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

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           <td class="pageName"><strong>August</strong></td>           
+
           <td class="pageName"><strong>September</strong></td>           
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           <td class="subHeader" bgcolor="#99CC66" id="04">2008-08-04</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="01">2008-09-01</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p><strong>Hill's cooperativity</strong><br />
+
<td class="bodyText"><div align="justify"><p> <strong>WET LAB</strong></p>
-
    <strong>5th Reaction </strong> <br />
+
<p><strong><u>Cultures</u></strong></p>
-
    <strong>Reminder:</strong> </p>
+
<ol>
-
<p>A + B &lt;--&gt; AB            <br />
+
  <li>One of the colonies obtained after we transformed the ligations (part1_part2_part 3 normal) + PRK415 and (part1_part2-part3Mutated)+PRK415 was  cultured. </li>
-
    <strong>Ka=Keq=[AB]/[A][B]=1/Kd</strong>     <br />
+
  <li>Cultures of 10 colonies of part1+part2+PRK415 in liquid broth were  prepared. </li>
-
  θ=[AB]/([AB]+[A])=[B]/([B]+Kd) </p>
+
  <li>1% Agarose Gel (PRK415+(parte1+parte2) repetition)<br />
-
<p><strong><u>MWC Model</u></strong> (Cooperativity)          <br />
+
    <blockquote>
-
  A + nB &lt;--&gt; ABn        <br />
+
      <p>1.-Molecular marker 2.5 μl<br />
-
  <strong>Ka=Keq=[ABn]/[A][B]n=1/Kd</strong>  <br />
+
        2.-[1]  part1_part2 in PRK415 extraction 5μl<br />
-
  θ=[B]n/([B]n+Kd)        <br />
+
        3.-[2] part1_part2 in PRK415 extraction 5μl<br />
-
  log(θ/(1- θ))=nlog(B)-log(kd)                        …Hill's equation</p>
+
        4.-[3] part1_part2 in PRK415 extraction 5μl<br />
-
<p> </p>
+
        5.-[4] part1_part2 in PRK415 extraction 5μl<br />
-
<p><strong>Suppression mediated by cI:</strong> <br />
+
        6.-[5] part1_part2 in PRK415 extraction 3 μl<br />
-
  ρ  + nCI &lt;--&gt; ρ:CIn    (k+,  k-)  <br />
+
        7.-[6] part1_part2 in PRK415 extraction 3 μl<br />
-
  <strong>Keq=Ka=[ρ:CIn]/[ρ][CI]n      <br />
+
        8.-[8] part1_part2 in PRK415 extraction 3 μl<br />
-
  </strong>Si ρ0=[ρ]+[ρ:CIn]      <br />
+
        9.-[9] part1_part2 in PRK415 extraction 3 μl<br />
-
  … ρ0=[ρ]+Keq[ρ][CI]n <br />
+
        10.-[13] part1_part2 in PRK415 extraction 3 μl<br />
-
  =&gt;  ρ= (ρ0/Keq)/((1/keq)+[CI]n) </p>
+
        11.-[15] part1_part2 in PRK415 extraction 3 μl<br />
-
<p>Flow=  k+[ρ][CI]n = K+((ρ0/Keq)/((1/Keq)+[CI]n))[CI]n </p>
+
      </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>
+
    </blockquote>
-
<p><strong>=&gt; </strong><strong>Vm=  k</strong><strong>+</strong>([ρ0]/Keq)<strong>  &amp;  Kp=1/Keq=K</strong>d </p>
+
    <p>The size of the plasmids does not correlate the size we are expecting.</p>
-
<p><strong>So:</strong> <br />
+
  </li>
-
  Keq = exp(  -ΔG / R T )          <br />
+
 
-
  k+ = (KB/h) T exp( -ΔG / R T ) = (KB/h) T Keq </p>
+
  <li>Step 2 was  repeated.</li>
-
<table bgcolor="#cc99ff" border="1" bordercolor="#000000" cellpadding="1" cellspacing="1" width="423">
+
  <li>Cultures with (Part1_part2_part3N)+PRK415  were prepared for extraction. <br />
-
  <tbody>
+
    <blockquote>1 PRK415 part1+part2<br />
-
    <tr>
+
    12 PRK415 part1+part2<br />
-
      <td width="53" nowrap="nowrap"><p>Keq= </p></td>
+
    13 PRK415 part1+part2<br />
-
      <td width="90" nowrap="nowrap"><p>2.89517E+17 </p></td>
+
    14 PRK415 part1+part2<br />
-
      <td width="52" nowrap="nowrap"><p>  </p></td>
+
    15 PRK415 part1+part2<br />
-
      <td width="39" nowrap="nowrap"><p><strong>K</strong><strong>B</strong><strong>=</strong> </p></td>
+
    16 PRK415 part1+part2<br />
-
      <td width="69" nowrap="nowrap"><p>1.38E-23 </p></td>
+
    17 PRK415 part1+part2<br />
-
      <td width="87" nowrap="nowrap"><p>J/K </p></td>
+
    18 PRK415 part1+part2<br />
-
    </tr>
+
    19 PRK415 part1+part2<br />
-
     <tr>
+
    20 PRK415 part1+part2<br />
-
      <td width="53" nowrap="nowrap"><p>k+= </p></td>
+
    21 PRK415 part1+part2</blockquote>
-
       <td width="90" nowrap="nowrap"><p>1.79764E+30 </p></td>
+
    </li>
-
       <td width="52" nowrap="nowrap"><p>/s </p></td>
+
</ol>
-
       <td width="39" nowrap="nowrap"><p><strong>h=</strong> </p></td>
+
</div></td>
-
       <td width="69" nowrap="nowrap"><p>6.63E-34 </p></td>
+
        </tr>  
-
       <td width="87" nowrap="nowrap"><p>J s </p></td>
+
<tr>
-
    </tr>
+
          <td class="subHeader" bgcolor="#99CC66" id="02">2008-09-02</td>  
-
    <tr>
+
        </tr>
-
       <td colspan="3" nowrap="nowrap"></td>
+
        <tr>
-
       <td width="39" nowrap="nowrap"><p><strong>R=</strong> </p></td>
+
<td class="bodyText"><div align="justify"><strong>WET LAB</strong><br />
-
       <td width="69" nowrap="nowrap"><p>1.9872 </p></td>
+
  Plasmid extraction of the cultures PRK415 +  p1_p2 y PRK415+ p1_p2_p3N was made.<br />
-
       <td width="87" nowrap="nowrap"><p>cal/(K mol) </p></td>
+
  <br />
-
     </tr>
+
  <strong><u>1%   Agarose gel.</u></strong></p>
-
     <tr>
+
<table border="1" cellspacing="0" cellpadding="0" width="100%">
-
      <td width="53" nowrap="nowrap"><p><strong>ΔG=</strong> </p></td>
+
  <tr>
-
       <td width="90" nowrap="nowrap"><p><strong>-23810</strong> </p></td>
+
     <td width="50%">     Gel (19    wells)<br />
-
       <td width="52" nowrap="nowrap"><p><strong>cal/mol</strong> </p></td>
+
       1.-Molecular-weight marker<br />
-
       <td width="39" nowrap="nowrap"><p><strong>T=</strong> </p></td>
+
      2.-[18]Part1_part2 extraction in PRK415<br />
-
       <td width="69" nowrap="nowrap"><p>298 </p></td>
+
       3.-[19]Part1_part2 extraction in PRK415<br />
-
      <td width="87" nowrap="nowrap"><p>K </p></td>
+
      4.-[20]Part1_parte2 extraction in PRK415<br />
-
    </tr>
+
       5.-[13] (part1_part2)_part3 extraction in PRK415<br />
-
  </tbody>
+
       6.-<br />
 +
      7.-<br />
 +
       8.-<br />
 +
      9.-<br />
 +
      10.-<br />
 +
      11.-[1] (part1_part2)_part3 extraction in PRK415<br />
 +
       12.-[3] (part1_part2)_part3 extraction in PRK415<br />
 +
       13.-[4] (part1_part2)_part3 extraction in PRK415<br />
 +
      14.-[5] (part1_part2)_part3 extraction in PRK415<br />
 +
      15.-[6] (part1_part2)_part3 extraction in PRK415<br />
 +
       16.-[8] (part1_part2)_part3 extraction in PRK415<br />
 +
      17.-[9] (part1_part2)_part3 extraction in PRK415<br />
 +
       18.-[11] (part1_part2)_part3 extraction in PRK415<br />
 +
     19.-[12] (part1_part2)_part3 extraction in PRK415 </td>
 +
     <td width="50%" valign="top"><p>Gel (9 wells extraction)<br />
 +
      1.- Molecular-weight marker<br />
 +
      2.-[11] part1_part2 extraction in PRK415<br />
 +
       3.-[12] part1_part2 extraction in PRK415<br />
 +
      4.-[13] part1_part2 extraction in PRK415<br />
 +
      5.-[14] part1_part2 extraction in PRK415<br />
 +
       6.-[15] part1_part2 extraction in PRK415<br />
 +
      7.-[16] part1_part2 extraction in PRK415<br />
 +
       8.-[17] part1_part2 extraction in PRK415<br />
 +
       <br />
 +
    &nbsp;</p></td>
 +
  </tr>
</table>
</table>
-
<p>&nbsp;</p>
+
<p><img width="400" src="https://static.igem.org/mediawiki/2008/a/a5/Gel02Sep08.png" /><br />
-
        </td>
+
    <br />
-
      </tr>  
+
  Dirty plasmid, in theory, it should not affect the  PCR because the genes occupied for this are not in the E.coli genome. We proceeded to do the PCR. (yo digo que no pongamos la foto del gel  esta muy sucio)</p>
 +
<p><strong><u>PCR</u></strong></p>
 +
<blockquote>
 +
  <p>1.-[17]  PRK415 + p1_p2 oligos 1upper 2 lower<br />
 +
    2.-[8] PRK415 + p1_p2_p3N oligos 2upper 3 lower<br />
 +
    3.-[9] PRK415 + p1_p2_p3N oligos 2upper 3 lower<br />
 +
    4.-CN1 H2O<br />
 +
    5.-CN2 PRK415 DR EcoR1 y BamH1 oligos 1upper 2lower<br />
 +
    6.-CN3 PRK415 DR EcoR1 y BamH1 oligos 2upper 3lower<br /></p>
 +
</blockquote></div></td>
 +
        </tr>  
 +
 
