
| − | </div>
| + | |
| − | <p>The achieved stoichiometric proportion of nifB: nifH: nifD: nifK: nifE: nifN: nifX: nifV=77.23: 46.93: 71.88: 62.10: 16.44: 16.04: 16.0: 15.94, which is close enough to the ideal 5:3:4:4:1:1:1:1. <br />
| + | |
| − | This model provided a potential strategy for the improvement of biological activity of nitrogenase expressed in our engineered <em>E.coli</em> strain and offered a great help to our further experiments.</p>
| + | |
| − | </div>
| + | |
| − | <div class="word" id="document">
| + | |
| − | Here is the codes we taped and used.
| + | |
| − | <object width="100%" height="600px" data="https://static.igem.org/mediawiki/2018/7/7d/T--Nanjing-China--model-code.pdf" type="application/pdf">
| + | |
| − | <param name="src" value="https://static.igem.org/mediawiki/2018/7/7d/T--Nanjing-China--model-code.pdf">
| + | |
| − | </object>
| + | |
| − | TXT downlaod:<a href="https://static.igem.org/mediawiki/2018/f/fe/T--Nanjing-China--model.txt">https://static.igem.org/mediawiki/2018/f/fe/T--Nanjing-China--model.txt</a>
| + | |
| − | <div id="code" align="left">
| + | |
| − | <p >The number we typed in:</p>
| + | |
| − | <ol><li>findSequence([7.8,47.4,24.2,4.48,1.66,1.62,1.01,3.22],[1,3,4,4,1,1,1,1],['nifB','nifH','nifD','nifK','nifE','nifN','nifX','nifV'])</li>
| + | |
| − | <li>findSequence([7.8,47.4,24.2,4.48,1.66,1.62,1.01,3.22],[5,3,4,4,1,1,1,1],['nifB','nifH','nifD','nifK','nifE','nifN','nifX','nifV'])</li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="word" id="reference" align="left">
| + | |
| − | <h2>References</h2>
| + | |
| − | <ol>
| + | |
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