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

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     <td colspan="3" rowspan="2"><img src="https://static.igem.org/mediawiki/igem.org/b/b3/LCG_copy.png" alt="Header image" width="524" height="143" border="0" /></td>
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     <td height="50" colspan="3" id="logo" valign="bottom" align="center" nowrap="nowrap">LCG-UNAM-Mexico</td>
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           <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/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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          <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 class="bodyText"><p>
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<td class="bodyText"><div align="justify"><p>
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<b><p>Session with our advisor Miguel A.<br /></b>
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<b><u>GROUP SESSION:</b></u><br>
 +
<b><p>Session with Dr. Miguel<br /></b>
   <br />
   <br />
-
• changes in pH due to colorimetry or electrodes, choose the electrodes wisely. <br />
+
We did some brainstorming in regards to the project. How to measure the efflux, and how to build the system. <br>
-
Nickel transporter in E.coli. * if they send the mutated strain: Transcriptional merger  with the trp operon; induce the repressor off.<br />
+
Monitor changes in pH using colorimetry or electrodes, choose the electrodes wisely. <br />
-
• Introducing the gene into a multicopy plasmid and select controls (-) and (+) <br />
+
If we use a nickel transporter in <i>E. coli</i> we can easily obtain the mutated strain (deleted trasporter). <br />
-
• Coli introduces at least 40% less zinc when it has the gene. While the flow is still measurable there is no problem.<br />
+
• Introducing the gene into a multicopy plasmid and establish positive and negative controls <br />
-
• An electrode is not really necessary, this can be done with conventional methods.<br />
+
• An electrode is not really necessary, measurements can be done with conventional methods.<br />
<br />
<br />
-
Research: Efflux pumps of metals.<br /><br />
+
<b>Researching Metal Efflux Pumps:</b><br />
-
(It must be ionized, maybe a simple salt, the anion doesn't matter). <br />
+
(The metal must be ionizable, maybe using a simple salt, the anion doesn't matter). <br />
</p>
</p>
-
<p>Team work: </p>
+
<p>We decided to research how are metals transported by <i> E. coli </i>, each member of the team chose a specific metal: </p>
<p>  • Cobalt - Mariana <br />
<p>  • Cobalt - Mariana <br />
   • Zinc - Jimena <br />
   • Zinc - Jimena <br />
-
   • Cadmium - Chicken <br />
+
   • Cadmium - Mariana GS <br />
   • Nickel - Libertad <br />
   • Nickel - Libertad <br />
   • Iron - Atahualpa <br />
   • Iron - Atahualpa <br />
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   • Pb - Martin <br />
   • Pb - Martin <br />
   <br />
   <br />
-
  * Primer design<br />
 
   </p>
   </p>
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<p>-- Documents /  E.coli pumps<br />
+
<p><b> Vectors we could use:</br></b></p>
-
</p>
+
<p><li>prk404 y7o prk415: resistant to tratracicline, maybe 4 - 9 copies.</li>
-
<p>-- Vectors we could use:</p>
+
   <li>pbbr1mcs5: resistant to kentamicine up to 10 copies.</li>
-
<p>prk404 y7o prk415 resistant to tratracicline, maybe 4 - 9 copies.<br />
+
   <li>puc: up to 20 copies resistant to ampicillin.</li>
-
   pbbr1mcs5 resistant kentamicine up to 10 copies.<br />
+
   <li>pjet: resistant to ampicillin up to 600 copies. </li>
-
   puc up to 20 copies resistant to ampicillin.<br />
+
<li>Plbb: clorma resistant, up to 12 copies with LacI in cis (it can be controlled with IPTG)</li>
-
   pjet, resistant to ampicillin up to 600 copies. <br />
+
<span class="font-size: small">REMEMBER not to combine the vector with the same replication origin.<br /> </span>
-
  REMEMBER not to combine the vector with the same replication origin.<br />
+
  <br />
-
  Plbb: clorma resistant, up to 12 copies with LacI in cis (it can be controlled with IPTG) <br />
+
   <b>Once the most suitable gene has been chosen, primers must be designed:<br /></b>
-
  <br />
+
   Tm = (2 (A + T)) + (4 * (C + G)) <br />
-
   • Choosing the most suitable gene and designing primers.<br />
+
   • Choose the single cut site. <br />
-
  <br />
+
   • Take in account compatibility of restriction sites.<br />
-
   Tm = (2 (A + T)) + (4 * (C + G)) <br />
+
-
  <br />
+
-