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="05">2008-08-05</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="03">2008-09-03</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"> <p>Hill's Cooperativity<br />
+
<td class="bodyText"><div align="justify"><p>
 +
  <strong><u>MODELING:</u></strong><br />Lac promoter synthesis rate:
 +
  <br />
 +
The effect of stochasticity on the Lac  Operon: An evolutionary perspective from van Hoek F <i>et al</i> (2007) <br />
 +
The article's objective, as you can infer from the title, is to evaluate the effect of stochasticity in the evolution of a Promoter. In  order to do so they built a comprehensive model including every parameter involved in transcription and translation. They measure some parameters but they depend mostly on literature to define them. <br />
 +
<br />
 +
They perform both a deterministic and a stochastic analysis. To generate a stochastic model they added one parameter, the average burst size of  protein translation (protein translation occurs in bursts, after an mRNA is synthesized, several proteins can be translated from the same  mRNA). This was possible because when an mRNA molecule is translated it can not be degraded. Therefore after each translation it can either be translated again (p) or be degraded (1-p). This suggests that protein production occurs in bursts with a burst size geometrically distributed. Afterwards, they compared the noise levels in their model with experimental noise measurements and found correlation. <br />
 +
<br />
 +
To model transcription they used a two-dimensional Hill-function  dependent on the cAMP and allolactose concentration. (repress the glucose and lactose operon via cAMP and activates the operon via allolactose). <br />
 +
<br />
 +
They use 11 biochemical parameters, including three of special importance for us: <br />
 +
<li>a, Transcription rate when the RNA Polymerase is bound to the DNA, but CRP and Laci are not. Initial value: 1.1 × 10-7 mM/min </li>
 +
<li>b, The transcription rate when both RNA Polymerase and CRP are bound,  but Laci is not bound to the DNA. Initial value: 2.2 × 10-5 mM/min </li>
 +
<li>c, <i>Leakiness</i>, the transcription rate when RNA Polymerase is not bound to the DNA. Initial value 5.5 × 10-10 mM/min </li>
 +
<br />
 +
 
 +
They modeled binomially protein degradation, assuming that when cells divide, their proteins are randomly divided between the cells. However in a population of non-Dividing cells this &quot;dilution&quot; can not be taken in  account. </p>
 +
<strong>WET LAB</strong></p>
 +
<p><strong><u>2% Agarose gel with PCR samples of 2/09/08</u></strong></p>
 +
<blockquote>
 +
  <p>  1.-Molecular marker<br />
 +
    2.- [1c2]&nbsp; [17] PRK415 + p1_p2 oligos 1upper 2 lower<br />
 +
    3.- [2]&nbsp; [8] PRK415 + p1_p2_p3N oligos 2upper 3 lower<br />
 +
    4.- [3]&nbsp; [9] PRK415 + p1_p2_p3N oligos 2upper 3 lower<br />
 +
    5.- [4]&nbsp; CN1 H2O<br />
 +
    6.- [5]&nbsp; CN2 PRK415 DR EcoR1 y BamH1 oligos 1upper 2lower<br />
 +
    7.- [6]&nbsp; CN3 PRK415 DR EcoR1 y BamH1 oligos 2upper 3lower<br />
 +
    8.- R6 Restriction EcoR1 HindIII 1_2_3 (5 μl)<br />
 +
    9.- R8 Restriction EcoR1 HindIII 1_2_3 (2 μl)<br />
 +
    10.- R9 Restriction EcoR1 HindIII 1_2_3 (2 μl)<br />
 +
    11.- R17 Restriction EcoR1 XbaI 1_2_3 (2 μl)<br />
 +
  </p>
 +
</blockquote>
 +
<p>No results.<br />
 +
  <br />
 +
  Cultures in 2ml of LbTc10 of PRK415 +part1+part2 and of DH5alfa 123 (3normal)  ligation were prepared.
</p>
</p>
-
<p>5th Reaction, conflict ...<br />
 
-
</p>
 
-
<p>If we consider that: <br />
 
-
</p>
 
-
<p>Keq = exp (-ΔG / R T) <br />
 
-
</p>
 
-
<p>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>  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>  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>
 
         </td>
         </td>
       </tr>  
       </tr>  
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="07">2008-08-07</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="04">2008-09-04</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p>Hill's Cooperativity: <br />
+
<td class="bodyText"><div align="justify"><strong>WET LAB</strong> <br />
-
  5th Reaction, resolving the conflict... <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 />
+
   <strong><u>Extraction of plasmid </u></strong><br />
 +
  Part1_2_3N-PRK415 <br />
 +
  Part1_2 PRK415 <br />
 +
  Part1_2_3N PRK415 Repeat</p>
 +
<p><strong><u>1%  Agarose gel with extractions</u></strong><br />
 +
  2.-[3] PRK415 part1_2_3N Ligation<br />
 +
  3.-[5] PRK415 part1_2_3N Ligation<br />
 +
  4.-[6] PRK415 part1_2_3N Ligation<br />
 +
  5.-[8] PRK415 part1_2_3N Ligation<br />
 +
  6.-[9]PRK415 part1_2_3N Ligation<br />
 +
  7.-[10] PRK415 part1_2_3N Ligation<br />
 +
  8.-[11] PRK415 part1_2_3N Ligation<br />
 +
  9.-[12] PRK415 part1_2_3N Ligation<br />
 +
  10.-[13] PRK415 part1_2_3N Ligation<br />
 +
  11.-[1.2] PRK415 part1_2_3N Ligation<br />
 +
  12.-[3.2] PRK415 part1_2_3N Ligation<br />
 +
  13.-[5.2] PRK415 part1_2_3N Ligation<br />
 +
  14.-[6.2] PRK415 part1_2_3N Ligation<br />
 +
  15.-[8.2] PRK415 part1_2_3N Ligation<br />
 +
  16.-[9.2] PRK415 part1_2_3N Ligation<br />
 +
  17.-[10.2] PRK415 part1_2_3N Ligation<br />
 +
  18.-[11.2] PRK415 part1_2_3N Ligation<br />
 +
  19.-[12.2] PRK415 part1_2_3N Ligation<br />
 +
  20.-[13.2] PRK415 part1_2_3N Ligation<br />
 +
  <img width="400" src="https://static.igem.org/mediawiki/2008/e/e6/Gel_04Sep08.png" /><br />
 +
  PCR ligation p1_p2_p3 in PRK415<br />
 +
  No positive results.<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>
+
  <strong><u>Electrophoresis ligation p1+p2 PRK415</u></strong><br />
-
<div id="urdd">
+
  </p>
-
   <div align="center"><img src="http://docs.google.com/File?id=dntmktb_99dz485zf8_b" alt="" name="sm1w6" id="sm1w6" /></div>
+
<blockquote>
-
</div>
+
   <p>1.-Molecular marker<br />
-
<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 />
+
    2.-[1] p1+p2 PRK415<br />
 +
    3.-[2] p1+p2 PRK415<br />
 +
    4.-[3] p1+p2 PRK415<br />
 +
    5.-[4] p1+p2 PRK415<br />
 +
    6.-[5] p1+p2 PRK415<br />
 +
    7.-[17] p1+p2 PRK415<br />
 +
    8.-[18] p1+p2 PRK415<br />
 +
    9.-[19] p1+p2 PRK415<br />
 +
    10.-[20] p1+p2 PRK415<br />
 +
    11.-[21] p1+p2 PRK415<br />
 +
    <br />
 +
    </p>
 +
</blockquote>
 +
<p>4 cultures of PRK415 were left.<br />
   <br />
   <br />
-
Thus: <br />
+
  <strong><u>Restrictions</u></strong></p>
-
       
+
<p><strong>part1_part2&nbsp; (PCR)<br />
-
Keq = exp (- ΔG 'º / R T) <br />
+
  part 3 mutated (PCR)</strong></p>
-
       
