Team:LCG-UNAM-Mexico/Notebook/2008-August

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           <td width="165" bgcolor="#5C743D">&nbsp;<br />
           <td width="165" bgcolor="#5C743D">&nbsp;<br />
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Team" class="navText">About Us</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Project" class="navText">Our project</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Project" class="navText">Our Project</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Modeling" class="navText">Modeling</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Experiments" class="navText">Experiments</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Experiments" class="navText">Wet Lab</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Modeling" class="navText">Modeling</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Notebook" class="navText">Notebook</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Notebook" class="navText">Notebook</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Story" class="navText">Our story</a></td>
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<tr>
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          <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Team" class="navText">About us</a></td>
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    </table> <br />
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          <td class="subHeader" bgcolor="#99CC66" id="01">2008-08-01</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><p><strong>WET LAB:</strong><br></p>
 +
<p>We left PCR of:</p>
 +
<ul>
 +
  <li>Part 1 of Biopart BBa_I79006</li>
 +
  <li>Biopart cI BBa_C0051</li>
 +
  <li>Biopart 3 (normal) ofBBa_I79006</li>
 +
  <li>Biopart 3 (mut) of BBa_I79006 </li>
 +
  <li>Operator of rcnR + RcnA </li>
 +
</ul>
 +
</div></td>
 +
        </tr> 
 +
<tr>
<tr>
           <td class="subHeader" bgcolor="#99CC66" id="04">2008-08-04</td>  
           <td class="subHeader" bgcolor="#99CC66" id="04">2008-08-04</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p><strong>Hill's cooperativity</strong><br />
+
<td class="bodyText"><div align="justify"><p><b>MODELING:</b><br>Hill cooperativity 5th Reaction Reminder: </p>
-
    <strong>5th Reaction </strong> <br />
+
-
    <strong>Reminder:</strong> </p>
+
<p>A  + B &lt;--&gt; AB            <br />
<p>A  + B &lt;--&gt; AB            <br />
     <strong>Ka=Keq=[AB]/[A][B]=1/Kd</strong>      <br />
     <strong>Ka=Keq=[AB]/[A][B]=1/Kd</strong>      <br />
-
   θ=[AB]/([AB]+[A])=[B]/([B]+Kd) </p>
+
   θ=[AB]/([AB]+[A])=[B]/([B]+Kd) </p><br>
<p><strong><u>MWC  Model</u></strong> (Cooperativity)          <br />
<p><strong><u>MWC  Model</u></strong> (Cooperativity)          <br />
   A + nB &lt;--&gt; ABn        <br />
   A + nB &lt;--&gt; ABn        <br />
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   θ=[B]n/([B]n+Kd)        <br />
   θ=[B]n/([B]n+Kd)        <br />
   log(θ/(1- θ))=nlog(B)-log(kd)                        …Hill's equation</p>
   log(θ/(1- θ))=nlog(B)-log(kd)                        …Hill's equation</p>
-
<p> </p>
+
<p> </p><br>
<p><strong>Suppression mediated by cI:</strong> <br />
<p><strong>Suppression mediated by cI:</strong> <br />
   ρ  + nCI &lt;--&gt; ρ:CIn    (k+,  k-)  <br />
   ρ  + nCI &lt;--&gt; ρ:CIn    (k+,  k-)  <br />
Line 140: Line 157:
   … ρ0=[ρ]+Keq[ρ][CI]n <br />
   … ρ0=[ρ]+Keq[ρ][CI]n <br />
   =&gt;  ρ= (ρ0/Keq)/((1/keq)+[CI]n) </p>
   =&gt;  ρ= (ρ0/Keq)/((1/keq)+[CI]n) </p>
-
<p>Flow=  k+[ρ][CI]n = K+((ρ0/Keq)/((1/Keq)+[CI]n))[CI]n </p>
+
<p>Flow=  k+[ρ][CI]n = K+((ρ0/Keq)/((1/Keq)+[CI]n))[CI]n </p><br>
<p><strong>Flow= k+</strong><strong>([ρ</strong><strong>0</strong><strong>]/K</strong><strong>eq</strong><strong>)</strong> <strong>[CI]n / ((1/Keq)+[CI]n)</strong></p>
<p><strong>Flow= k+</strong><strong>([ρ</strong><strong>0</strong><strong>]/K</strong><strong>eq</strong><strong>)</strong> <strong>[CI]n / ((1/Keq)+[CI]n)</strong></p>
<p><strong>=&gt; </strong><strong>Vm=  k</strong><strong>+</strong>([ρ0]/Keq)<strong>  &amp;  Kp=1/Keq=K</strong>d </p>
<p><strong>=&gt; </strong><strong>Vm=  k</strong><strong>+</strong>([ρ0]/Keq)<strong>  &amp;  Kp=1/Keq=K</strong>d </p>
-
<p><strong>So:</strong> <br />
+
<p><strong>Therefore:</strong> <br />
   Keq = exp(  -ΔG / R T )          <br />
   Keq = exp(  -ΔG / R T )          <br />
   k+ = (KB/h) T exp( -ΔG / R T ) = (KB/h) T Keq </p>
   k+ = (KB/h) T exp( -ΔG / R T ) = (KB/h) T Keq </p>
-
<table bgcolor="#cc99ff" border="1" bordercolor="#000000" cellpadding="1" cellspacing="1" width="423">
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<table bgcolor="#cc99ff" border="0" bordercolor="#000000" cellpadding="1" cellspacing="1" width="423">
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<p>&nbsp;</p>
<p>&nbsp;</p>
-
         </td>
+
<p><strong>WET LAB:</strong></p>
 +
<p><strong><u>Gel</u></strong></p>
 +
<p>We run a gel with the PCR products obtained the day 01-08-08</p>
 +
<p><strong><u>Purification</u></strong></p>
 +
<p>From the PCR of the 01-08-08 we took 120 μl to purify DNA in a low fusion point agarose gel  in line with the kit.</p>
 +
<p>We run an agarose gel to verify the status of the purified PRC products.</p>
 +
<p><img src="https://static.igem.org/mediawiki/2008/1/17/Gel_04Ago08.png" alt="Gel_04Ago08" width="400" /></p>
 +
<ol>
 +
  <li>Molecular Marker</li>
 +
  <li> Part 1 of biopart BBa_I79006</li>
 +
  <li> Biopart cI BBa_C0051</li>
 +
  <li> Biopart 3 (normal) of BBa_I79006</li>
 +
  <li> Biopart 3 (mut) ofBBa_I79006</li>
 +
  <li> Operator of  rcnR + RcnA</li>
 +
</ol>
 +
<p><u><strong>Restrictions</strong></u><br />
 +
</p>
 +
<p>We left restrictions over night(double and simple) of each biopart.</p>
 +
<p><strong>First Simple Restriction</strong></p>
 +
<p>The bioparts PCR product was cut with 2 simple consecutive restrictions. The reactives and volumes used in the first reaction were the following:</p>
 +
<ul>
 +
  <li><strong>Part 1; BamH1</strong>
 +
    <blockquote>
 +
      <p>    Buffer U.... 4 microlts<br />
 +
        BSA.......... 4 microlts<br />
 +
        BamH1...... 2 microlts<br />
 +
        DNA PCR... 15 microlts<br />
 +
        <u>H2O......... 15 microlts &nbsp;&nbsp; </u><br />
 +
        <strong>Total......... 40 microlts</strong><br />
 +
      </p>
 +
    </blockquote>
 +
  </li>
 +
  <li><strong>Part 2_cI; BamH1</strong>
 +
    <blockquote>
 +
      <p>    Buffer U.... 4 microlts<br />
 +
        BSA.......... 4 microlts<br />
 +
        BamH1...... 2 microlts<br />
 +
        DNA PCR... 15 microlts<br />
 +
        <u>H2O......... 15 microlts &nbsp;&nbsp; </u><br />
 +
        <strong>Total......... 40 microlts</strong><br />
 +
      </p>
 +
    </blockquote>
 +
  </li>
 +
  <li><strong>Part 3 Normal; Xba1</strong>
 +
    <blockquote>
 +
      <p>    Buffer U.... 7 microlts<br />
 +
        BSA.......... 7 microlts<br />
 +
        Xba1......... 3 microlts<br />
 +
        DNA PCR... 28 microlts<br />
 +
        <u>H2O......... 25 microlts &nbsp;&nbsp; </u><br />
 +
      <strong>Total......... 70 microlts</strong></p>
 +
    </blockquote>
 +
  </li>
 +
  <li><strong>Part 3 Mutated; Xba1</strong>
 +
    <blockquote>
 +
      <p>    Buffer U.... 7 microlts<br />
 +
        BSA.......... 7 microlts<br />
 +
        Xba1......... 3 microlts<br />
 +
        DNA PCR... 28 microlts<br />
 +
        <u>H2O......... 25 microlts &nbsp;&nbsp; </u><br />
 +
      <strong>Total......... 70 microlts</strong></p>
 +
    </blockquote>
 +
  </li>
 +
  <li><strong>Part 4 RcnA; Xba1</strong>
 +
    <blockquote>
 +
      <p>    Buffer U.... 7 microlts<br />
 +
        BSA.......... 7 microlts<br />
 +
        Xba1......... 3 microlts<br />
 +
        DNA PCR... 28 microlts<br />
 +
        <u>H2O......... 25 microlts &nbsp;&nbsp; </u><br />
 +
        <strong>Total......... 70 microlts</strong><br />
 +
      </p>
 +
    </blockquote>
 +
  </li>
 +
</ul>
 +
 