   • Choose the single cut site! <br />
+
-
   • Compatibility of restriction sites.<br />
+
   • Read more to start writing.</p>
   • Read more to start writing.</p>
-
<br />
 
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<td class="bodyText"><p><b>Expositions: Choosing the bomb! (efflux pump)<br /></b>
+
<td class="bodyText"><div align="justify"><p><b><u>GROUP SESSION:</b></u><br><b>Expositions: Choosing the efflux pump:<br /></b>
   <br />
   <br />
   <strong>Nickel and cobalt </strong><br />
   <strong>Nickel and cobalt </strong><br />
• Very specific<br />
• Very specific<br />
• We don't know how to get the Co inside the cell.<br />
• We don't know how to get the Co inside the cell.<br />
-
• We can leave the natural entrance, regulating the output. <br />
+
• We can keep the wild type entry, and regulate the efflux. <br />
-
• All this in E. coli. <br />
+
• All this in <i>E. coli</i>. <br />
-
• Co is very toxic, it can hurt many things ... We better use Nickel only. <br />
+
• Co is very toxic, it can damage the cell, we better only use Nickel. <br />
• We have two pumps we can test. <br />
• We have two pumps we can test. <br />
-
It can hold up to 2 minimolar of Ni. <br />
+
The cell hold up to 2 millimolar of Ni. <br />
-
• It has more than one system to get Ni in.<br />
+
• It has more than one system for Ni entry.<br />
-
Help getting out Ni, RcnA &amp; RcnR (~200 &amp; 300 aa). <br />
+
• RcnA &amp; RcnR (~200 &amp; ~300 aa) are used during Ni efflux. <br />
-
• Pending: How Co enters and the mechanism to get Ni in.<br />
+
• Pending: How does Co enter the cell and the mechanism of Ni entry.<br />
<br />
<br />
<strong>Zinc </strong><br />
<strong>Zinc </strong><br />
-
Getting in ZnuABC, Zupt, ZntB. Getting out ZntA and ZitB. <br />
+
Uses ZnuABC, Zupt and ZntB to enter the cell. To leave the cell it uses ZntA and ZitB. <br />
-
• The ones that get them in are not specific for Zn (they also work for other metals), except ZnuABC. <br />
+
• The only specific entry for Zn is ZnuABC, the others also transport other metals. <br />
-
• Regulated by Zurt joining Zn. <br />
+
• Regulated by Zurt, which binds Zn. <br />
-
• To draw: ZntA only works at high concentrations, but is not specific to Zn; also, ZitB is not specific, it only operates at low concentrations. <br />
+
• To withdraw Zn: ZntA only works at high concentrations, but is not specific for Zn; ZitB is not specific either and it only operates at low concentrations. <br />
-
• Problem: It is very small... it is difficult to regulate their extrusion. <br />
+
• Problem: It is difficult to regulate their extrusion. <br />
-
It is essential.<br />
+
Zn is essential.<br />
<br />
<br />
<strong>Cadmium </strong><br />
<strong>Cadmium </strong><br />
-
• Its entry is a transportation system of divalent ions, it is cotransported with Manganese, which is essential for the cell, so the entry would not be regulated.<br />
+
• Its entry is mediated by a transportation system of divalent ions, it is cotransported with Manganese, which is essential for the cell, so the entry is not tightly regulated.<br />
-
• It is toxic to the cell, but it seems that nothing too serious.<br />
+
• It is toxic to the cell, but it doesn't seem too serious.<br />
-
• The output can be mediated by multiple systems, all present in E. coli (CzcD, CzcCBA, CadA, ...). <br />
+
• The efflux can be mediated by multiple systems, all found in <i>E. coli</i> (CzcD, CzcCBA, CadA, ...). <br />
-
• Legatzki et al. (2003) make an experiment in which they use a mutant of E  coli GG48 ((delta) zntA &amp; (delta) zitB) that accumulates both Zn as  Cd, but when they transform it with a plasmid with zntA &amp; cadA of R.  metallidurans, it recover resistance quite well. It is true that we will not regulate all  systems involved, but according to their experiment, change is quite significant. It could be useful. <br />
+
• Legatzki et al. (2003) made an experiment in which they use a mutant <i>E  coli</i> GG48 ((delta) zntA &amp; (delta) zitB) that accumulates both Zn and Cd, but when they transform it with a plasmid with zntA &amp; cadA of R.  metallidurans, it regained resistance. It is true that we will not regulate all  systems involved, but according to their experiment, change is quite significant. It could be useful. <br />
• Genes are large, up to ~800aa. <br />
• Genes are large, up to ~800aa. <br />
• Pending: What concentration can the cell hold? <br />
• Pending: What concentration can the cell hold? <br />
<br />  
<br />  
<strong>Iron</strong><br />
<strong>Iron</strong><br />
-
