Difference between revisions of "Team:Nanjing-China/Model"

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      <h2>Overview</h2>
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      <p> Biosafety is the precaution of large-scale loss of biological integrity in terms of ecology and human’s health.<sup>[1]</sup>These precaution mechanisms include conducting regular reviews of the biosafety in laboratory settings,following strict guidelines,employing an ongoing risk management assessment and so on. Downplaying or failing to fulfill such protocols can lead to augmented risk of exposure to biohazards or pathogens.</p>
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      <h2>Risk assessment& Precautions:</h2>
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      <p>Our project is to develop a sound whole-cell  photocatalytic nitrogen fixation system, using engineered <em>E.coli</em> BL21 or JM109.<br />
 +
        We believe that risks are primarily embodied in three  aspects and we have figured out appropriate solutions to all of them. Firstly,  some chemical and molecular biology reagents we use in experiments might be  harmful and noxious. In order to reduce the adverse effect of these reagents on  the environment and our health, we put the hazardous reagents in proper  storage, and carry out special procedures when dealing with them. For example,  cadmium ion, a toxic substance, is involved for CdS precipitation. We rigorously  recycle the sewage containing Cd ion every time handling it. <br />
 +
        Another safety concern comes from the inflammable and  explosive acetylene we use to test the activity of nitrogenase. In order to  avoid gas leakage, the gas cylinders which contains acetylene are operated correctly  and inspected regularly by special security staffs.<br />
 +
        Thirdly, although both <em>E.coli</em> BL21 and JM109 are in Risk Group 1 and can cause no  disease to healthy adults, the genetically modified(GM) organisms can  potentially pose threats to the welfare of people and the environment if  released to real world. So we have very strict rules to prevent this from  happening. The waste produced in the lab are periodically collected, sterilized  and categorized by our team members and then recycled by professional chemical waste  recycling companies. These standard procedures guarantee that the GM organisms  would never escape from our labs.<br />
 +
      We have inspectors to ensure our operation  correctness and all team  members have received systematical biosafety training from relevant courses in  advance. We all possess the skills in waste disposal, accident prevention,  emergency measures(such as  how to tackle fire, electric leakage and negligent wounds) and so on. During our experiments, we  stringently observed the biosafety guidelines issued both by our university and  WHO. </p>
 
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     <p><em>Biosafety and the environment:An introduction to the Cartagena Protocol on Biosafety. GE.03-01836/E. United Nations Environment Programme. p. 8.</em></p>
     <p>This year our team created a mathematical  model to optimize the arrangement of the nif gene cluster. This model helped we  optimized our design and provided some new perspectives of our  nitrogen-fixation system in transcriptional level.<br />
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We developed this model with two goals in  mind:<br />
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1.We want to achieve the 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 />
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2.We want our system as simple as possible, that means  minimizing the number of promoters and copy number of each nif gene.<br />
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We made the following assumptions:<br />
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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 />
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2.One promoter is stronger (called H) while  the other is relatively weak(called L). Under promoter H, each gene&rsquo;s  transcription level is double that of under promoter L.<br />
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3.The order of genes has little influence  on their transcriptional level.<br />
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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>
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    <div class="word-1">
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      <tr>
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        <td width="191" valign="top">
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          <p>&nbsp;</p>
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          <p>gene</p></td>
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        <td width="181" valign="top"><p>Average value of Cq</p></td>
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        <td width="181" valign="top"><p>Relative expression level</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>16S DNA</p></td>
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        <td width="181" valign="top"><p>6.33</p></td>
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        <td width="181" valign="top"><p>&nbsp;</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>nifB</p></td>
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        <td width="181" valign="top"><p>19.97</p></td>
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        <td width="181" valign="top"><p>7.80E-05</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>nifH</p></td>
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        <td width="181" valign="top"><p>17.37</p></td>
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        <td width="181" valign="top"><p>4.74E-04</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>nifD</p></td>
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        <td width="181" valign="top"><p>18.34</p></td>
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        <td width="181" valign="top"><p>2.42E-04</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>nifK</p></td>
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        <td width="181" valign="top"><p>20.77</p></td>
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        <td width="181" valign="top"><p>4.48E-05</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>nifE</p></td>
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        <td width="181" valign="top"><p>22.20</p></td>
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        <td width="181" valign="top"><p>1.66E-05</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>nifN</p></td>
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        <td width="181" valign="top"><p>22.24</p></td>
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        <td width="181" valign="top"><p>1.62E-05</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>nifX</p></td>
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        <td width="181" valign="top"><p>22.92</p></td>
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        <td width="181" valign="top"><p>1.01E-05</p></td>
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      </tr>
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      <tr>
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        <td width="191" valign="top"><p>nifV</p></td>
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        <td width="181" valign="top"><p>21.25</p></td>
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        <td width="181" valign="top"><p>3.22E-05</p></td>
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      </tr>
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    </table>
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    <p><font size="-1">Table1  The result of qPCR </font></p>
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     </div>
 