+
<table width="400" border="1" cellspacing="0" cellpadding="0">
-
k + = (KB / h) T exp (- ΔG ‡ / RT) ≠ (KB / h) T Keq</p>
+
  <tr>
-
<p>&nbsp; </p>
+
    <td>H2O</td>
-
        </td>
+
    <td>3 μl</td>
-
      </tr>   
+
  </tr>
 +
  <tr>
 +
    <td>Buffer</td>
 +
    <td>5 μl</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BSA</td>
 +
    <td>5 μl</td>
 +
  </tr>
 +
  <tr>
 +
    <td>DNA</td>
 +
    <td>35 μl</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Xba</td>
 +
    <td>2 μl</td>
 +
  </tr>
 +
  <tr>
 +
    <td>&nbsp;</td>
 +
    <td><strong>50 μl</strong></td>
 +
  </tr>
 +
</table>
 +
<p>Part1_part2 ligation sep-09</p>
 +
<table width="400" border="1" cellspacing="0" cellpadding="0">
 +
  <tr>
 +
    <td>H2O</td>
 +
    <td>2 μl</td>
 +
  </tr>
 +
   <tr>
 +
    <td>Buffer</td>
 +
    <td>3 μl</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BSA</td>
 +
    <td>3 μl</td>
 +
  </tr>
 +
  <tr>
 +
    <td>DNA</td>
 +
    <td>20 μl</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Xba</td>
 +
    <td>1 μl</td>
 +
  </tr>
 +
  <tr>
 +
    <td>&nbsp;</td>
 +
    <td><strong>30 μl</strong></td>
 +
  </tr>
 +
</table></div></td>
 +
        </tr>
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="11">2008-08-11</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="05">2008-09-05</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p><strong>GROUP MEETING </strong><br />
+
<td class="bodyText"><div align="justify"><strong>WET LAB</strong><br />
-
   Experimental work<br />
+
    <br />
-
   <strong><br />
+
   PCRs were run in gels obtained from three different oligo sets from the  extractions of PRK415 part1_part2 and PRK415 part1_part2_part3N. <br />
-
  Objectives: </strong><br />
+
   Similarly, double restrictions were made to see the size of the insert. The  size of the insert is not the one desired.</p></div></td>
-
  - Build the bioparts. <br />
+
        </tr>
-
   - Transform the bacteria with the construction that we have. <br />
+
<tr>
-
  - Design the experiments to test our construction. <br />
+
          <td class="subHeader" bgcolor="#99CC66" id="09">2008-09-09</td>
-
  - Build the system. <br />
+
        </tr>
-
  - Collaborate with the modeling group. <br />
+
        <tr>
 +
<td class="bodyText"><div align="justify"><strong>WET LAB</strong><br />
 +
    <br />
 +
   PCRs and restrictions were rectified. There is no ligation of part 1 + part 2 + part 3 N in the PRK415 samples.</p></div></td>
 +
        </tr>  
 +
<tr>
 +
          <td class="subHeader" bgcolor="#99CC66" id="10">2008-09-10</td>  
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><strong>WET LAB</strong></p>
 +
<p>  PCR ligation part1 + part2 with RttH. <br />
   <br />
   <br />
-
   <strong>To do: </strong><br />
+
   <strong><u>Ligation and transformation of part1_part2 in pJET. (check kit)</u></strong> <br />
-
  - Extract DNA of the strain to get RcnA. <br />
+
-
  - Get the bioparts catalog. <br />
+
-
  - We need to have a large number of plasmids that we can use, amplifying the bioparts. <br />
+
-
  - Transformation of the bacteria with bioparts. <br />
+
   <br />
   <br />
-
   <strong>Currently:</strong><br />
+
   PBB + RcnA was streaked again. (1,2,5 and 9) <br />
-
  - There are plasmids. <br />
+
-
  - There are parts already amplified and in a plasmid. <br />
+
   <br />
   <br />
-
   <strong>Problems: </strong><br />
+
   PBB + RcnA restriction<br />
-
   - There was no DNA that we needed in the catalog. <br />
+
   DNA 10 μl<br />
-
   - The oligos were delayed 2 week and a half. <br />
+
   H2O 12 μl<br />
-
   - Issues to extract the plasmid from the colonies. <br />
+
   BSA 3 μl<br />
-
   - Make a PCR ligation with the three parts and amplify with the ends (it did not work). <br />
+
   XbaI 1 ml<br />
-
   - With the enzyme used: Increased frequency of spontaneous mutation of all the enzymes that exist. <br />
+
   HindIII 1 ml<br />
-
   An error every thousand base pairs. <br />
+
   30 μl<br />
-
   - There is a problem with tetracycline. You get false positives. <br />
+
   <br />
-
   <strong><br />
+
   <strong><u>1% Agarose gel </u></strong><br />
-
  Can be done: </strong><br />
+
   </p>
-
   - A part with RcnA and can be linked to the plasmid. <br />
+
<blockquote>
-
   - 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 />
+
   <p>1 .- molecular marker<br />
-
  -  HindIII can be used with the big biopart to verify the sequence. <br />
+
    2.-DR1 pBB+RcnA<br />
-
  <strong><br />
+
    3.-DR2 pBB+RcnA<br />
-
  Electrodes: </strong><br />
+
    4.-DR5 pBB+RcnA<br />
-
  - Are they specific for Nickel?</p>
+
    5.-DR9 pBB+RcnA<br />
-
<p>&nbsp;</p>
+
    6.-DR PRK415 2<br />
-
        </td>
+
    7.-DR PRK415 3</p>
-
      </tr>  
+
</blockquote>
 +
  <p><img width="400" src="https://static.igem.org/mediawiki/2008/2/2b/Gel_10Sep08.png" /></p></div></td>
 +
        </tr>  
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="20">2008-08-20</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="11">2008-09-11</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p><strong>Our response to the IPN team:<br />
+
<td class="bodyText"><div align="justify"><strong>WET LAB</strong></p>
-
</strong><br />
+
<p><strong><u>Plasmids were purified from  the samples: </u></strong><br />
-
   Hello,<br />
+
   1.-pBB+Rcna[2]<br />
-
   We apologize for the late reply, but we had to discuss carefully our answer.<br />
+
   2.-pBB+Rcna[5]<br />
-
   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 />
+
   3.-pBB+Rcna[9]<br />
-
   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 />
+
  4.-part1_part2 PRK415[6]<br />
-
   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 />
+
  5.-part1_part2 PRK415[8]<br />
 +
  6.-part1_part2 PRK415[9]<br />
 +
   7.-part1_part2+part3N PRK415[5]<br />
 +
  8.-part1_part2+part3N PRK415[8]<br />
 +
   9.-part1_part2+part3N PRK415[9]<br />
   <br />
   <br />
-
   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 />
+
   <strong><u>1%  Agarose gel</u></strong><br />
-
  We are doing small moves. At first, we only want to make one successful assayWe 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 />
+
  Samples were run through an agarose gel at 1%</p>
-
   We hope this letter answers your questions,<br />
+
<blockquote>
 +
  <p> 1.-molecular-weight marker <br />
 +
    2.-[1]pBB+Rcna[2]<br />
 +
    3.-[2]pBB+Rcna[5]<br />
 +
    4.-[3]pBB+Rcna[9]<br />
 +
    5.-[4]part1_part2 PRK415[6]<br />
 +
    6.-[5]part1_part2 PRK415[8]<br />
 +
    7.-[6]part1_part2 PRK415[9]<br />
 +
    8.-[7]part1_part2+parteN PRK415[5]<br />
 +
    9.-[8]part1_part2+parteN PRK415[8]<br />
 +
    10.-[9]part1_part2+part3N PRK415[9]<br />
 +
    11.-PCR rTth part1_part2<br />
 +
    12.-molecular marker</p>
 +
</blockquote>
 +
<p><img width="400" src="https://static.igem.org/mediawiki/2008/c/ca/Gel_11Sep08.png" /> </p>
 +
<p>Restrictions were made of the previous samples.</p>
 +
<p><strong>RcnA</strong></p>
 +
<table border="1" cellspacing="0" cellpadding="0" width="421">
 +
  <tr>
 +
    <td width="25%" valign="top"><br />
 +
    &nbsp; </td>
 +
    <td width="25%" valign="top"><p><strong>[1]pBB+Rcna[2]</strong></p></td>
 +
    <td width="25%" valign="top"><p><strong>[2]pBB+Rcna[5]</strong></p></td>
 +
    <td width="25%"><table border="0" cellspacing="0" cellpadding="0" width="136">
 +
      <tr>
 +
        <td width="25%" valign="top" class="style2"><p>[2]pBB+Rcna[5]</p></td>
 +
      </tr>
 +
    </table></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="25%" valign="top"><p>H2O </p></td>
 +
    <td width="25%" valign="top"><p>7μl </p></td>
 +
    <td width="25%" valign="top"><p>0 μl </p></td>
 +
    <td width="25%"><p>0 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="25%" valign="top"><p>Buffer 2 10X </p></td>
 +
    <td width="25%" valign="top"><p>3 μl </p></td>
 +
    <td width="25%" valign="top"><p>3 μl </p></td>
 +
    <td width="25%"><p>3 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="25%"><p>BSA</p></td>
 +
    <td width="25%"><p>3μl </p></td>
 +
    <td width="25%"><p>3μl </p></td>
 +
    <td width="25%"><p>3μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="25%" valign="top"><p>DNA </p></td>
 +
    <td width="25%" valign="top"><p>15 μl </p></td>
 +
    <td width="25%" valign="top"><p>20 μl </p></td>
 +
    <td width="25%"><p>20 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="25%" valign="top"><p>Xba</p></td>
 +
    <td width="25%" valign="top"><p>1 μl </p></td>
 +
    <td width="25%" valign="top"><p>2 μl </p></td>
 +
    <td width="25%"><table border="0" cellspacing="0" cellpadding="0" width="138">
 +
      <tr>
 +
        <td width="25%" valign="top"><p>2 μl </p></td>
 +
      </tr>
 +
    </table></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="25%" valign="top"><p>HindIII</p></td>
 +
    <td width="25%" valign="top"><p>1 μl </p></td>
 +
    <td width="25%" valign="top"><p>2 μl </p></td>
 +
    <td width="25%"><p>2 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="25%" valign="top"><p>&nbsp; </p></td>
 +
    <td width="25%" valign="top"><p><strong>30μl </strong></p></td>
 +
    <td width="25%" valign="top"><p><strong>30 μl </strong></p></td>
 +
    <td width="25%"><p><strong>30 μl </strong></p></td>
 +
  </tr>
 +
</table>
 +
<p><strong>Part1+part2 in PRK415</strong></p>
 +
<table border="1" cellspacing="0" cellpadding="0" width="280">
 +
  <tr>
 +
    <td width="50%" valign="top"><p>H2O </p></td>
 +
    <td width="50%" valign="top"><p>12μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%"><p>BSA</p></td>
 +
    <td width="50%"><p>3 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%" valign="top"><p>Buffer 10X </p></td>
 +
    <td width="50%" valign="top"><p>3 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%" valign="top"><p>DNA </p></td>
 +
    <td width="50%" valign="top"><p>10 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%" valign="top"><p>XbaI</p></td>
 +
    <td width="50%" valign="top"><p>1 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%" valign="top"><p>EcoRI </p></td>
 +
    <td width="50%" valign="top"><p>1 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="50%" valign="top"><p>&nbsp; </p></td>
 +
    <td width="50%" valign="top"><p><strong>30μl </strong></p></td>
 +
  </tr>
 +
</table>
 +
<p><strong>Part1+part2+part3N in PRK415</strong></p>
 +
<table border="1" cellspacing="0" cellpadding="0" width="266">
 +
  <tr>
 +
    <td width="155" valign="top"><p>H2O </p></td>
 +
    <td width="157" valign="top"><p>15μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>Buffer 10X </p></td>
 +
    <td width="157" valign="top"><p>3 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>DNA </p></td>
 +
    <td width="157" valign="top"><p>10 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>Pst1 </p></td>
 +
    <td width="157" valign="top"><p>1 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>EcoR1 </p></td>
 +
    <td width="157" valign="top"><p>1 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>&nbsp; </p></td>
 +
    <td width="157" valign="top"><p>30μl </p></td>
 +
  </tr>
 +
</table>
 +
<p><strong><u>1%  Agarose gel </u></strong><br />
 +
  Gel with the bioparts restrictions </p>
 +
<blockquote>
 +
  <p> 1.-molecular-weight marker <br />
 +
    2 .- [1] PBB + Rcna [2] double restriction with XbaI and HindIII <br />
 +
    3 .- [2] PBB + Rcna [5] double restriction with XbaI and HindIII <br />
 +
    4 .- [3] PBB + Rcna [9] double restriction with XbaI and HindIII <br />
 +
    5 .- [4] part1_part2 PRK415 [6] double restriction with EcoRI and Xba I <br />
 +
    6 .- [5] part1_part2 PRK415 [8] double restriction with EcoRI and Xba I <br />
 +
    7 .- [6] part1_part2 PRK415 [9] double restriction with EcoRI and Xba I <br />
 +
    8 .- [7] part1_part2 + parteN PRK415 [5] double restriction with EcoRI and PstI <br />
 +
    9 .- [8] part1_part2 + parteN PRK415 [8] double restriction with EcoRI and PstI <br />
 +
    10 .- [9] part1_part2 + part3N PRK415 [9] double restriction with EcoRI and PstI <br />
 +
    11.-Molecular Marker</p>
 +
</blockquote>
 +
<p><img width="400" src="https://static.igem.org/mediawiki/2008/4/48/Gel_11Sep08_2.png" /> </p>
 +
<p><strong><u>Plating</u></strong><br />
 +
  Striated colonies resulting from cloning in PJet <br />
 +
  Cloning in PJet of ligation Part1 + Part2 <br />
 +
   Transformation according to the manual<br />
   <br />
   <br />
-
   LCG-UNAM-Mexico Team<br />
+
   <strong><u>Restrictions with XbaI of</u></strong>: </p>
-
Cuernavaca, Morelos</p>
+
<blockquote>
-
<p>&nbsp;</p>
+
  <p>  1.-Part 1 + part2 <br />
-
<p>&nbsp;</p>
+
    2.-Normal Part 3</p>
-
<p>&nbsp;</p>
+
</blockquote>
-
<p><b>AHL: LuxR <br /></b>
+
<table border="1" cellspacing="0" cellpadding="0" width="581">
 +
  <tr>
 +
    <td width="155" valign="top"><p>H2O </p></td>
 +
    <td width="157" valign="top"><p>13μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>Buffer 10X </p></td>
 +
    <td width="157" valign="top"><p>3 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>DNA </p></td>
 +
    <td width="157" valign="top"><p>10 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>BSA</p></td>
 +
    <td width="157" valign="top"><p>3 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>XbaI</p></td>
 +
    <td width="157" valign="top"><p>1 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>&nbsp; </p></td>
 +
    <td width="157" valign="top"><p>30μl </p></td>
 +
  </tr>
 +
</table>
 +
<p>The restrictions were left alone for 2:30 hrs. <br />
 +
    <br />
 +
    <strong><u>Ligation </u></strong><br />
 +
  Ligations were made of [part1 + part2] + parte3N and [part1 + part2] + parte3M  in a final volume of 50μl </p>
 +
<table border="1" cellspacing="0" cellpadding="0" width="389">
 +
  <tr>
 +
    <td width="155" valign="top"><p>&nbsp;Part 3N or 3M </p></td>
 +
    <td width="157" valign="top"><p>15 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>Ligation of part1+part2</p></td>
 +
    <td width="157" valign="top"><p>15 μl</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>Buffer 5X </p></td>
 +
    <td width="157" valign="top"><p>10 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>H2O</p></td>
 +
    <td width="157" valign="top"><p>8 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>DNA ligase T4</p></td>
 +
    <td width="157" valign="top"><p>2 μl </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="155" valign="top"><p>&nbsp; </p></td>
 +
    <td width="157" valign="top"><p>50μl </p></td>
 +
  </tr>
 +
</table></div></td>
 +
        </tr> 
 +
 