• There are many ways to get iron. Through siderophores! <br />
+
• There are many ways to get iron, including through siderophores. <br />
-
• Problem: On entering the cell, it forms a complex with an overall  regulator (fur) involved in many important functions. Essential. <br />
+
• Problem: On entering the cell, it forms a complex with a global regulator (fur) involved in many important functions. Essential. <br />
-
• The pump is ok, unique and the only way to remove the iron. About ~ 920kb, Fief. <br />
+
• The pump Fief (~920kb) is ok, highly specific and the only way to remove the iron. . <br />
<br />
<br />
<strong>Tellurium</strong> <br />
<strong>Tellurium</strong> <br />
• Not so much an extrusion pump, because there is a transformation by means of an enzyme, which is not well known. <br />
• Not so much an extrusion pump, because there is a transformation by means of an enzyme, which is not well known. <br />
• All resistance genes are in two plasmids. <br />
• All resistance genes are in two plasmids. <br />
-
Admission is a potential difference of ions in membranes. <br />
+
Entry to the cell is based on a ionic potential in membranes. <br />
-
• It is not necessary, toxic. <br />
+
• It is not necessary, rather toxic. <br />
-
• We can not regulate the entry and when the Tellurium enters, unless there is resistance, the cell dies immediately. <br />
+
• We can not regulate the entry, and when the Tellurium enters, unless there is resistance, the cell dies immediately. <br />
• The role of the genes involved in resistance is not well understood. <br />
• The role of the genes involved in resistance is not well understood. <br />
<br />
<br />
<strong>Copper </strong><br />
<strong>Copper </strong><br />
-
• When it enters, it is reduced from 2+ to +, because the extrusion systems only recognize this. <br />
+
• When it enters, it is reduced from 2+ to 1+, because the extrusion systems only recognize Cu1+. <br />
-
Hold up to 3.5 miniMolar inside the cell. <br />
+
Cells hold up to 3.5 miniMolar. <br />
-
• CusCFAB operon is responsible for extrusion regulated transcriptionally by cusRS. There is probably a biopart. Known in  E. coli. <br />
+
• CusCFAB operon is responsible for efflux, regulated transcriptionally by cusRS. There is probably a biopart. Known in  <i>E. coli.</i> <br />
-
• CusRS ~1000 aa. Cus CFAB ~2000aa. <br />
+
• CusRS is ~1000 aa. Cus CFAB ~2000aa. <br />
-
• Problem: It's size! --&gt; Bioparts<br />
+
• Problem: Its size! <br />
-
• Admission is ATPase dependent... by bombs? Described in yeast and  animals, it is known that it enters to E. coli, but how can we regulate it in  E. coli? <br />
+
• Admission is ATPase dependent... by pumps? Described in yeast and  animals, it is known to enter to <i>E. coli</i>, but how can we regulate it? <br />
• It is not essential, it is highly toxic. <br />
• It is not essential, it is highly toxic. <br />
-
• Pending: The entry? <br />
+
• Pending: How is the entry mediated? <br />
<br />
<br />
<strong>Arsenic</strong> <br />
<strong>Arsenic</strong> <br />
-
It is in a plasmid in E. coli. Five genes (Ars [RABCD] ~ 1.4Kb), the  plasmid is in total ~ 4.4kb. Some genes on chromosome are also involved; they are not necessary, but reduced from 10 to 100 times the  resistance if they are absent. <br />
+
Resistance is in a plasmid in <i>E. coli</i>. Five genes (Ars [RABCD] ~ 1.4Kb), the  plasmid is in total ~ 4.4kb. Some genes on the chromosome are also involved; they are not necessary but the resistance is reduced from 10 to 100 times if they are absent. <br />
• Do they have a translational control? <br />
• Do they have a translational control? <br />
• The pump works with ATP. <br />
• The pump works with ATP. <br />
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<br />
<br />
<strong>Mercury </strong><br />
<strong>Mercury </strong><br />
-
• Free admission... and three carrier assets are known.<br />
+
• Free diffusion, and three carriers are known.<br />
-
• It is highly toxic, but it is not drawn as such, because it is reduced... So there is no nice way to remove it. <br />
+
• It is highly toxic, to reduce its toxicity it is reduced. <br />
-
It is not a well-known system of entry. <br />
+
Ther is no well-known system of entry. <br />
• The pumps are quite specific.<br />
• The pumps are quite specific.<br />
-
• Also toxic to the cells environment, that's why the cell eats it, for processing... <br />
+
• Also toxic in the cells environment, therefore cells absorb it, for processing. <br />
-
• Pending: Getting it out? <br />
+