     </div>
    <p> Method:<br />
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  </div>
      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 introduced some parameters shown in table2. </p>
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    <div class="word-1">
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    <table border="1" cellspacing="0" cellpadding="0">
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      <tr>
+
        <td width="200" valign="top">
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          <p>Parameters/data</p> </td>
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        <td width="200" valign="top"><p>Meanings</p></td>
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      </tr>
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      <tr>
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        <td width="277" valign="top"><p>weak[ ]</p></td>
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        <td width="277" valign="top"><p>the expression level of each nif gene    under the weak promoter</p></td>
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      </tr>
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      <tr>
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        <td width="277" valign="top"><p>strong[ ]</p></td>
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        <td width="277" valign="top"><p>the expression level of each nif gene    under the strong promoter</p></td>
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      </tr>
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      <tr>
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        <td width="277" valign="top"><p>expected[ ]</p></td>
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        <td width="277" valign="top"><p>the ideal stoichiometric proportion</p></td>
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      </tr>
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      <tr>
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        <td width="277" valign="top"><p>d</p></td>
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        <td width="277" valign="top"><p>deviation between the expected expression    level and the actual expression level</p></td>
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      </tr>
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    </table>
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    <p align="center"><font size="-1">Table  2</font></p>
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    </div>
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    <p> Then we did some necessary preprocessing.  Firstly, we presumed the smallest element in each array was 1 and normalized  all the other data accordingly. In addition, to ensure there is at least one  solution, we adjusted expected[] to make each element greater than or equal to  the smallest expression level of the corresponding gene.<br />
+
      After that, we began the organization. In  order to minimize the total number of genes, we arranged the strong promoter  group first, and considered the weak group later. For each gene, we constantly  added one copy of it to the strong promoter group, calculated the current deviation  after each addition and compared the current deviation with the last one. If  the deviation was decreasing ,we added one more copy and repeated the operation  until the last deviation was smaller than the current one. In that way, we were  able to determine the number of each gene with which the deviations were the  smallest and completed the arrangement of the strong group. Similarly, we  arranged the weak group and finally received the result.</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%"/>
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    <p><font size="-1">Fig 1. A flow diagram describing the idea of our modeling process</font><br />
+
      </div>
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      <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><br />
+
      </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 most close to the ideal proportion among all the solutions.</p>
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    </div>
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    <div class="word">
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    Here is the codes we taped and used.
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    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>
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Revision as of 11:14, 11 October 2018

Nanjing-China2018

Overview

Biosafety is the precaution of large-scale loss of biological integrity in terms of ecology and human’s health.[1]These precaution mechanisms include conducting regular reviews of the biosafety in laboratory settings,following strict guidelines,employing an ongoing risk management assessment and so on. Downplaying or failing to fulfill such protocols can lead to augmented risk of exposure to biohazards or pathogens.

Risk assessment& Precautions:

Our project is to develop a sound whole-cell photocatalytic nitrogen fixation system, using engineered E.coli BL21 or JM109.
We believe that risks are primarily embodied in three aspects and we have figured out appropriate solutions to all of them. Firstly, some chemical and molecular biology reagents we use in experiments might be harmful and noxious. In order to reduce the adverse effect of these reagents on the environment and our health, we put the hazardous reagents in proper storage, and carry out special procedures when dealing with them. For example, cadmium ion, a toxic substance, is involved for CdS precipitation. We rigorously recycle the sewage containing Cd ion every time handling it.
Another safety concern comes from the inflammable and explosive acetylene we use to test the activity of nitrogenase. In order to avoid gas leakage, the gas cylinders which contains acetylene are operated correctly and inspected regularly by special security staffs.
Thirdly, although both E.coli BL21 and JM109 are in Risk Group 1 and can cause no disease to healthy adults, the genetically modified(GM) organisms can potentially pose threats to the welfare of people and the environment if released to real world. So we have very strict rules to prevent this from happening. The waste produced in the lab are periodically collected, sterilized and categorized by our team members and then recycled by professional chemical waste recycling companies. These standard procedures guarantee that the GM organisms would never escape from our labs.
We have inspectors to ensure our operation correctness and all team members have received systematical biosafety training from relevant courses in advance. We all possess the skills in waste disposal, accident prevention, emergency measures(such as how to tackle fire, electric leakage and negligent wounds) and so on. During our experiments, we stringently observed the biosafety guidelines issued both by our university and WHO.

Biosafety and the environment:An introduction to the Cartagena Protocol on Biosafety. GE.03-01836/E. United Nations Environment Programme. p. 8.