 +
 
 +
<tr>
 +
          <td class="subHeader" bgcolor="#99CC66" id="13">2008-09-13</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><strong>WET LAB</strong></p>
 +
<p>10 colonies  from every LB Petri dish Amp with Part1_Part2_part3mutated and  Part1_Part2_Part3normal were scratched. </p></div></td>
 +
        </tr>
 +
<tr>
 +
          <td class="subHeader" bgcolor="#99CC66" id="15">2008-09-15</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><strong>WET  LAB</strong><br />
 +
  Cultures of transformed colonies in pJet  were done. </p>
 +
<table border="1" cellspacing="0" cellpadding="0" width="500">
 +
  <tr>
 +
    <td width="143" valign="top"><br />
 +
    Part1_part2 pJet </td>
 +
    <td width="467" valign="top"><p>Petri dish 1 34; Petri dish 2 2; Petri    dish 2 21; Petri dish 1 1; 8; Petri dish 1 29; Petri dish 1 36; 11; Petri    dish 2 10</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="143" valign="top"><p>Part1_part2_part3N pJet </p></td>
 +
    <td width="467" valign="top"><p>v1 3; v1    11; 12; Petri dish 2 6; Petri dish 2 5</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="143" valign="top"><p>Part1_part2_part3M pJet </p></td>
 +
    <td width="467" valign="top"><p>Petri dish 1 6; Petri dish 1 4; Petri    dish 1 3; Petri dish 2 5 ; Petri dish 2 2</p></td>
 +
  </tr>
 +
</table>
 +
<p>RcnA [2] Big flask alkaline lysis<br />
 +
  RcnA[1] Purification tuve with kit<br />
   <br />
   <br />
-
Reaction 3<br />
+
  <strong><u>Plasmid extraction gel</u></strong></p>
-
<br />
+
<blockquote>
-
Conflict: k3 (ON) &lt;k3 (OFF)? <br />
+
  <p>  1.-Molecular marker<br />
-
Reference: Goryachev et al. (2006) <br />
+
    2.-part1 + part2 pJet 2<br />
-
<br />
+
    3.-part1 + part2 pJet 34<br />
 +
    4.-part1 + part2 pJet 21<br />
 +
    5.-part1 + part2 pJet 1<br />
 +
    6.-part1 + part2 pJet 8<br />
 +
    7.-part1 + part2 pJet 29<br />
 +
    8.-part1 + part2 pJet 36<br />
 +
    9.-part1 + part2 + part3M&nbsp; pJet 6<br />
 +
    10.-part1 + part2 + part3M&nbsp; pJet 4<br />
 +
    11.-part1 + part2 + part3M&nbsp; pJet 3<br />
 +
    12.-part1 + part2 + part3N&nbsp; pJet 3<br />
 +
    13.-part1 + part2 + part3N&nbsp; pJet 11<br />
 +
    14.-part1 + part2 + part3N&nbsp; Petri dish2 pJet 5<br />
 +
    15.-part1 + part2 + part3N&nbsp; Petri dish2 pJet 12<br />
 +
    16.-part1 + part2 + part3M&nbsp; Petri dish2 pJet 2<br />
 +
    17.-part1 + part2 + part3M&nbsp; Petri dish2 pJet 5<br />
 +
    18.-part1 + part2 + part3M&nbsp; 6<br />
 +
    19.-part1 + part2 10 Petri dish 2<br />
 +
    20.-part1 + part2 21<br />
 +
    </p>
 +
</blockquote>
 +
<p>    There is no need to make a gel. Nothing definite yet.</p>
 +
<p>4 tubes of part3 normal  were restricted, and 2 of ligation part1 + part2.</p></div></td>
 +
        </tr>  
-
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>
-
-&gt; Check whether the article mentions how they got the parameter, or search through the references.<br />
+
          <td class="subHeader" bgcolor="#99CC66" id="17">2008-09-17</td>
-
<br />
+
        </tr>
-
<br />
+
        <tr>
 +
<td class="bodyText"><div align="justify"><p>
 +
  <strong><u>MODELING: </u></strong><br />
 +
Exploring Sensibility Analysis:<br />
 +
Normalizing sensitivity <br />
 +
- None: dx(t)/dt <br />
 +
- Medium: 1/x(t)*dx(t)/dt<br />
 +
- Complete: k/x(t)*dx(t)/dt (Allows you to compare dimensionless.) <br />
 +
  <br />
 +
  How is it measured? Are the k values moved in a range? Or is it a property of the system? <br />
 +
   <br />
 +
  <i>Wilkinson (1978). </i><br />
 +
  The parameters are systematically perturbed from their given values…    <br />
 +
  …  change from the given value…  (although it is recommended to define them in each system). <br />
 +
  <br />
 +
   <i> Ingalls &amp; Sauro (2002) </i><br />
 +
  - Before the analysis, it is recommended that you detect the 'preserved structures' (linear units, eg moieties). <br />
</p>
</p>
-
<p>They do explain why in the model, the k3 (ON) is in principle &quot;very small&quot;: <br />
+
<p>How to reduce the system:<br />
-
   &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 />
+
</p><p>
 +
- The response coefficient, defined above, provides a measure of the difference between this ‘‘perturbed trajectory’’ and the ‘‘nominal’’ (unperturbed) trajectory at each time t: As time tends to infinity, each trajectory will converge to its steady state, and so the response coefficient will converge to the steady-state response of MCA.</p>
 +
<p>- At steady state, these coefficients reduce to their standard MCA counterparts—flux responses.</p>
 +
<p>  NOTE: The sensitivity analysis is sensitive to the initial concentrations of metabolites. <br />
 +
</p>
 +
        </div></td>
 +
      </tr>
 +
<tr>
 +
          <td class="subHeader" bgcolor="#99CC66" id="18">2008-09-18</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"> 
 +
<b><u>TO-DO LIST:</u></b> 
 +
<li>Electrodes and measurement method:</li>
 +
<p>  There are, broadly speaking, four options to choose from: </p>
 +
<p> 1) The faculty of physiology at UNAM has a sensor for variations of voltages of orders that could be useful (Question:  If it is not specific for nickel, is there a way to filter the noise?). <br />
 +
   Among the benefits versus the other  possibilities: sensitivity appears to be very good and we know this because similar experiments have been done previously.  This in turn gives us the assurance that the sensor has already been  tested in other biological systems in line with the results expected.  Plus, a member of our team already knows how to use this  system. On the other hand, the fact that the Insitute is part of the UNAM has the advantage of working with people from the same team, not  counting the enormous advantage of being physically close.<br />
 +
</p>
 +
<p> 2) At the University of  Guadalajara, there is a device that measures the medium resistivity in the orders of 10^-9 Molar. We have not checked with sufficient detail the operation of this system, but at least the sensitivity offered is very promising. Furthermore, we believe that the metal used  in a phase of measurement reacts specifically with nickel also producing a easily measurable and identifiable optical effect. <br />
 +
  Among the advantages this option provides are: that it is sensitive  and that the software used to process and record each measurement is very comprehensive and drops the noise reliably. The main disadvantage, is that the apparatus is in a laboratory of the UdG, which  means that we would have to carry biological material.</p>
 +
<p> 3) Someone offered to buy the specific sensor for nickel and lend it to us during the measuring stage. We need to contact him and describe the project and what we need at the time of sensing. His only requirement is that he appears as a collaborator in the experimental publication resulting from this research. </p>
 +
<p> 4) Finally, most certainly not least, Trejo is still building up the sensor as we had planned initially. He has progressed well and in about two weeks it will be ready. <br />
   <br />
   <br />
 +
  - We decided to wait for Option 4 and, as a backup, the support of Dr. Pena (1), but this does not rule out the  option 3, which will be investigated for further details such as shipping time and specificity of the device. <br />
 +
  - We need to define the requirements for the bioparts and make the oligos. The oligos are being designed and probably by Friday they will be sent.<br />
 +
  - Design of experiments to estimate parameters (probable date September 10-12). <br />
 +
  They are still working on the buildings, but there will be a first meeting on Tuesday, Sept. 23, at 4:00 pm. <br />
   <br />
   <br />
-
   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 />
+
   <li>Wiki:</li>
-
   &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>
+
  - Update the notebook.<br />
-
<p>&nbsp;</p>
+
   - Update the section of the model. <br />
-
         </td>
+
  - We need to solve the problem of space. <br />
 +
  - Correct the image format. <br />
 +
  <br />
 +
  <li>Model: </li>
 +
  - Simulation. <br />
 +
  - Pending data. <br />
 +
  - Analysis (... stochastic processes?). <br />
 +
  - We are working on it... We've had some problems with the simulation, and  we are doing sensitivity analysis and parameter's scanning.</p>
 +
         </div></td>
       </tr>   
       </tr>   
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="21">2008-08-21</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="19">2008-09-19</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p></p>
+
<td class="bodyText"><div align="justify"><p>
-
         </td>
+
  <strong><u> MODELING:</u></strong><br />Converting units:<br>
 +
  <li>Reaction 1.</li>
 +
  3.723mM = ? Molecules <br />
 +
  M = mole/liter <br />
 +