• Pending: Getting Hg out of the cells? <br />
<br />
<br />
<strong>Lead </strong><br />
<strong>Lead </strong><br />
• It enters together with manganese, Zn or Co. <br />
• It enters together with manganese, Zn or Co. <br />
-
• It is highly toxic to E. coli because it affects membranes. <br />
+
• It is highly toxic to <i>E. coli</i> because it affects membranes. <br />
-
• Calcium pumps that can help it get in were found, but they are animals...<br />
+
• Calcium pumps that transport it into the cells are known, but they are animals.<br />
-
• To remove it, it uses the Cd systems, there are no specific system.<br />
+
• To remove it, it uses the Cd detoxification systems, there are no specific system.<br />
-
• Pending: Finding a target, Concentration that endures? </p>
+
• Pending: Finding a target, Concentration that cells can endures? </p>
<p><strong><br />
<p><strong><br />
-
   Not useful</strong><br />
+
   Not useful for our purposes</strong><br />
   • Iron <br />
   • Iron <br />
   • Lead <br />
   • Lead <br />
Line 229: Line 256:
   • Tellurium <br />
   • Tellurium <br />
   <br />
   <br />
-
   <strong>More or less</strong> <br />
+
   <strong>Probably useful</strong> <br />
-
   • Zinc -&gt; Against: It is essential. <br />
+
   • Zinc (Cons: It is essential.) <br />
-
   • Copper -&gt; Against: It is very big.<br />
+
   • Copper (Cons: It is very big.)<br />
   <br />
   <br />
   <strong>Favourites </strong><br />
   <strong>Favourites </strong><br />
   • Cobalt &amp; Nickel <br />
   • Cobalt &amp; Nickel <br />
   • Cadmium <br />
   • Cadmium <br />
-
arsenic </p>
+
Arsenic </p>
-
         </td>
+
         </div></td>
       </tr>  
       </tr>  
<tr>
<tr>
Line 243: Line 270:
         </tr>
         </tr>
         <tr>
         <tr>
-
<td class="bodyText"><p><strong>Project Design</strong></p>
+
<td class="bodyText"><div align="justify"><p><b><u>GROUP SESSION:</b></u><br><strong>Project Design</strong></p>
-
<p><strong>  <em>Experimental </em></strong></p>
+
<p><strong> Experimental</strong></p>
<p>
<p>
-
  <em>Pump we will use: </em>Nickel. </p>
+
<b>Pump we will use: </b>Nickel. </p>
-
<p><em>  Articles: </em></p>
+
<p>Articles:</p>
-
<p>  -- Complex Transcriptional Control Links NikABCDE-HYDROGEN with Dependent Nickel Transport Expression in E. coli (2005). <br />
+
<p>  <li>Complex Transcriptional Control Links NikABCDE-HYDROGEN with Dependent Nickel Transport Expression in <i>E. coli</i> (2005).</li>
-
   -- Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function (2006). <br />
+
   <li>Nickel homeostasis in <i>Escherichia coli</i> - the rcnR-rcnA efflux pathway and its linkage to NikR function (2006).</li>
-
   -- Identification of rcnA (yohM), Nickel and Cobalt Resistance Gene in Escherichia coli (2005). </p>
+
   <li>Identification of rcnA (yohM), Nickel and Cobalt Resistance Gene in <i>Escherichia coli</i> (2005).</li> </p>
-
<p><em>  Pending: </em></p>
+
<p><b>  Pending: </b></p>
-
<p>  -- Check bioparts. <br />
+
<p>  <li>Check bioparts</li>
-
   -- Design vectors. <br />
+
   <li>Design vectors</li>
-
   -- Design primers. <br />
+
   <li>Design primers</li>
-
   -- Strain with deletion of rcnA. <br />
+
   <li>Strain with deletion of rcnA.</li>
-
  <br />
+
-
  <em>Tentative design</em>: <br />
+
   <br />
   <br />
 +
  <b>Preliminary design</b>: <br />
   •  The mechanism of entry of Nickel will remain wildtype. <br />
   •  The mechanism of entry of Nickel will remain wildtype. <br />
-
   • In the absence of Nickel, RcnR (whose gene will remain  in the plasmid with its normal regulation) will repress rcnA (which will be deleted from chromosome and put into a plasmid). <br />
+
   • In the absence of Nickel, RcnR (whose gene will remain  in the chromosome with its normal regulation) will repress rcnA (which will be deleted from the chromosome and inserted into a plasmid). <br />
-
   •  By putting (*) we will repress transcription of rcnA, even in the presence of Nickel, so this will be will be our signal to retain the metal in the cell and modify the concentration of the medium (if it is not enough to turn off the pump, it will be necessary to find a new level of regulation). <br />
+
   •  By adding (*) to the system we will repress transcription of rcnA, even in the presence of Nickel, so this will be will be our signal to retain the metal inside the cell and modify the concentration of the medium (if it is not enough to turn off the pump, it will be necessary to find a new level of regulation). <br />
   • How will we turn off the signal (*)? </p>