 +
         </div></td>
       </tr>  
       </tr>  
<tr>
<tr>
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-
<td class="bodyText"> <p>Hill's Cooperativity<br />
+
<td class="bodyText"> <div align="justify"><p><b>MODELING:</b><br>Hill Cooperativity<br />
</p>
</p>
-
  <p>5th Reaction, conflict ...<br />
+
  <p>5th Reaction, conflict<br />
  </p>
  </p>
  <p>If we consider that: <br />
  <p>If we consider that: <br />
  </p>
  </p>
-
  <p>Keq = exp (-ΔG / R T) <br />
+
  <p>&nbsp;&nbsp;- Keq = exp (-ΔG / R T) <br />
  </p>
  </p>
-
  <p>k + = (KB / h) T exp (-ΔG / R T) = (KB / h) T Keq</p>
+
  <p>&nbsp;&nbsp;- k + = (KB / h) T exp (-ΔG / R T) = (KB / h) T Keq</p>
  <p> and given that the flow is (k + / Keq) [ρ0] [CI] n / ((1/Keq) + [CI]  n), the value of the maximum speed of the flow loses its meaning. </p>
  <p> and given that the flow is (k + / Keq) [ρ0] [CI] n / ((1/Keq) + [CI]  n), the value of the maximum speed of the flow loses its meaning. </p>
  <p>  The speed limit is being determined by (k + / Keq) [ρ0], but k + / Keq  = (KB / h) * T, and we know that [ρ0] is arbitrary, i.e., Vmax is no longer  based on the reaction as such, which does not make sense. </p>
  <p>  The speed limit is being determined by (k + / Keq) [ρ0], but k + / Keq  = (KB / h) * T, and we know that [ρ0] is arbitrary, i.e., Vmax is no longer  based on the reaction as such, which does not make sense. </p>
  <p>  For  example: Take the same reaction that we are considering, the maximum  speed of the flow of the reaction would be the same with the promoter  that has the operators of CI, that if you used one with a random sequence,  so, whether we repeated the experiment, with the same temperature and  the same concentration of DNA and an equal number of copies of the sequence, the  maximum speed reached by the flow would be the same for the real  promoter as for for any sequence, without taking any consideration with their affinity for their substrates... That does not makes sense! </p>
  <p>  For  example: Take the same reaction that we are considering, the maximum  speed of the flow of the reaction would be the same with the promoter  that has the operators of CI, that if you used one with a random sequence,  so, whether we repeated the experiment, with the same temperature and  the same concentration of DNA and an equal number of copies of the sequence, the  maximum speed reached by the flow would be the same for the real  promoter as for for any sequence, without taking any consideration with their affinity for their substrates... That does not makes sense! </p>
-
  <p>  The proposed explanation is that the equation used to determine k + does not fit our model, we should explore other possibilities. </p>
+
  <p>  The proposed explanation is that the equation used to determine k + does not fit our model. We should explore other possibilities. </p>
  <p>&nbsp;</p>
  <p>&nbsp;</p>
-
         </td>
+
<p><strong>WET LAB:</strong></p>
 +
<p><strong><u>Restrictions</u></strong></p>
 +
<p><strong>Second simple restriction</strong></p>
 +
<p>Before the second simple restriction we cleaned the product oof the first restriction with the purification Kit.<br />
 +
Due to the purification protocol we knew that the DNA was clean and diluted in 40 μl of buffer, and in order to obtain an efficient restriction we try to dilute the less the DNA-Buffer mix, obtaining the following volumes.</p>
 +
<ul>
 +
  <li><strong>Part 1; EcoR1</strong>
 +
    <blockquote>
 +
      <p>Buffer  U....... 5 μl<br />
 +
        BamH1........ 2.5 μl<br />
 +
        Clean DNA  ... 40 μl<br />
 +
        <u>H2O............2.5 μl</u><br />
 +
      <strong>Total........... 50 μ</strong><strong>l</strong><br />
 +
      </p>
 +
    </blockquote>
 +
  </li>
 +
  <li><strong>Part 2_cI; Xba1</strong>
 +
    <blockquote>
 +
      <p>Buffer  2....&nbsp; 5 μl<br />
 +
        BSA.......... 2.5 μl<br />
 +
        BamH1......  2.5 μl<br />
 +
        <u>DNA  PCR... 40 μl&nbsp; </u><br />
 +
        <strong>Total......... 50  μl</strong></p>
 +
    </blockquote>
 +
  </li>
 +
 