  The volume of a bacterium is 10^-15L <br />
 +
3.723mM = 37.23x10-18 mol at 10-15  liters <br />
 +
37.23 x10-18 mol = 224.20427x105 molecules <br />
 +
<span class="font-size: small">* 1 mol = 6.02214x1023 molecules  <br></span>
 +
<br>
 +
<li> Reaction 6. </li>
 +
The flow in 20 plasmids is 20mM/h <br />
 +
Therefore, in 10 plasmids it would be 10mM/h <br />
 +
Flow = 10 mM/h = 10mM/3600s = 0.00278mM/s <br />
 +
0.00278mM = 0.0278x10-18mol in 10-15 liters <br />
 +
0.0278x10-18mol = 1.67415x10^5 molecules. <br />
 +
The flow in the cell is 1.67415x10^4 molecules / s with 10 copies (plasmids). <br />
 +
ν = k * [promoter] <br />
 +
1.67415x10^4 molecules / s = k * 10 molecules <br />
 +
-&gt; k = 1.67415x10^3 molecules / s <br />
 +
<br />
 +
<li>Reaction 5. </li></p>
 +
<p>  ΔG ° =- 23.81 kcal / mol <br />
 +
  Keq = exp (-ΔG º / RT) <br />
 +
  <br />
 +
  <span class="font-size: small">*Units supposedly do not affect this formula's usage  <br></span>
 +
  <br />
 +
  Correction of the synthesis reaction of cI: <br />
 +
  Units of the k3ON, estimated in reference 3 are 1/molecules*seconds, which means that the reaction is of second order. <br />
 +
  In that same article, they suggest that the sole presence of the dimer ensures the production of cI with k3ON rate (that is, that bonding is efficient). Since the estimated values do not consider the intermediate step of the promoter's union and the complex, we should not consider it. <br />
 +
  <br />
 +
  3.1 ρcI + (AHL: LuxR): (AHL: LuxR) -&gt; CI + ρcI + (AHL: LuxR): (AHL: LuxR) <br />
 +
  k3ON <br />
 +
  <br />
 +
  Unknown parameters:<br />
 +
  </p>
 +
<table width="266" border="2">
 +
  <tr>
 +
    <td width="147">cI Dimerization</td>
 +
    <td width="101"> k4.1 &amp; k-4.1</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Suppression by CI</td>
 +
    <td>V5max or k5 </td>
 +
  </tr>
 +
  <tr>
 +
    <td>Nickel Extrusion </td>
 +
    <td>k7</td>
 +
  </tr>
 +
  <tr>
 +
    <td>RcnA Degradation </td>
 +
    <td> k8</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Nickel Internalization </td>
 +
    <td><p>k9</p>
 +
    </td>
 +
  </tr>
 +
</table>
 +
<p><br /></p>
 +
         </div></td>
       </tr>   
       </tr>   
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="26">2008-08-26</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="22">2008-09-22</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p></p>
+
<td class="bodyText"><div align="justify"><p>
-
         </td>
+
  <strong> <u>MODELING:</u></strong><br />
 +
Dimerization of cI<br>
 +
  k4.1 &amp; k-4.1? <br />
 +
  2 cI &lt;-&gt; cI: cI <br />
 +
  <br />
 +
  ¿Quasi-equilibrium? <br />
 +
  The initial concentration of cI varies over time, but the proportion is preserved. <br />
 +
  <i>...avoid kinetic analysis of the fast reactions, ie to take into  consideration only their equilibrium constants instead of considering  their rates</i> (Kholodenko et al. 1998). <br />
 +
  <br />
 +
  Keq = [cI:cI]/[cI][cI] -&gt; [cI:cI] = Keq [cI] 2 <br />
 +
  <br />
 +
  d(cI:cI 2cI)/dt = v+ - v- <br />
 +
  d(Keq[cI]^2+2cI)/dt = v+ - v-</p>
 +
         </div></td>
       </tr>   
       </tr>   
<tr>
<tr>
-
           <td class="subHeader" bgcolor="#99CC66" id="28">2008-08-28</td>  
+
           <td class="subHeader" bgcolor="#99CC66" id="23">2008-09-23</td>  
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p></p>
+
<td class="bodyText"><div align="justify"><p>
-
         </td>
+
<b><u>MODELING:</u></b><br>
 +
<li>Simulation:</li> The previous crisis was overcome... Things seem to be working, however, there are still some parameters missing, but it has been decided that they will be estimated by adjusting the model and experimentally when we have the opportunity.<br />
 +
<li>Data:</li> There are a few missing parameters and we know that most of them are not available, so we have given up the search. What has yet to be defined in terms of data are concentrations of AiiA and LuxR, considering that they are constant in the model.</p>
 +
<p>We will also design  experiments to obtain the missing parameters through the experimental measurements (viable). <br />
 +
<li>Analysis:</li> - Stoichiometric matrix: There was a meeting today in the morning with Osbaldo to review its analysis, we will share the information as soon as possible. </p>
 +
<p> - Sensitivity  analysis: Although we don't yet understand the particular units in which SimBiology returns the results, the first graphics that display the basic parameters of the model are ready and they are the ones involved in the  degradation of AHL and its dimerization with LuxR (the beginning of the cascade) and the ones regarding the entry and exit of Nickel. We must do this analysis later, as we have seen that this is sensitive to the initial concentrations of metabolites, which are not yet fully defined. </p>
 +
<p>- Stationary States and Jacobian: They were stopped briefly because we need the parameters for further analysis.
 +
  <br /><br>
 +
  <strong><u>WET LAB:</u></strong><br />
 +
  <li>Requirements:</li> Urgent! We need to send the oligos required to be synthesized; We are working on it, and it is our priority. <br />
 +
  <li>Electrodes:</li> The device is not ready.<br />
 +
  <li>Design of experiments:</li> The first meeting will be today. <br />
 +
</p>
 +
<p><li>Funds &amp; Jamboree:</li> We are still waiting for some sponsors to reply.<br />
 +
</p>
 +
         </div></td>
 +
      </tr> 
 +
<tr>    </td>
 +
<td class="subHeader" bgcolor="#99CC66" id="24">2008-09-24</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><p><b><u>MODELING:</u></b><br> Correcting reaction 5 <br />
 +
    <br />
 +
    The Keq is dimensionless, but for formality in the reaction, it is sometimes necessary to add units. <br />
 +
  <br />
 +
    In the case of the reaction 5, the only information we have is the ΔGº of the reaction, so the Keq is calculated as exp (-ΔG º / RT),  which returns a value without units. <br />
 +
  <br />
 +
    As the definition of  Keq is given in concentration, we interpret this value in terms of  molarity. We know that once defined, they are completely  interconvertibles: Molar &lt;-&gt; moles &lt;-&gt; molecules and since we are working on molecules for our model, we make the relevant adjustments.    </div></dt>
       </tr>  
       </tr>  
 +
<tr>
 +
          <td class="subHeader" bgcolor="#99CC66" id="26">2008-09-26</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><strong>WET LAB</strong>
 +
<p>We purified the  PCR products of RcnA and the promoter region of RcnA.<br />
 +
  4 reactions of  RcnA<br />
 +
  We prepared a 1%  low melting point agarose gel, let it cool down for about 20min at the freezer.  We use 2.5 μl of loading dye Buffer.<br />
 +
  We run the gel  at 4°C,  130 Volts for about 40min.<br />
 +
  Total  volume of the loaded samples:<br />
 +
  45μ per  sample of RcnA (1,2,3,4<br />
 +
  We put  the gel in a recipient with 100ml of distilled water and 120μl of Ethidium  Bromide   for 10min.<br />
 +
  We cut  from the gel the 900bp band using a sterilized scalpel. <br />
 +
  We  purified the gel band using the <strong>QIAquick Gel Extraction Kit.</strong> <br />
 +
  We ran a   1% agarose gel to verify if we have the purified PCR product. <br />
 +
  <img src="https://static.igem.org/mediawiki/2008/3/39/Gel_26Sep08_neneVI.jpg" alt="Nene_VI" width="400" /> </p>
 +
<p><strong>September 29 2008</strong><br />
 +
<strong>WET LAB</strong></p>
 +
<p>We Cloned  the RcnA PCR fragment according to the ColoneJet protocol of the  <strong>CloneJET  PCR cloning Kit by Fermentas,</strong> and let the cells grow all the night.</p></div></td>
 +
        </tr> 
 +
<tr>
 +
          <td class="subHeader" bgcolor="#99CC66" id="29">2008-09-29</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><strong>WET LAB</strong>
 +
<p>We Cloned  the RcnA PCR fragment according to the ColoneJet protocol of the  <strong>CloneJET  PCR cloning Kit by Fermentas,</strong> and let the cells grow all the night.</p>
 +
</div></td>
 +
        </tr> 
 +
<tr>
 +
          <td class="subHeader" bgcolor="#99CC66" id="30">2008-09-30</td>
 +
        </tr>
 +
        <tr>
 +
<td class="bodyText"><div align="justify"><strong>WET LAB</strong>
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<p>We streak 18  colonies on an LB Am100 agar plate.</p></div></td>
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 +
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<a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Notebook/2008-October" onMouseOver="hiLite ('Next','b2','Next')" onMouseOut="hiLite('Next','b1','')"> <img name="Next" src="https://static.igem.org/mediawiki/igem.org/c/c8/BOTON_Next1.jpg" border=0 width="200" height="40"/></a></p>
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Latest revision as of 02:06, 30 October 2008