   • How will we turn off the signal (*)? </p>
-
<p>* we don't know what can the (*) be.<br />
+
<p><strong>Task:</strong> Suggest a molecule for (*)! <br />
   <br />
   <br />
-
   <strong>Task:</strong> Find (*)! <br />
+
   <strong>Modeling</strong> </p>
-
  <br />
+
-
  <strong><em>Modeling</em></strong> </p>
+
<p>  Pending: </p>
<p>  Pending: </p>
-
<p>  -- Responding vs. Concentration (experimental part). <br />
+
<p>  <li>Response vs. Concentration (experimental part).</li>
-
   -- Set thresholds &amp; limitations. <br />
+
   <li> Set thresholds &amp; limitations. </li>
-
   -- Efficiency of interactions? <br />
+
   <li> Efficiency of interactions? </li>
-
   -- Defining variables: <br />
+
   <li>Defining variables: </li>
-
   &gt; Metal concentration.<br />
+
   - Metal concentration.<br />
-
   &gt; Repressor concentration.<br />
+
   - Repressor concentration.<br />
-
   &gt; (*) concentration.</p>
+
   - (*) concentration.</p>
-
         </td>
+
         </div></td>
       </tr>   
       </tr>   
-
<tr>
+
      <tr>
-
          <td class="subHeader" bgcolor="#99CC66" id="18">2008-06-18</td>  
+
      <td colspan="6">
-
        </tr>
+
<p align="center"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Notebook/2008-May" onMouseOver="hiLite ('Back','a2','Back')" onMouseOut="hiLite('Back','a1','')"> <img name="Back" src="https://static.igem.org/mediawiki/igem.org/5/57/BOTON_BACK1.jpg" border=0 width="200" height="40"/></a>
-
        <tr>
+
 
-
<td class="bodyText"><p><strong>Final design</strong></p>
+
<a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Notebook/2008-June_2" 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>
-
<p>Scheme</p>
+
      </td>
-
<p><div id="pbfw">
+
-
  <div id="ub_r"><img src="http://docs.google.com/File?id=dntmktb_46fd48j4fx_b" width="580" alt="" id="ckrm" /></div>
+
-
</div>
+
-
<p align="center"><br /></p>
+
-
<p>The first plasmid contains the efflux pump for Nickel (rcnA), which  will maintain its natural regulation dependent of metal (by RcnR) and  additionally, it will contain a promoter regulated by the repressor of lambda  phage, cI. Besides, of course, a resistance as a marker of the plasmid. </p>
+
-
<p>   
+
-
  The second contains everything needed for regulating the power down  from an external signal (AHL). Both luxR as aiiA will occur  constitutively, the first one, with a strong promoter (pTetR), as we do  not want the presence of LuxR to be limiting, and the second one, having a moderate promoter  or weak (pLacZ), to give us space to play with concentrations  of AHL without aiiA always wining. And cI *, cI modified with a queue of  LVA for rapid degradation, regulated by a promoter dependent of LuxR +  AHL. It will also contain an equal resistance as a marker.  </p>
+
-
<p> 
+
-
  In the presence  of AHL, this joins with LuxR and induces the production of cI *, which in  turn represses the transcription of rcnA. Like cI *, signal AHL has to  be short-lived since aiiA is degradating constantly, so the system quickly  returns to its initial state once it ceases to manage AHL.  <br />
+
-
  <br />
+
-
  <strong>Parts </strong><br />
+
-
  <br />
+
-
  Defining bioparts we will use or where to get what is necessary. <br />
+
-
  <em><br />
+
-
  <strong>Part: BBa_I729006</strong></em></p>
+
-
<p><div id="pbfw">
+
-
  <div id="ub_r">
+
-
    <div id="w-cp"><img src="http://docs.google.com/File?id=dc5zwbn5_6cfx5c956_b" width="580" alt="" id="yp7t" /> </div>
+
-
  </div>
+
-
</div>
+
-
<p align="center"><br /></p>
+
-
<p>Part of Quorum sensing used by the team Chiba in iGEM2007. Both tetR  and LacI + pL are constitutive promoters, but since LacI + pL is  a very strong promoter, it will probably be replaced. This biopart will be  responsible for the regulation by luxR  and the action of the system by AHL.  Instead of GFP (Subpart E0040), the BBa_C0051 part that  codes for the protein cI + LVA will be inserted, which will join the regulatory region of cI  (biopart BBa_R0051) in the other plasmid.</p>
+
-
<p>(Previous experience: none)</p>
+
-
<p><strong><em>Part:BBa_C0051</em></strong></p>
+
-
<p><div id="pbfw">
+
-
  <div id="ub_r">
+
-
    <div id="w-cp">
+
-
      <div id="sebl"><img src="http://docs.google.com/File?id=dc5zwbn5_8hrpddscq_b" alt="" width="580" id="qu3v" /> </div>
+
-
    </div>
+
-
  </div>
+
-
</div>
+
-
<p align="center"><br /></p>
+
-
<p>Region coding for the repressor cI based on the repressor cI of lambda  phage with modified LVA with a queue for rapid degradation. cI joins the  regulator cI (BBa_R0051)</p>
+
-
<p>(Previous experience: none)</p>