 +
  <li><strong>    Part 3 (Normal); Pst1</strong>  </li>
 +
  <blockquote>
 +
    <p>    Buffer 3....  5 μl<br />
 +
      Pst1......... 2.5  μl<br />
 +
      DNA PCR...  40 μl<br />
 +
      <u>H2O......... 2.5  μl &nbsp;&nbsp; </u><br />
 +
      <strong>Total......... 50 μl</strong></p>
 +
  </blockquote>
 +
  <li><strong>Part 3 Mutated; Pst1</strong>  </li>
 +
</ul>
 +
<ul>
 +
  <blockquote>
 +
    <p>    Buffer 3....  5 μl<br />
 +
      Xba1.........  2.5 μl<br />
 +
      DNA PCR...  40 μl<br />
 +
      <u>H2O......... 2.5  μl &nbsp;&nbsp; </u><br />
 +
    <strong>Total......... 50 μl</strong></p>
 +
  </blockquote>
 +
  <li><strong> Part 4 RcnA; HindIII</strong>
 +
    <blockquote>
 +
      <p>    Buffer 2....  5 μl<br />
 +
        HindIII.....  2.5 μl<br />
 +
        DNA PCR...  40 μl<br />
 +
        <u>H2O......... 2.5  μl &nbsp;&nbsp; </u><br />
 +
        <strong>  Total......... 50 μl</strong></p>
 +
    </blockquote>
 +
      </li>
 +
</ul>
 +
<p><strong><u>Extraction</u></strong></p>
 +
<p>Plasmids pRK415 and pBBR1MCS-5 were extracted with the Roche kit(see Techniques).</p>
 +
<p><strong><u>Cultures</u></strong></p>
 +
<p>We cultured DH5alfa cells tranfromed with pJet+biopart</p>
 +
 