LCG-UNAM-Mexico:Notebook/September

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September

2008-09-01

WET LAB

Cultures

  1. One of the colonies obtained after we transformed the ligations (part1_part2_part 3 normal) + PRK415 and (part1_part2-part3Mutated)+PRK415 was cultured.
  2. Cultures of 10 colonies of part1+part2+PRK415 in liquid broth were prepared.
  3. 1% Agarose Gel (PRK415+(parte1+parte2) repetition)

    1.-Molecular marker 2.5 μl
    2.-[1] part1_part2 in PRK415 extraction 5μl
    3.-[2] part1_part2 in PRK415 extraction 5μl
    4.-[3] part1_part2 in PRK415 extraction 5μl
    5.-[4] part1_part2 in PRK415 extraction 5μl
    6.-[5] part1_part2 in PRK415 extraction 3 μl
    7.-[6] part1_part2 in PRK415 extraction 3 μl
    8.-[8] part1_part2 in PRK415 extraction 3 μl
    9.-[9] part1_part2 in PRK415 extraction 3 μl
    10.-[13] part1_part2 in PRK415 extraction 3 μl
    11.-[15] part1_part2 in PRK415 extraction 3 μl

    The size of the plasmids does not correlate the size we are expecting.

  4. Step 2 was repeated.
  5. Cultures with (Part1_part2_part3N)+PRK415 were prepared for extraction.
    1 PRK415 part1+part2
    12 PRK415 part1+part2
    13 PRK415 part1+part2
    14 PRK415 part1+part2
    15 PRK415 part1+part2
    16 PRK415 part1+part2
    17 PRK415 part1+part2
    18 PRK415 part1+part2
    19 PRK415 part1+part2
    20 PRK415 part1+part2
    21 PRK415 part1+part2
2008-09-02
WET LAB
Plasmid extraction of the cultures PRK415 + p1_p2 y PRK415+ p1_p2_p3N was made.

1%  Agarose gel.

Gel (19 wells)
1.-Molecular-weight marker
2.-[18]Part1_part2 extraction in PRK415
3.-[19]Part1_part2 extraction in PRK415
4.-[20]Part1_parte2 extraction in PRK415
5.-[13] (part1_part2)_part3 extraction in PRK415
6.-
7.-
8.-
9.-
10.-
11.-[1] (part1_part2)_part3 extraction in PRK415
12.-[3] (part1_part2)_part3 extraction in PRK415
13.-[4] (part1_part2)_part3 extraction in PRK415
14.-[5] (part1_part2)_part3 extraction in PRK415
15.-[6] (part1_part2)_part3 extraction in PRK415
16.-[8] (part1_part2)_part3 extraction in PRK415
17.-[9] (part1_part2)_part3 extraction in PRK415
18.-[11] (part1_part2)_part3 extraction in PRK415
19.-[12] (part1_part2)_part3 extraction in PRK415

Gel (9 wells extraction)
1.- Molecular-weight marker
2.-[11] part1_part2 extraction in PRK415
3.-[12] part1_part2 extraction in PRK415
4.-[13] part1_part2 extraction in PRK415
5.-[14] part1_part2 extraction in PRK415
6.-[15] part1_part2 extraction in PRK415
7.-[16] part1_part2 extraction in PRK415
8.-[17] part1_part2 extraction in PRK415

 



Dirty plasmid, in theory, it should not affect the PCR because the genes occupied for this are not in the E.coli genome. We proceeded to do the PCR. (yo digo que no pongamos la foto del gel esta muy sucio)

PCR

1.-[17] PRK415 + p1_p2 oligos 1upper 2 lower
2.-[8] PRK415 + p1_p2_p3N oligos 2upper 3 lower
3.-[9] PRK415 + p1_p2_p3N oligos 2upper 3 lower
4.-CN1 H2O
5.-CN2 PRK415 DR EcoR1 y BamH1 oligos 1upper 2lower
6.-CN3 PRK415 DR EcoR1 y BamH1 oligos 2upper 3lower

2008-09-03

MODELING:
Lac promoter synthesis rate:
The effect of stochasticity on the Lac Operon: An evolutionary perspective from van Hoek F et al (2007)
The article's objective, as you can infer from the title, is to evaluate the effect of stochasticity in the evolution of a Promoter. In order to do so they built a comprehensive model including every parameter involved in transcription and translation. They measure some parameters but they depend mostly on literature to define them.

They perform both a deterministic and a stochastic analysis. To generate a stochastic model they added one parameter, the average burst size of protein translation (protein translation occurs in bursts, after an mRNA is synthesized, several proteins can be translated from the same mRNA). This was possible because when an mRNA molecule is translated it can not be degraded. Therefore after each translation it can either be translated again (p) or be degraded (1-p). This suggests that protein production occurs in bursts with a burst size geometrically distributed. Afterwards, they compared the noise levels in their model with experimental noise measurements and found correlation.

To model transcription they used a two-dimensional Hill-function dependent on the cAMP and allolactose concentration. (repress the glucose and lactose operon via cAMP and activates the operon via allolactose).

They use 11 biochemical parameters, including three of special importance for us:

  • a, Transcription rate when the RNA Polymerase is bound to the DNA, but CRP and Laci are not. Initial value: 1.1 × 10-7 mM/min
  • b, The transcription rate when both RNA Polymerase and CRP are bound, but Laci is not bound to the DNA. Initial value: 2.2 × 10-5 mM/min
  • c, Leakiness, the transcription rate when RNA Polymerase is not bound to the DNA. Initial value 5.5 × 10-10 mM/min

  • They modeled binomially protein degradation, assuming that when cells divide, their proteins are randomly divided between the cells. However in a population of non-Dividing cells this "dilution" can not be taken in account.