+
-
<p><strong><em>Part:BBa_R0051</em></strong></p>
+
-
<p><div id="dmii"><img src="http://docs.google.com/File?id=dc5zwbn5_7fsbr26rq_b" alt="" width="580" id="zrt-" /></div>
+
-
<p align="center">&nbsp;<br />
+
-
</p>
+
-
<p>Promoter regulated by cI based on the pR promoter of lambda phage. The  promoter has two binding sites to cI repressor of lambda phage  (BBa_C0051). The union of cI results in the suppression of the  transcript. </p>
+
-
<p>(Previous experience: it works) <br />
+
-
  <br />
+
-
  Of the previous 3  bioparts, the sequence is in the registration of biological  parts and according to this page, DNA is available. </p>
+
-
<p><strong><em>  Part: BBa_G00510</em></strong></p>
+
-
<p> This is the forward primer of C0051 that has 24 pb. <br />
+
-
  (No DNA in the bank, but we know that it works) <br />
+
-
  gatttctgcatagccagacttggg </p>
+
-
<p><strong><em>  Part: BBa_G00511 </em></strong></p>
+
-
<p>  Reverse primer for C0051 that has 26 pb. <br />
+
-
  cactgactagcgataactttccccac <br />
+
-
  (No DNA in the bank but we know that it works)</p>
+
-
<p><strong>Vectors</strong></p>
+
-
<p>We need to define the vectors we can use. </p>
+
-
<p>  Possibilities: <br />
+
-
  *The ones recorded in the spreadsheet (courses). <br />
+
-
  <br />
+
-
  In bioparts:</p>
+
-
<table width="500" border="2">
+
-
  <tr>
+
-
    <td width="225"><div align="center"><strong>Name</strong></div></td>
+
-
    <td width="257"><div align="center"><strong>Description</strong></div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td><a href="http://partsregistry.org/wiki/index.php?title=Part:pSB3C5">pSB3C5</a></td>
+
-
    <td>Low to medium copy    BioBrick standard vector</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td><a href="http://partsregistry.org/wiki/index.php?title=Part:pSB3T5">pSB3T5</a></td>
+
-
    <td>Low to medium copy    BioBrick standard vector</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td><a href="http://partsregistry.org/wiki/index.php?title=Part:pSB4A3">pSB4A3</a></td>
+
-
    <td>pSB4A3</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td><a href="http://partsregistry.org/wiki/index.php?title=Part:pSB4C5">pSB4C5</a></td>
+
-
    <td>Low copy BioBrick    standard vector</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td><a href="http://partsregistry.org/wiki/index.php?title=Part:pSB4A1">pSB4A1</a></td>
+
-
    <td>pSB4A1</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td><a href="http://partsregistry.org/wiki/index.php?title=Part:pSB4A5">pSB4A5</a></td>
+
-
    <td>Low copy BioBrick    standard vector</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td><a href="http://partsregistry.org/wiki/index.php?title=Part:pSB4T5">pSB4T5</a></td>
+
-
    <td>Low copy BioBrick    standard vector </td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I739202">BBa_I739202</a></td>
+
-
    <td>pCK01BB1</td>
+
-
  </tr>
+
-
</table>
+
-
<p><strong>Primers</strong></p>
+
-
<p>  Build or find oligos that we could use for our constructions.</p>
+
-
<p>  We need: <br />
+
-
  • rcnA (with its regulatory region; no promoter).<br />
+
-
  • cI *.<br />
+
-
  • Constitutive promoter for luxR (tetR is proposed, it is a strong promoter). <br />
+
-
  • Constitutive promoter for aiiA (lacZ is proposed, it is a moderate promoter). <br />
+
-
  • aiiA.<br />
+
-
  • Promoter dependent of cI.<br />
+
-
  ~ In all cases, we have to check whether they already exist (in biopartes or something) and evaluate them.</p>
+
-
<table border="2" cellspacing="0" cellpadding="0" width="500">
+
-
  <tr>
+
-
    <td width="107" valign="bottom"><p align="center">&nbsp;</p></td>
+
-
    <td width="56" valign="bottom"><p align="center">&nbsp;</p></td>
+
-
    <td width="319" valign="bottom"><p align="center"><strong>Sequence</strong></p></td>
+
-
    <td width="94" valign="bottom"><p align="center"><strong>Tm</strong></p></td>
+
-
    <td width="35" valign="bottom"><p align="center"><strong>Deg.</strong></p></td>
+
-
    <td width="67" valign="bottom"><p align="center"><strong>Restr. S</strong></p></td>
+
-
    <td width="100" valign="bottom"><p align="center"><strong>Bioparts</strong></p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="107" valign="bottom"><div>
+
-
      <p align="center">(pTetR)luxR/(p. </p>
+
-
    </div></td>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Upper </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">&nbsp;</p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">62.5 ºC</p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div>
+
-
      <p align="center">None </p>