 +
         </div></td>
       </tr>  
       </tr>  
<tr>
<tr>
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         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p>Hill's Cooperativity: <br />
+
<td class="bodyText"><div align="justify"><p><b>MODELING:</b><br>Hill Cooperativity: <br />
-
   5th Reaction, resolving the conflict... <br />
+
   5th Reaction, solving the problem: <br />
   <br />
   <br />
-
   The error in the previous approach is that we were considering ΔG to be the same for both equations (for Keq &amp;  k+).<br />
+
   The error in the previous approach was that we were considering ΔG to be the same for both equations (for Keq &amp;  k+).<br />
   <br />
   <br />
-
The explanation of why these two values are different is very clear  when we look at the graph below. Recalling what the two  constants represent:</p>
+
<p>We know that the equilibrium depends solely on the difference in the free energy of Gibbs between the substrate and the product (ΔG 'th). The one with less energy will be favored in the balance, while the rate of reaction depends on the activation energy needed for the conversion (ΔG ‡). A  reaction reaches equilibrium faster or slower depending on the rate of  reaction (depending on the magnitude of its ΔG ‡), but the balance itself does not change. <br />
-
<div id="urdd">
+
-
  <div align="center"><img src="http://docs.google.com/File?id=dntmktb_99dz485zf8_b" alt="" name="sm1w6" id="sm1w6" /></div>
+
-
</div>
+
-
<p>We know that the balance depends solely on the difference between Gibbs  free energy of the substrate and the product (ΔG 'th), The one with less energy will be favored in the balance, while the rate of reaction depends on the activation energy needed for the conversion (ΔG ‡). A  reaction reaches equilibrium faster or slower depending on the rate of  reaction (depending on how big is ΔG ‡), but the balance of it as such does not change. <br />
+
   <br />
   <br />
Thus: <br />
Thus: <br />
Line 224: Line 372:
k + = (KB / h) T exp (- ΔG ‡ / RT) ≠ (KB / h) T Keq</p>
k + = (KB / h) T exp (- ΔG ‡ / RT) ≠ (KB / h) T Keq</p>
<p>&nbsp; </p>
<p>&nbsp; </p>
-
         </td>
+
         </div></td>
       </tr>   
       </tr>   
<tr>
<tr>
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         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p><strong>GROUP MEETING </strong><br />
+
<td class="bodyText"><div align="justify"><p><strong>GROUP MEETING </strong><br />
-
   Experimental work<br />
+
   Wet Lab Statusk<br />
   <strong><br />
   <strong><br />
   Objectives: </strong><br />
   Objectives: </strong><br />
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   <br />
   <br />
   <strong>To do: </strong><br />
   <strong>To do: </strong><br />
-
   - Extract DNA of the strain to get RcnA. <br />
+
   - Extract DNA from the wild type strain to obtain RcnA. <br />
   - Get the bioparts catalog. <br />
   - Get the bioparts catalog. <br />
-
   - We need to have a large number of plasmids that we can use, amplifying the bioparts. <br />
+
   - Obtain a large amount of plasmid that we can use, and amplify the bioparts. <br />
   - Transformation of the bacteria with bioparts. <br />
   - Transformation of the bacteria with bioparts. <br />
   <br />
   <br />
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   <br />
   <br />
   <strong>Problems: </strong><br />
   <strong>Problems: </strong><br />
-
   - There was no DNA that we needed in the catalog. <br />
+
   - The DNA that we needed was not in the registry. <br />
-
   - The oligos were delayed 2 week and a half. <br />
+
   - The oligos were delayed 2 weeks and a half. <br />
   - Issues to extract the plasmid from the colonies. <br />
   - Issues to extract the plasmid from the colonies. <br />
   - Make a PCR ligation with the three parts and amplify with the ends (it did not work). <br />
   - Make a PCR ligation with the three parts and amplify with the ends (it did not work). <br />
-
   - With the enzyme used: Increased frequency of spontaneous mutation of all the enzymes that exist. <br />
+
   - With the enzyme used the frequency of spontaneous mutation was increased to about an error every thousand base pairs. <br />
-
  An error every thousand base pairs. <br />
+
   - There is a problem with tetracycline. You get false positives. <br />
   - There is a problem with tetracycline. You get false positives. <br />
   <strong><br />
   <strong><br />
-
   Can be done: </strong><br />
+
   What can be done: </strong><br />
-
   - A part with RcnA and can be linked to the plasmid. <br />
+
   - The biopart with RcnA can already be linked to the plasmid. <br />
-
   - In the others we have to link and restrict, and re-link and restrict once more and re-connect the last time in the final plasmid. <br />
+
   - For the other construction we will have to link two parts and digest them, then link them with the third part and digest once more, then insert into the final plasmid. <br />
-
   -  HindIII can be used with the big biopart to verify the sequence. <br />
+
   -  HindIII can be used with the large biopart to verify the sequence. <br />
   <strong><br />
   <strong><br />
   Electrodes: </strong><br />
   Electrodes: </strong><br />
-
   - Are they specific for Nickel?</p>
+
   - Will they be specific for Nickel?</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
-
         </td>
+
         </div></td>
       </tr>  
       </tr>  
 +
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="20">2008-08-20</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="12">2008-08-12</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p><strong>Our response to the IPN team:<br />
+
<td class="bodyText"><div align="justify"><p><strong>WET LAB:</strong></p>
-
</strong><br />
+
<p><u><strong>Cultures</strong></u></p>
-
  Hello,<br />
+
<p>We left cultures of Biopart 1 in pJEt</p>
-
  We apologize for the late reply, but we had to discuss carefully our answer.<br />
+
<p><strong><u>Plasmid Extraction</u></strong></p>
-
   First of all, we think you are confused about what our project really is. We  want to make bacteria to modify the extracellular nickel concentration in  response to an external signal (AHL in this case), and of course, be able to predict to what extent the concentration of the input signal will affect the  amount of nickel in the medium. To achieve this, it is true we have to  synchronize our cell population at the beginning. This is easy to do and  doesn't represent any technical problems.<br />
+
<p>Plasmid RcnA was extracted by alkaline lysis</p>
-
   We are very conscious of the facts you tell us, first: we know the half-life of  the lactones is relatively long (24 hrs as you say). That's why we are  including AiiA under a constitutive promoter in our model, which degrades AHL  very efficiently. This will ensure AHL does not saturate the medium. Second, we  know AiiA does not diffuse freely through the cell membrane. However, we don't  need that to happen, as each cell will degrade its own AHL (yes, we are  assuming that all AHL will enter a cell within a window of time).<br />
+
<p><strong><u>Gels</u></strong></p>
-
   In other words, we do not need to  synchronize the bacterial population more than in the first step. We are  considering that some cells may respond earlier than others. However, we are  assuming that, as we are not changing the physical nor chemical conditions, the  proportion of cells responding &quot;earlier&quot; will remain constant, thus  allowing us to draw some conclusions of the behaviour of the population as a whole. We hope you see why the synchrony is no longer important for our  project. <br />
+
<p>We run a 2% Agarose gel with the following samples:</p>
-
   <br />
+
<ol>
-
   To summarize what we plan to do, AHL will enter the cell and form a dimer with  LuxR (which is under a constitutive promoter, so AHL is the only limiting  step). This will start the transcription of cI*, which will repress the  expression of RcnA. RcnA is the nickel efflux pump, and thus we are aiming to predict the amount of AHL necessary to get the desired extracellular nickel  concentration.<br />
+
  <li>Molecular Marker (2.5 μl)</li>
-
   We are doing small moves. At first, we only want to make one successful assay. We hope that in the near future we will be able to use the response time of the  system to generate a succession of desired nickel concentrations, thus  generating a song.<br />
+
   <li> Restriction of part 1_3 (5 μl)</li>
-
   We hope this letter answers your questions,<br />
+
  <li> Double Restriction of RcnA_3 (5 μl) no purified to verify.</li>
-
   <br />
+
   <li> RcnA (purified 5 μl)</li>
-
   LCG-UNAM-Mexico Team<br />
+
  <li> CI (5 μl)</li>
-
Cuernavaca, Morelos</p>
+
   <li> Part 3 Normal (5 μl)</li>
-
<p>&nbsp;</p>
+
  <li> Part 3 Mutated (5 μl)</li>
-
<p>&nbsp;</p>
+
</ol>
-
<p>&nbsp;</p>
+
<p>With this gel the parts were verified</p>
-
<p><b>AHL: LuxR <br /></b>
+
<p><strong><u>PCR </u></strong></p>
-
   <br />
+
<p>We performed a PCR reaction for RcnA and part 1 using Taq pol.</p>
-
Reaction 3<br />
+
<p><strong><u>Transformation y ligation</u></strong></p>
 +
<p>We took cut RcnA and then it was ligated at vector PBBIMCS_5</p>
 +
<table width="30%" border="1" cellpadding="0" cellspacing="0">
 +
   <tr>
 +
    <td width="45%"><p>2 μl</p></td>
 +
    <td width="55%"><p>vector</p></td>
 +
   </tr>
 +
  <tr>
 +
    <td width="45%"><p>5 μl</p></td>
 +
    <td width="55%"><p>Cut DNA </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="45%"><p>4 μl</p></td>
 +
    <td width="55%"><p>buffer</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="45%"><p>1 μl</p></td>
 +
    <td width="55%"><p>enzyme (T4 ligase)</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="45%"><p>8 μl</p></td>
 +
    <td width="55%"><p>H2O</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="45%"><p>20 μl</p></td>
 +
    <td width="55%"><p>Total</p></td>
 +
  </tr>
 +
</table>
 +
<p>The transformation was performed by the previously mentioned technique and the strain was cultured in two Gentamycine cages(Gm 20)</p>
 +
<p><strong><u>PCR Cleaning </u></strong></p>
 +
<p>We cleaned the PCR and it was filled up to 40 μl</p>
 +
</div></td>
 +
        </tr> 
 +
 