    WET LAB

    2% Agarose gel with PCR samples of 2/09/08

    1.-Molecular marker
    2.- [1c2]  [17] PRK415 + p1_p2 oligos 1upper 2 lower
    3.- [2]  [8] PRK415 + p1_p2_p3N oligos 2upper 3 lower
    4.- [3]  [9] PRK415 + p1_p2_p3N oligos 2upper 3 lower
    5.- [4]  CN1 H2O
    6.- [5]  CN2 PRK415 DR EcoR1 y BamH1 oligos 1upper 2lower
    7.- [6]  CN3 PRK415 DR EcoR1 y BamH1 oligos 2upper 3lower
    8.- R6 Restriction EcoR1 HindIII 1_2_3 (5 μl)
    9.- R8 Restriction EcoR1 HindIII 1_2_3 (2 μl)
    10.- R9 Restriction EcoR1 HindIII 1_2_3 (2 μl)
    11.- R17 Restriction EcoR1 XbaI 1_2_3 (2 μl)

    No results.

    Cultures in 2ml of LbTc10 of PRK415 +part1+part2 and of DH5alfa 123 (3normal) ligation were prepared.

    2008-09-04
    WET LAB

    Extraction of plasmid
    Part1_2_3N-PRK415
    Part1_2 PRK415
    Part1_2_3N PRK415 Repeat

    1% Agarose gel with extractions
    2.-[3] PRK415 part1_2_3N Ligation
    3.-[5] PRK415 part1_2_3N Ligation
    4.-[6] PRK415 part1_2_3N Ligation
    5.-[8] PRK415 part1_2_3N Ligation
    6.-[9]PRK415 part1_2_3N Ligation
    7.-[10] PRK415 part1_2_3N Ligation
    8.-[11] PRK415 part1_2_3N Ligation
    9.-[12] PRK415 part1_2_3N Ligation
    10.-[13] PRK415 part1_2_3N Ligation
    11.-[1.2] PRK415 part1_2_3N Ligation
    12.-[3.2] PRK415 part1_2_3N Ligation
    13.-[5.2] PRK415 part1_2_3N Ligation
    14.-[6.2] PRK415 part1_2_3N Ligation
    15.-[8.2] PRK415 part1_2_3N Ligation
    16.-[9.2] PRK415 part1_2_3N Ligation
    17.-[10.2] PRK415 part1_2_3N Ligation
    18.-[11.2] PRK415 part1_2_3N Ligation
    19.-[12.2] PRK415 part1_2_3N Ligation
    20.-[13.2] PRK415 part1_2_3N Ligation

    PCR ligation p1_p2_p3 in PRK415
    No positive results.

    Electrophoresis ligation p1+p2 PRK415

    1.-Molecular marker
    2.-[1] p1+p2 PRK415
    3.-[2] p1+p2 PRK415
    4.-[3] p1+p2 PRK415
    5.-[4] p1+p2 PRK415
    6.-[5] p1+p2 PRK415
    7.-[17] p1+p2 PRK415
    8.-[18] p1+p2 PRK415
    9.-[19] p1+p2 PRK415
    10.-[20] p1+p2 PRK415
    11.-[21] p1+p2 PRK415

    4 cultures of PRK415 were left.

    Restrictions

    part1_part2  (PCR)
    part 3 mutated (PCR)

    H2O 3 μl
    Buffer 5 μl
    BSA 5 μl
    DNA 35 μl
    Xba 2 μl
      50 μl

    Part1_part2 ligation sep-09

    H2O 2 μl
    Buffer 3 μl
    BSA 3 μl
    DNA 20 μl
    Xba 1 μl
      30 μl
    2008-09-05
    WET LAB

    PCRs were run in gels obtained from three different oligo sets from the extractions of PRK415 part1_part2 and PRK415 part1_part2_part3N.
    Similarly, double restrictions were made to see the size of the insert. The size of the insert is not the one desired.

    2008-09-09
    WET LAB

    PCRs and restrictions were rectified. There is no ligation of part 1 + part 2 + part 3 N in the PRK415 samples.

    2008-09-10
    WET LAB

    PCR ligation part1 + part2 with RttH.

    Ligation and transformation of part1_part2 in pJET. (check kit)

    PBB + RcnA was streaked again. (1,2,5 and 9)

    PBB + RcnA restriction
    DNA 10 μl
    H2O 12 μl
    BSA 3 μl
    XbaI 1 ml
    HindIII 1 ml
    30 μl

    1% Agarose gel

    1 .- molecular marker
    2.-DR1 pBB+RcnA
    3.-DR2 pBB+RcnA
    4.-DR5 pBB+RcnA
    5.-DR9 pBB+RcnA
    6.-DR PRK415 2
    7.-DR PRK415 3

    2008-09-11
    WET LAB

    Plasmids were purified from the samples:
    1.-pBB+Rcna[2]
    2.-pBB+Rcna[5]
    3.-pBB+Rcna[9]
    4.-part1_part2 PRK415[6]
    5.-part1_part2 PRK415[8]
    6.-part1_part2 PRK415[9]
    7.-part1_part2+part3N PRK415[5]
    8.-part1_part2+part3N PRK415[8]
    9.-part1_part2+part3N PRK415[9]

    1%  Agarose gel
    Samples were run through an agarose gel at 1%

    1.-molecular-weight marker
    2.-[1]pBB+Rcna[2]
    3.-[2]pBB+Rcna[5]
    4.-[3]pBB+Rcna[9]
    5.-[4]part1_part2 PRK415[6]
    6.-[5]part1_part2 PRK415[8]
    7.-[6]part1_part2 PRK415[9]
    8.-[7]part1_part2+parteN PRK415[5]
    9.-[8]part1_part2+parteN PRK415[8]
    10.-[9]part1_part2+part3N PRK415[9]
    11.-PCR rTth part1_part2
    12.-molecular marker

    Restrictions were made of the previous samples.

    RcnA


     

    [1]pBB+Rcna[2]

    [2]pBB+Rcna[5]

    [2]pBB+Rcna[5]

    H2O

    7μl

    0 μl

    0 μl

    Buffer 2 10X

    3 μl

    3 μl

    3 μl

    BSA

    3μl

    3μl

    3μl

    DNA

    15 μl

    20 μl

    20 μl

    Xba

    1 μl

    2 μl

    2 μl

    HindIII

    1 μl

    2 μl

    2 μl

     

    30μl

    30 μl

    30 μl

    Part1+part2 in PRK415

    H2O

    12μl

    BSA

    3 μl

    Buffer 10X

    3 μl

    DNA

    10 μl

    XbaI

    1 μl

    EcoRI

    1 μl

     

    30μl

    Part1+part2+part3N in PRK415

    H2O

    15μl

    Buffer 10X

    3 μl

    DNA

    10 μl

    Pst1

    1 μl

    EcoR1

    1 μl

     

    30μl

    1%  Agarose gel
    Gel with the bioparts restrictions

    1.-molecular-weight marker
    2 .- [1] PBB + Rcna [2] double restriction with XbaI and HindIII
    3 .- [2] PBB + Rcna [5] double restriction with XbaI and HindIII
    4 .- [3] PBB + Rcna [9] double restriction with XbaI and HindIII
    5 .- [4] part1_part2 PRK415 [6] double restriction with EcoRI and Xba I
    6 .- [5] part1_part2 PRK415 [8] double restriction with EcoRI and Xba I
    7 .- [6] part1_part2 PRK415 [9] double restriction with EcoRI and Xba I
    8 .- [7] part1_part2 + parteN PRK415 [5] double restriction with EcoRI and PstI
    9 .- [8] part1_part2 + parteN PRK415 [8] double restriction with EcoRI and PstI
    10 .- [9] part1_part2 + part3N PRK415 [9] double restriction with EcoRI and PstI
    11.-Molecular Marker

    Plating
    Striated colonies resulting from cloning in PJet
    Cloning in PJet of ligation Part1 + Part2
    Transformation according to the manual

    Restrictions with XbaI of:

    1.-Part 1 + part2
    2.-Normal Part 3

    H2O

    13μl

    Buffer 10X

    3 μl

    DNA

    10 μl

    BSA

    3 μl

    XbaI

    1 μl

     

    30μl

    The restrictions were left alone for 2:30 hrs.

    Ligation
    Ligations were made of [part1 + part2] + parte3N and [part1 + part2] + parte3M in a final volume of 50μl

     Part 3N or 3M

    15 μl

    Ligation of part1+part2

    15 μl

    Buffer 5X

    10 μl

    H2O

    8 μl

    DNA ligase T4

    2 μl

     

    50μl

    2008-09-13
    WET LAB

    10 colonies from every LB Petri dish Amp with Part1_Part2_part3mutated and Part1_Part2_Part3normal were scratched.

    2008-09-15
    WET LAB
    Cultures of transformed colonies in pJet were done.


    Part1_part2 pJet

    Petri dish 1 34; Petri dish 2 2; Petri dish 2 21; Petri dish 1 1; 8; Petri dish 1 29; Petri dish 1 36; 11; Petri dish 2 10

    Part1_part2_part3N pJet

    v1 3; v1 11; 12; Petri dish 2 6; Petri dish 2 5

    Part1_part2_part3M pJet

    Petri dish 1 6; Petri dish 1 4; Petri dish 1 3; Petri dish 2 5 ; Petri dish 2 2

    RcnA [2] Big flask alkaline lysis
    RcnA[1] Purification tuve with kit

    Plasmid extraction gel

    1.-Molecular marker
    2.-part1 + part2 pJet 2
    3.-part1 + part2 pJet 34
    4.-part1 + part2 pJet 21
    5.-part1 + part2 pJet 1
    6.-part1 + part2 pJet 8
    7.-part1 + part2 pJet 29
    8.-part1 + part2 pJet 36
    9.-part1 + part2 + part3M  pJet 6
    10.-part1 + part2 + part3M  pJet 4
    11.-part1 + part2 + part3M  pJet 3
    12.-part1 + part2 + part3N  pJet 3
    13.-part1 + part2 + part3N  pJet 11
    14.-part1 + part2 + part3N  Petri dish2 pJet 5
    15.-part1 + part2 + part3N  Petri dish2 pJet 12
    16.-part1 + part2 + part3M  Petri dish2 pJet 2
    17.-part1 + part2 + part3M  Petri dish2 pJet 5
    18.-part1 + part2 + part3M  6
    19.-part1 + part2 10 Petri dish 2
    20.-part1 + part2 21

    There is no need to make a gel. Nothing definite yet.