+
-
    </div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">? </p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="107" valign="bottom"><div>
+
-
      <p align="center">c.fuerte)aiiA </p>
+
-
    </div></td>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Lower </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">&nbsp;</p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">63.8 ºC </p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div>
+
-
      <p align="center">None</p>
+
-
    </div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">? </p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="107" rowspan="2"><div>
+
-
      <p align="center">pcI </p>
+
-
    </div></td>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Upper </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">&nbsp;</p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">61.9-76.2 ºC </p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">864 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div align="center">None</div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">? </p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Lower </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">&nbsp;</p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">66.5 ºC </p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div>
+
-
      <p align="center">None </p>
+
-
    </div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">? </p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="107" rowspan="2"><div>
+
-
      <p align="center">pLacZ </p>
+
-
    </div></td>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Upper </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">5' GCACCCAGGCTTTACACTTT 3' </p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">64.7 ºC </p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div align="center">None</div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">? </p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Lower </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">5' TGTTATCCGCTCACAATTCCA 3' </p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">60.3 ºC </p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div>
+
-
      <p align="center">None</p>
+
-
    </div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">? </p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="107" rowspan="2"><div>
+
-
      <p align="center">cI* </p>
+
-
    </div></td>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Upper </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">5' GATTTCTGCATAGCCAGACTTGGG 3'</p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">62.9 ºC </p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div align="center">None</div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">BBa_G00510 </p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Lower </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">5' CACTGACTAGCGATAACTTTCCCCAC 3' </p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">61.9 ºC </p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div align="center">None</div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">BBa_G00511 </p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="107" rowspan="2"><div>
+
-
      <p align="center">rcnA </p>
+
-
    </div></td>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Upper </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">5' CACTATTAATCTACTGGGGGGTAG3' </p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">64.2ºC </p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div align="center">None</div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">&nbsp;</p>
+
-
    </div></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="56" valign="bottom"><div>
+
-
      <p align="center">Lower </p>
+
-
    </div></td>
+
-
    <td width="319" valign="bottom"><div>
+
-
      <p align="center">5' AGTTATCGCATTATGCCCATG 3' </p>
+
-
    </div></td>
+
-
    <td width="94" valign="bottom"><div>
+
-
      <p align="center">65.8ºC</p>
+
-
    </div></td>
+
-
    <td width="35" valign="bottom"><div>
+
-
      <p align="center">1 </p>
+
-
    </div></td>
+
-
    <td width="67" valign="bottom"><div align="center">None</div></td>
+
-
    <td width="100" valign="bottom"><div>
+
-
      <p align="center">&nbsp;</p>
+
-
    </div></td>
+
-
  </tr>
+
-
</table>
+
-
<p><strong>Promoters</strong></p>
+
-
<p>Investigate a little more about the proposed promoters and define whether they are the most optimal.</p>
+
-
<table border="2" cellspacing="0" cellpadding="0" width="580">