 +
 
 +
<tr>
 +
          <td class="subHeader" bgcolor="#99CC66" id="13">2008-08-13</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><p><strong>WET LAB:</strong></p>
 +
<p>4μl of each sample were charged in the following order:<br />
 +
</p>
 +
<ol>
 +
   <li>Molecular Marker</li>
 +
  <li> RcnA 1</li>
 +
  <li> RcnA 3</li>
 +
  <li> RcnA 4</li>
 +
  <li> RcnA 5</li>
 +
  <li> RcnA 6</li>
 +
  <li> Part 1_1</li>
 +
  <li> Part 1_3</li>
 +
  <li> Part 1_6</li>
 +
  <li> Part 1_7</li>
 +
  <li> Part 1_9</li>
 +
</ol>
 +
<p>&lt;Falta pegar gel segun liber...&gt;</p>
 +
<p><u><strong>Part 1_1 restriction (Double digestion)</strong></u></p>
 +
<table width="23%" border="1" cellpadding="0" cellspacing="0">
 +
  <tr>
 +
    <td width="50%"><p>DNA</p></td>
 +
    <td width="50%"><p>36 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>EcoR1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BamH1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BSA</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Buffer</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
</table>
 +
<p>After double digestion of part 1 we left massive ligation of part 1,2 and 3.</p>
 +
<p>Ligation Recipe:</p>
 +
<table width="23%" border="1" cellpadding="0" cellspacing="0">
 +
   <tr>
 +
    <td width="50%"><p>Part 1</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
   </tr>
 +
   <tr>
 +
    <td width="50%"><p>Part 2</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Part 3</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Buffer 5x</p></td>
 +
    <td width="50%"><p>4 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Ligase</p></td>
 +
    <td width="50%"><p>1 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p><strong>Total </strong></p></td>
 +
    <td width="50%"><p>20 μl</p></td>
 +
  </tr>
 +
</table>
 +
<p>Restriction of the other bioparts was performed</p>
 +
<table border="1" cellspacing="0" cellpadding="0" width="198">
 +
  <tr>
 +
    <td width="50%"><p><strong>RcnA_4</strong></p></td>
 +
    <td width="50%"><p>&nbsp;</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>H2O</p></td>
 +
    <td width="50%"><p>6 μl</p></td>
 +
   </tr>
 +
  <tr>
 +
    <td width="50%"><p>Buffer 2</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BSA</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>DNA</p></td>
 +
    <td width="50%"><p>30 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Xba1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>HindiIII</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
</table>
<br />
<br />
-
Conflict: k3 (ON) &lt;k3 (OFF)? <br />
+
<table border="1" cellspacing="0" cellpadding="0" width="198">
-
Reference: Goryachev et al. (2006) <br />
+
  <tr>
 +
    <td width="50%"><p><strong>RcnA_6</strong></p></td>
 +
    <td width="50%"><p>&nbsp;</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>H2O</p></td>
 +
    <td width="50%"><p>11 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Buffer 2</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BSA</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>DNA</p></td>
 +
    <td width="50%"><p>25 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Xba1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>HindiIII</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
</table>
<br />
<br />
-
 
+
<table border="1" cellspacing="0" cellpadding="0" width="198">
-
The references they use where they obtained parameters were not specific for this parameters (?) In fact, one mentions the rate of RNA polymerase in HUMAN! <br />
+
  <tr>
-
    