    4 tubes of part3 normal were restricted, and 2 of ligation part1 + part2.

    2008-09-17

    MODELING:
    Exploring Sensibility Analysis:
    Normalizing sensitivity
    - None: dx(t)/dt
    - Medium: 1/x(t)*dx(t)/dt
    - Complete: k/x(t)*dx(t)/dt (Allows you to compare dimensionless.)

    How is it measured? Are the k values moved in a range? Or is it a property of the system?

    Wilkinson (1978).
    The parameters are systematically perturbed from their given values…
    … change from the given value… (although it is recommended to define them in each system).

      Ingalls & Sauro (2002)
    - Before the analysis, it is recommended that you detect the 'preserved structures' (linear units, eg moieties).

    How to reduce the system:

    - The response coefficient, defined above, provides a measure of the difference between this ‘‘perturbed trajectory’’ and the ‘‘nominal’’ (unperturbed) trajectory at each time t: As time tends to infinity, each trajectory will converge to its steady state, and so the response coefficient will converge to the steady-state response of MCA.

    - At steady state, these coefficients reduce to their standard MCA counterparts—flux responses.

    NOTE: The sensitivity analysis is sensitive to the initial concentrations of metabolites.

    2008-09-18
      TO-DO LIST: 
  • Electrodes and measurement method:
  • There are, broadly speaking, four options to choose from:

    1) The faculty of physiology at UNAM has a sensor for variations of voltages of orders that could be useful (Question: If it is not specific for nickel, is there a way to filter the noise?).
    Among the benefits versus the other possibilities: sensitivity appears to be very good and we know this because similar experiments have been done previously. This in turn gives us the assurance that the sensor has already been tested in other biological systems in line with the results expected. Plus, a member of our team already knows how to use this system. On the other hand, the fact that the Insitute is part of the UNAM has the advantage of working with people from the same team, not counting the enormous advantage of being physically close.

    2) At the University of Guadalajara, there is a device that measures the medium resistivity in the orders of 10^-9 Molar. We have not checked with sufficient detail the operation of this system, but at least the sensitivity offered is very promising. Furthermore, we believe that the metal used in a phase of measurement reacts specifically with nickel also producing a easily measurable and identifiable optical effect.
    Among the advantages this option provides are: that it is sensitive and that the software used to process and record each measurement is very comprehensive and drops the noise reliably. The main disadvantage, is that the apparatus is in a laboratory of the UdG, which means that we would have to carry biological material.

    3) Someone offered to buy the specific sensor for nickel and lend it to us during the measuring stage. We need to contact him and describe the project and what we need at the time of sensing. His only requirement is that he appears as a collaborator in the experimental publication resulting from this research.

    4) Finally, most certainly not least, Trejo is still building up the sensor as we had planned initially. He has progressed well and in about two weeks it will be ready.

    - We decided to wait for Option 4 and, as a backup, the support of Dr. Pena (1), but this does not rule out the option 3, which will be investigated for further details such as shipping time and specificity of the device.
    - We need to define the requirements for the bioparts and make the oligos. The oligos are being designed and probably by Friday they will be sent.
    - Design of experiments to estimate parameters (probable date September 10-12).
    They are still working on the buildings, but there will be a first meeting on Tuesday, Sept. 23, at 4:00 pm.

  • Wiki:
  • - Update the notebook.
    - Update the section of the model.
    - We need to solve the problem of space.
    - Correct the image format.

  • Model:
  • - Simulation.
    - Pending data.
    - Analysis (... stochastic processes?).
    - We are working on it... We've had some problems with the simulation, and we are doing sensitivity analysis and parameter's scanning.

    2008-09-19

    MODELING:
    Converting units:

  • Reaction 1.
  • 3.723mM = ? Molecules
    M = mole/liter
    The volume of a bacterium is 10^-15L
    3.723mM = 37.23x10-18 mol at 10-15 liters
    37.23 x10-18 mol = 224.20427x105 molecules
    * 1 mol = 6.02214x1023 molecules

  • Reaction 6.
  • The flow in 20 plasmids is 20mM/h
    Therefore, in 10 plasmids it would be 10mM/h
    Flow = 10 mM/h = 10mM/3600s = 0.00278mM/s
    0.00278mM = 0.0278x10-18mol in 10-15 liters
    0.0278x10-18mol = 1.67415x10^5 molecules.
    The flow in the cell is 1.67415x10^4 molecules / s with 10 copies (plasmids).
    ν = k * [promoter]
    1.67415x10^4 molecules / s = k * 10 molecules
    -> k = 1.67415x10^3 molecules / s

  • Reaction 5.
  • ΔG ° =- 23.81 kcal / mol
    Keq = exp (-ΔG º / RT)

    *Units supposedly do not affect this formula's usage

    Correction of the synthesis reaction of cI:
    Units of the k3ON, estimated in reference 3 are 1/molecules*seconds, which means that the reaction is of second order.
    In that same article, they suggest that the sole presence of the dimer ensures the production of cI with k3ON rate (that is, that bonding is efficient). Since the estimated values do not consider the intermediate step of the promoter's union and the complex, we should not consider it.

    3.1 ρcI + (AHL: LuxR): (AHL: LuxR) -> CI + ρcI + (AHL: LuxR): (AHL: LuxR)
    k3ON

    Unknown parameters:

    cI Dimerization k4.1 & k-4.1
    Suppression by CI V5max or k5
    Nickel Extrusion k7
    RcnA Degradation k8
    Nickel Internalization

    k9


    2008-09-22

    MODELING:
    Dimerization of cI
    k4.1 & k-4.1?
    2 cI <-> cI: cI

    ¿Quasi-equilibrium?
    The initial concentration of cI varies over time, but the proportion is preserved.
    ...avoid kinetic analysis of the fast reactions, ie to take into consideration only their equilibrium constants instead of considering their rates (Kholodenko et al. 1998).

    Keq = [cI:cI]/[cI][cI] -> [cI:cI] = Keq [cI] 2

    d(cI:cI 2cI)/dt = v+ - v-
    d(Keq[cI]^2+2cI)/dt = v+ - v-

    2008-09-23

    MODELING:

  • Simulation:
  • The previous crisis was overcome... Things seem to be working, however, there are still some parameters missing, but it has been decided that they will be estimated by adjusting the model and experimentally when we have the opportunity.
  • Data:
  • There are a few missing parameters and we know that most of them are not available, so we have given up the search. What has yet to be defined in terms of data are concentrations of AiiA and LuxR, considering that they are constant in the model.

    We will also design experiments to obtain the missing parameters through the experimental measurements (viable).

  • Analysis:
  • - Stoichiometric matrix: There was a meeting today in the morning with Osbaldo to review its analysis, we will share the information as soon as possible.

    - Sensitivity analysis: Although we don't yet understand the particular units in which SimBiology returns the results, the first graphics that display the basic parameters of the model are ready and they are the ones involved in the degradation of AHL and its dimerization with LuxR (the beginning of the cascade) and the ones regarding the entry and exit of Nickel. We must do this analysis later, as we have seen that this is sensitive to the initial concentrations of metabolites, which are not yet fully defined.

    - Stationary States and Jacobian: They were stopped briefly because we need the parameters for further analysis.

    WET LAB:

  • Requirements:
  • Urgent! We need to send the oligos required to be synthesized; We are working on it, and it is our priority.
  • Electrodes:
  • The device is not ready.
  • Design of experiments:
  • The first meeting will be today.

  • Funds & Jamboree:
  • We are still waiting for some sponsors to reply.

    2008-09-24

    MODELING:
    Correcting reaction 5

    The Keq is dimensionless, but for formality in the reaction, it is sometimes necessary to add units.

    In the case of the reaction 5, the only information we have is the ΔGº of the reaction, so the Keq is calculated as exp (-ΔG º / RT), which returns a value without units.

    As the definition of Keq is given in concentration, we interpret this value in terms of molarity. We know that once defined, they are completely interconvertibles: Molar <-> moles <-> molecules and since we are working on molecules for our model, we make the relevant adjustments.

    2008-09-26
    WET LAB

    We purified the PCR products of RcnA and the promoter region of RcnA.
    4 reactions of RcnA
    We prepared a 1% low melting point agarose gel, let it cool down for about 20min at the freezer. We use 2.5 μl of loading dye Buffer.
    We run the gel at 4°C, 130 Volts for about 40min.
    Total volume of the loaded samples:
    45μ per sample of RcnA (1,2,3,4
    We put the gel in a recipient with 100ml of distilled water and 120μl of Ethidium Bromide   for 10min.
    We cut from the gel the 900bp band using a sterilized scalpel.
    We purified the gel band using the QIAquick Gel Extraction Kit.
    We ran a  1% agarose gel to verify if we have the purified PCR product.
    Nene_VI

    September 29 2008
    WET LAB

    We Cloned the RcnA PCR fragment according to the ColoneJet protocol of the  CloneJET PCR cloning Kit by Fermentas, and let the cells grow all the night.

    2008-09-29
    WET LAB

    We Cloned the RcnA PCR fragment according to the ColoneJet protocol of the  CloneJET PCR cloning Kit by Fermentas, and let the cells grow all the night.

    2008-09-30
    WET LAB

    We streak 18 colonies on an LB Am100 agar plate.