+
    <td width="50%"><p><strong>Part 1_3</strong></p></td>
-
-&gt; Check whether the article mentions how they got the parameter, or search through the references.<br />
+
    <td width="50%"><p>&nbsp;</p></td>
 +
   </tr>
 +
  <tr>
 +
    <td width="50%"><p>H2O</p>    </td>
 +
    <td width="50%"><p>6 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Buffer&nbsp; EcoR1</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BSA</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>DNA</p></td>
 +
    <td width="50%"><p>30 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>EcoR1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BamH1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
</table>
<br />
<br />
 +
<table border="1" cellspacing="0" cellpadding="0" width="198">
 +
  <tr>
 +
    <td width="50%"><p><strong>Part 1_6</strong></p></td>
 +
    <td width="50%"><p>&nbsp;</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>H2O</p></td>
 +
    <td width="50%"><p>6 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>Buffer&nbsp; EcoR1</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BSA</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>DNA</p></td>
 +
    <td width="50%"><p>30 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>EcoR1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BamH1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
</table>
<br />
<br />
-
</p>
+
<table border="1" cellspacing="0" cellpadding="0" width="198">
-
<p>They do explain why in the model, the k3 (ON) is in principle &quot;very small&quot;: <br />
+
  <tr>
-
   &lt;&lt;common to all models considered here, is that the stability of  the state &quot;off&quot; defined by the constitutive Transcription levels of I  and R comes at a price of high value for the critical self  Extracellular concentration.&gt;&gt; <br />
+
    <td width="50%"><p><strong>Part 1_9</strong></p></td>
-
   <br />
+
    <td width="50%"><p>&nbsp;</p></td>
-
   <br />
+
   </tr>
-
   And it seems that  this explains a bit the criteria that determined the parameters  used, although it does not appear in references such as: <br />
+
  <tr>
-
   &lt;&lt;For each layout we attempted to identify a set of parameters  that optimize the functional fitness of the network. The search in the  parameter space is constrained by requesting that the kinetic  parameters must remain in the biologically realistic range and the  resulting network should demonstrate the behavior compatible with our  present understanding of the phenomenon quorum sensing.&gt;&gt;</p>
+
    <td width="50%"><p>H2O</p></td>
-
<p>&nbsp;</p>
+
    <td width="50%"><p>1 μl</p></td>
-
        </td>
+
  </tr>
-
      </tr>   
+
  <tr>
 +
    <td width="50%"><p>Buffer&nbsp; EcoR1</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
   </tr>
 +
   <tr>
 +
    <td width="50%"><p>BSA</p></td>
 +
    <td width="50%"><p>5 μl</p></td>
 +
   </tr>
 +
   <tr>
 +
    <td width="50%"><p>DNA</p></td>
 +
    <td width="50%"><p>35 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>EcoR1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BamH1</p></td>
 +
    <td width="50%"><p>2 μl</p></td>
 +
  </tr>
 +
</table></div></td>
 +
        </tr>   
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="21">2008-08-21</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="14">2008-08-14</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p><br />
+
<td class="bodyText"><div align="justify"><p><strong>WET LAB:</strong></p>
-
  <strong><br />
+
<p><strong><u>GEL</u></strong></p>
-
  Natural degradation of cI * </strong><br />
+
<p>2% Agarose gel was run with the product of the massive ligation of the three parts</p>
-
  <br />
+
<p>(we didn't obtain the desired product)</p>
-
  <em>Reaction 4</em> <br />
+
<p><strong><u>PCR</u></strong></p>
-
  <br />
+
<p>We pick up a PCR product with the folowing oligos:</p>
-
   Real life time of cI? <br />
+
<blockquote>
-
   <br />
+
   <p>a) 1up and 2low<br />
-
   The half-life of modified cI is 4 minutes, according to Elowitz &amp; Leibler (2000). They analyze the tail LAA, and JB Andersen et al (1998)  conclude that the queues LAA and LVA confer about the same time of life  to GFP. <br />
+
   b) 2up and 2low<br />
-
   <br />
+
   c) control</p>
-
   Reaction rate? <br />
+
</blockquote>
-
   <br />
+
<p>The PCR reaction was prepared in the following way:</p>
-
   Once we get the half-life time of the protein, how do we calculate the rate of reaction and the flow? <br />
+
<table border="1" cellspacing="0" cellpadding="0" width="204">
-
   <br />
+
  <tr>
-
   The half-life of a reaction (t1 / 2) is the time it takes for half of the reagents to become  products. In a first order reaction, t1 / 2 is a  constant and can be calculated from the rate constant, as follows: <br />
+
    <td width="50%"><p><strong>Reaction 1</strong></p></td>
-
   <br />
+
    <td width="50%"><p>&nbsp;</p></td>
-
   t1 / 2 =-ln (0.5) / k = 0.693 / k <br />
+
   </tr>
-
   <br />
+
   <tr>
-
   This reciprocal relationship between the half life time and  the rate  constant is very useful to make an estimate of the timea given reaction will take place  in. Thus, for k = 0.01 s-1, the half life time would  be about 70 s. For k = 10 s-1, the half life time would be about 0.07 s  or 70 milliseconds. The average life time of the reactions of the first  order is also independent of the initial concentration. If the first  half of the molecules  react in aprox 20 s, half of the remaining  molecules will also take 20 s to react, and so on. The fact that the  lifetime average in an unimolecular reaction is a constant means that, at any  time of the reaction, a constant fraction of reactive molecules have enough  energy to overcome the kinetic barrier and become  molecules of product.  This makes sense because the energy of a set of molecules is  distributed randomly according to a Boltzmann distribution. <br />
+
    <td width="50%"><p>H2O</p></td>
-
   <br />
+
    <td width="50%"><p>10 μl</p></td>
-
   RT Sauer (1999); http://mit.ocw.universia.net/7.51/f01/pdf/fa01-lec02.pdf. <br />
+
   </tr>
-
  <br />
+
   <tr>
-
   NOTE: A first order reaction is the type A → B.
+
    <td width="50%"><p>Buffer 3.3x</p></td>
 +
    <td width="50%"><p>6 μl</p></td>
 +
   </tr>
 +
   <tr>
 +
    <td width="50%"><p>dNTPs</p></td>
 +
    <td width="50%"><p>4 μl</p></td>
 +
   </tr>
 +
   <tr>
 +
    <td width="50%"><p>Oligo up</p></td>
 +
    <td width="50%"><p>2.5 μl</p></td>
 +
   </tr>
 +
   <tr>
 +
    <td width="50%"><p>Oligo low</p></td>
 +
    <td width="50%"><p>2.5 μl</p></td>
 +
   </tr>
 +
   <tr>
 +
    <td width="50%"><p>Mg (Ac)2</p></td>
 +
    <td width="50%"><p>3 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>DNA</p></td>
 +
    <td width="50%"><p>2 μl </p></td>
 +
   </tr>
 +
</table>
<br />
<br />
-
</p>
+
<table width="27%" border="1" cellpadding="0" cellspacing="0">
-
        </td>
+
  <tr>
-
      </tr>
+
    <td width="50%"><p><strong>Reaction 2</strong></p></td>
-
<tr>
+
    <td width="50%"><p>&nbsp;</p></td>
-
          <td class="subHeader" bgcolor="#99CC66" id="26">2008-08-26</td>  
+
  </tr>
-
        </tr>
+
  <tr>
-
        <tr>
+
    <td width="50%"><p>H2O</p></td>
-
<td class="bodyText"><p> <br />
+
    <td width="50%"><p>9 μl</p></td>
-
  <br />
+
   </tr>
-
  <strong>Checking parameters </strong><br />
+
   <tr>
-
   <br />
+
    <td width="50%"><p>Buffer 3.3x</p></td>
-
   <em>Reaction 6 </em><br />
+
    <td width="50%"><p>9 μl</p></td>
-
  <br />
+
  </tr>
-
  The value that had been found before (reference 7 from the model document) k6  (Pl) = 0.20mM / h is really the flow of the reaction (which is why the  units are mM / h). That is, how many mRNA molecules are being produced per  unit of time. The system in measuring this parameter is a derivative of  pBR322 plasmid, pTrc99A. It has the same origin of replication and the  number of copies is estimated at 15-20 (doi: 10.1016/S0264-410X (02)  00292-X) but under certain conditions where replication is limited (?)  it appears to be between 3-5 copies. <br />
+
  <tr>
-
  <br />
+
    <td width="50%"><p>rTth</p></td>
-
  From here we can  calculate the value of k+ of the reaction in our system if we consider  that each promoter acts independently and we multiply by the ratio between  the number of copies of our plasmid and theirs. <br />
+
    <td width="50%"><p>2 μl</p></td>
-
  <br /></p>
+
   </tr>
-
        </td>
+
</table>
-
      </tr>  
+
<p><strong><u>GEL </u></strong></p>
-
<tr>
+
<p>We run for 1 hr, an 2% Agarose Gel </p>
-
          <td class="subHeader" bgcolor="#99CC66" id="28">2008-08-28</td>  
+
<ol>
-
        </tr>
+
   <li>Molecular Marker (2.5 μl)</li>
-
        <tr>
+
   <li> P1_P2 (oligo 1 up y 2 low) ligation with part 3 (normal) (5μl)</li>
-
<td class="bodyText"><p><br />
+
   <li> P2_P3 normal (oligo 2 up y 3 low) (5 μl)</li>
-
   <br />
+
   <li> Negative Control(oligo 1 up y 2 low) (5 μl)</li>
-
  <strong>Tasks </strong><br />
+
   <li> P1_P2 (oligo 1 up y 2 low) ligation with part 3 (mutated) (5 μl)</li>
-
  <br />
+
   <li> P2_P3 mutated (oligo 2 up y 3 low) (5μl)</li>
-
  1. Parameters. <br />
+
   <li> Negative Control (oligo 1 up y 2 low) (5μl)</li>
-
  - cI Dimerization. <br />
+
</ol>
-
  - Nickel extrusion . (Estimate,?). <br />
+
<p>*The results suggest inspecific primer join.</p>
-
  - RcnA degradation. <br />
+
<p><u><strong>Cultures</strong></u></p>
-
   - Nickel Internalization (Experimentally,?). <br />
+
<p>We left cultures of the RcnA +  PBBRIMCS_5 ligation</p>
-
   - Initial concentrations (aiiA, LuxR? (Define arbitrary medium), ? (by  number of copies of the plasmid; change to molar), Niext (experimental)). <br />
+
</div></td>
-
   <br />
+
        </tr>  
-
  2. Reviewing tools in SimBiology (sensitivity analysis, parameter estimation, moiety conservation). <br />
+
 
-
   <br />
+
-
  3. Stationary states of the system (if there is multistationarity). <br />
+
-
   <br />
+
-
  4. Jacobian. <br />
+
-
   <br />
+
-
  5. Analysis of the stechiometric matrix (also analyze the null space and its transposed). <br />
+
-
   <br />
+
-
  6. Electrochemical theory (the difference between potential and the concentration of nickel). <br />
+
-
  <br />
+
-
  7. Electrodes. <br />
+
-
  -&gt;We need to check how the measurement device we will use is going.<br />
+
-
  -&gt; Take into account the possibility of asking for support to Dr. Peña. <br />
+
-
  <br />
+
-
  <br /></p>
+
-
        </td>
+
-
      </tr>  
+
       <tr>
       <tr>
       <td colspan="6">
       <td colspan="6">
-
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+
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Latest revision as of 03:45, 29 October 2008

LCG-UNAM-Mexico:Notebook/August

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iGEM 2008 TEAM
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August

2008-08-01

WET LAB:

We left PCR of:

  • Part 1 of Biopart BBa_I79006
  • Biopart cI BBa_C0051
  • Biopart 3 (normal) ofBBa_I79006
  • Biopart 3 (mut) of BBa_I79006
  • Operator of rcnR + RcnA
2008-08-04

MODELING:
Hill cooperativity 5th Reaction Reminder:

A + B <--> AB
Ka=Keq=[AB]/[A][B]=1/Kd
θ=[AB]/([AB]+[A])=[B]/([B]+Kd)


MWC Model (Cooperativity)
A + nB <--> ABn
Ka=Keq=[ABn]/[A][B]n=1/Kd
θ=[B]n/([B]n+Kd)
log(θ/(1- θ))=nlog(B)-log(kd) …Hill's equation


Suppression mediated by cI:
ρ + nCI <--> ρ:CIn (k+, k-)
Keq=Ka=[ρ:CIn]/[ρ][CI]n
Si ρ0=[ρ]+[ρ:CIn]
… ρ0=[ρ]+Keq[ρ][CI]n
=> ρ= (ρ0/Keq)/((1/keq)+[CI]n)

Flow= k+[ρ][CI]n = K+((ρ0/Keq)/((1/Keq)+[CI]n))[CI]n


Flow= k+([ρ0]/Keq) [CI]n / ((1/Keq)+[CI]n)

=> Vm= k+([ρ0]/Keq) & Kp=1/Keq=Kd

Therefore:
Keq = exp( -ΔG / R T )
k+ = (KB/h) T exp( -ΔG / R T ) = (KB/h) T Keq

Keq=

2.89517E+17

KB=

1.38E-23

J/K

k+=

1.79764E+30

/s

h=

6.63E-34

J s

R=

1.9872

cal/(K mol)

ΔG=

-23810

cal/mol

T=

298

K

 

WET LAB:

Gel

We run a gel with the PCR products obtained the day 01-08-08

Purification

From the PCR of the 01-08-08 we took 120 μl to purify DNA in a low fusion point agarose gel in line with the kit.

We run an agarose gel to verify the status of the purified PRC products.

Gel_04Ago08

  1. Molecular Marker
  2. Part 1 of biopart BBa_I79006
  3. Biopart cI BBa_C0051
  4. Biopart 3 (normal) of BBa_I79006
  5. Biopart 3 (mut) ofBBa_I79006
  6. Operator of rcnR + RcnA

Restrictions

We left restrictions over night(double and simple) of each biopart.

First Simple Restriction

The bioparts PCR product was cut with 2 simple consecutive restrictions. The reactives and volumes used in the first reaction were the following:

  • Part 1; BamH1

    Buffer U.... 4 microlts
    BSA.......... 4 microlts
    BamH1...... 2 microlts
    DNA PCR... 15 microlts
    H2O......... 15 microlts   
    Total......... 40 microlts

  • Part 2_cI; BamH1

    Buffer U.... 4 microlts
    BSA.......... 4 microlts
    BamH1...... 2 microlts
    DNA PCR... 15 microlts
    H2O......... 15 microlts   
    Total......... 40 microlts

  • Part 3 Normal; Xba1

    Buffer U.... 7 microlts
    BSA.......... 7 microlts
    Xba1......... 3 microlts
    DNA PCR... 28 microlts
    H2O......... 25 microlts   
    Total......... 70 microlts

  • Part 3 Mutated; Xba1

    Buffer U.... 7 microlts
    BSA.......... 7 microlts
    Xba1......... 3 microlts
    DNA PCR... 28 microlts
    H2O......... 25 microlts   
    Total......... 70 microlts

  • Part 4 RcnA; Xba1

    Buffer U.... 7 microlts
    BSA.......... 7 microlts
    Xba1......... 3 microlts
    DNA PCR... 28 microlts
    H2O......... 25 microlts   
    Total......... 70 microlts

2008-08-05

MODELING:
Hill Cooperativity

5th Reaction, conflict

If we consider that:

  - Keq = exp (-ΔG / R T)

  - k + = (KB / h) T exp (-ΔG / R T) = (KB / h) T Keq

and given that the flow is (k + / Keq) [ρ0] [CI] n / ((1/Keq) + [CI] n), the value of the maximum speed of the flow loses its meaning.

The speed limit is being determined by (k + / Keq) [ρ0], but k + / Keq = (KB / h) * T, and we know that [ρ0] is arbitrary, i.e., Vmax is no longer based on the reaction as such, which does not make sense.

For example: Take the same reaction that we are considering, the maximum speed of the flow of the reaction would be the same with the promoter that has the operators of CI, that if you used one with a random sequence, so, whether we repeated the experiment, with the same temperature and the same concentration of DNA and an equal number of copies of the sequence, the maximum speed reached by the flow would be the same for the real promoter as for for any sequence, without taking any consideration with their affinity for their substrates... That does not makes sense!

The proposed explanation is that the equation used to determine k + does not fit our model. We should explore other possibilities.

 

WET LAB:

Restrictions

Second simple restriction

Before the second simple restriction we cleaned the product oof the first restriction with the purification Kit.
Due to the purification protocol we knew that the DNA was clean and diluted in 40 μl of buffer, and in order to obtain an efficient restriction we try to dilute the less the DNA-Buffer mix, obtaining the following volumes.

  • Part 1; EcoR1

    Buffer U....... 5 μl
    BamH1........ 2.5 μl
    Clean DNA ... 40 μl
    H2O............2.5 μl
    Total........... 50 μl

  • Part 2_cI; Xba1

    Buffer 2....  5 μl
    BSA.......... 2.5 μl
    BamH1...... 2.5 μl
    DNA PCR... 40 μl 
    Total......... 50 μl

  • Part 3 (Normal); Pst1
  • Buffer 3.... 5 μl
    Pst1......... 2.5 μl
    DNA PCR... 40 μl
    H2O......... 2.5 μl   
    Total......... 50 μl

  • Part 3 Mutated; Pst1

    Buffer 3.... 5 μl
    Xba1......... 2.5 μl
    DNA PCR... 40 μl
    H2O......... 2.5 μl   
    Total......... 50 μl

  • Part 4 RcnA; HindIII

    Buffer 2.... 5 μl
    HindIII..... 2.5 μl
    DNA PCR... 40 μl
    H2O......... 2.5 μl   
    Total......... 50 μl

Extraction

Plasmids pRK415 and pBBR1MCS-5 were extracted with the Roche kit(see Techniques).

Cultures

We cultured DH5alfa cells tranfromed with pJet+biopart

2008-08-07

MODELING:
Hill Cooperativity:
5th Reaction, solving the problem:

The error in the previous approach was that we were considering ΔG to be the same for both equations (for Keq & k+).

We know that the equilibrium depends solely on the difference in the free energy of Gibbs between the substrate and the product (ΔG 'th). The one with less energy will be favored in the balance, while the rate of reaction depends on the activation energy needed for the conversion (ΔG ‡). A reaction reaches equilibrium faster or slower depending on the rate of reaction (depending on the magnitude of its ΔG ‡), but the balance itself does not change.

Thus:
Keq = exp (- ΔG 'º / R T)
k + = (KB / h) T exp (- ΔG ‡ / RT) ≠ (KB / h) T Keq

 

2008-08-11

GROUP MEETING
Wet Lab Statusk

Objectives:

- Build the bioparts.
- Transform the bacteria with the construction that we have.
- Design the experiments to test our construction.
- Build the system.
- Collaborate with the modeling group.

To do:
- Extract DNA from the wild type strain to obtain RcnA.
- Get the bioparts catalog.
- Obtain a large amount of plasmid that we can use, and amplify the bioparts.
- Transformation of the bacteria with bioparts.

Currently:
- There are plasmids.
- There are parts already amplified and in a plasmid.

Problems:
- The DNA that we needed was not in the registry.
- The oligos were delayed 2 weeks and a half.
- Issues to extract the plasmid from the colonies.
- Make a PCR ligation with the three parts and amplify with the ends (it did not work).
- With the enzyme used the frequency of spontaneous mutation was increased to about an error every thousand base pairs.
- There is a problem with tetracycline. You get false positives.

What can be done:

- The biopart with RcnA can already be linked to the plasmid.
- For the other construction we will have to link two parts and digest them, then link them with the third part and digest once more, then insert into the final plasmid.
- HindIII can be used with the large biopart to verify the sequence.

Electrodes:

- Will they be specific for Nickel?

 

2008-08-12

WET LAB:

Cultures

We left cultures of Biopart 1 in pJEt

Plasmid Extraction

Plasmid RcnA was extracted by alkaline lysis

Gels

We run a 2% Agarose gel with the following samples:

  1. Molecular Marker (2.5 μl)
  2. Restriction of part 1_3 (5 μl)
  3. Double Restriction of RcnA_3 (5 μl) no purified to verify.
  4. RcnA (purified 5 μl)
  5. CI (5 μl)
  6. Part 3 Normal (5 μl)
  7. Part 3 Mutated (5 μl)

With this gel the parts were verified

PCR

We performed a PCR reaction for RcnA and part 1 using Taq pol.

Transformation y ligation

We took cut RcnA and then it was ligated at vector PBBIMCS_5

2 μl

vector

5 μl

Cut DNA

4 μl

buffer

1 μl

enzyme (T4 ligase)

8 μl

H2O

20 μl

Total

The transformation was performed by the previously mentioned technique and the strain was cultured in two Gentamycine cages(Gm 20)

PCR Cleaning

We cleaned the PCR and it was filled up to 40 μl

2008-08-13

WET LAB:

4μl of each sample were charged in the following order:

  1. Molecular Marker
  2. RcnA 1
  3. RcnA 3
  4. RcnA 4
  5. RcnA 5
  6. RcnA 6
  7. Part 1_1
  8. Part 1_3
  9. Part 1_6
  10. Part 1_7
  11. Part 1_9

<Falta pegar gel segun liber...>

Part 1_1 restriction (Double digestion)

DNA

36 μl

EcoR1

2 μl

BamH1

2 μl

BSA

5 μl

Buffer

5 μl

After double digestion of part 1 we left massive ligation of part 1,2 and 3.

Ligation Recipe:

Part 1

5 μl

Part 2

5 μl

Part 3

5 μl

Buffer 5x

4 μl

Ligase

1 μl

Total

20 μl

Restriction of the other bioparts was performed

RcnA_4

 

H2O

6 μl

Buffer 2

5 μl

BSA

5 μl

DNA

30 μl

Xba1

2 μl

HindiIII

2 μl


RcnA_6

 

H2O

11 μl

Buffer 2

5 μl

BSA

5 μl

DNA

25 μl

Xba1

2 μl

HindiIII

2 μl


Part 1_3

 

H2O

6 μl

Buffer  EcoR1

5 μl

BSA

5 μl

DNA

30 μl

EcoR1

2 μl

BamH1

2 μl


Part 1_6

 

H2O

6 μl

Buffer  EcoR1

5 μl

BSA

5 μl

DNA

30 μl

EcoR1

2 μl

BamH1

2 μl


Part 1_9

 

H2O

1 μl

Buffer  EcoR1

5 μl

BSA

5 μl

DNA

35 μl

EcoR1

2 μl

BamH1

2 μl

2008-08-14

WET LAB:

GEL

2% Agarose gel was run with the product of the massive ligation of the three parts

(we didn't obtain the desired product)

PCR

We pick up a PCR product with the folowing oligos:

a) 1up and 2low
b) 2up and 2low
c) control

The PCR reaction was prepared in the following way:

Reaction 1

 

H2O

10 μl

Buffer 3.3x

6 μl

dNTPs

4 μl

Oligo up

2.5 μl

Oligo low

2.5 μl

Mg (Ac)2

3 μl

DNA

2 μl


Reaction 2

 

H2O

9 μl

Buffer 3.3x

9 μl

rTth

2 μl

GEL

We run for 1 hr, an 2% Agarose Gel

  1. Molecular Marker (2.5 μl)
  2. P1_P2 (oligo 1 up y 2 low) ligation with part 3 (normal) (5μl)
  3. P2_P3 normal (oligo 2 up y 3 low) (5 μl)
  4. Negative Control(oligo 1 up y 2 low) (5 μl)
  5. P1_P2 (oligo 1 up y 2 low) ligation with part 3 (mutated) (5 μl)
  6. P2_P3 mutated (oligo 2 up y 3 low) (5μl)
  7. Negative Control (oligo 1 up y 2 low) (5μl)

*The results suggest inspecific primer join.

Cultures

We left cultures of the RcnA + PBBRIMCS_5 ligation