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center"><strong>Promoter</strong></p></td>
+
-
    <td width="129" valign="bottom"><p align="center"><strong>Biopart</strong></p></td>
+
-
    <td width="215" valign="bottom"><p align="center"><strong>Constitutive</strong>?</p></td>
+
-
    <td width="135" valign="bottom"><p align="center"><strong>Strength </strong></p></td>
+
-
    <td width="246" valign="bottom"><p align="center"><strong>Notes</strong></p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">pTetR </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_R0040 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">In  tetracycline presence or  TetR absence</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">medium</p></td>
+
-
    <td width="246" valign="bottom"><p align="center">Recomended by our advisor Miguel</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">pLuxR-HSL </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_R0062 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Over-regulated by LuxR-HSL (increases its expression). </p></td>
+
-
    <td width="135" valign="bottom"><p align="center">weak (constitutive)/medium (LuxR-HSL) </p></td>
+
-
    <td width="246" valign="bottom"><p align="center"> luxR could bring some trouble if it becomes a part of the sistem</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">pLacIQ </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBA_I14032</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">high</p></td>
+
-
    <td width="246" valign="bottom"><p align="center">¿Is there a biopart? It could be  the promoter for luxR</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">pCyc </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_I766555 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes </p></td>
+
-
    <td width="135" valign="bottom"><p align="center">medium</p></td>
+
-
    <td width="246" valign="bottom"><p align="center">Yeast promoter</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23112 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">1 </p></td>
+
-
    <td width="246" valign="bottom"><p>&nbsp;</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23103 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">17 </p></td>
+
-
    <td width="246"><p>&nbsp;</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23113 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">21 </p></td>
+
-
    <td width="246"><p>&nbsp;</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23109 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">106</p></td>
+
-
    <td width="246"><p>&nbsp;</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23117</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">162</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23114</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">256</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23115</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">387</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23116</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">396</p></td>
+
-
    <td width="246" rowspan="2" valign="bottom"><p align="center">Constitutive promoters family</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23105</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">62</p></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23110</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">844</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23107</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">908</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23106</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">1185</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23108</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">1303</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23118</p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113</p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">1429</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23111 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">1487</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23101 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">1791</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23104 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">1831</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23102 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">2179</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td width="74" valign="bottom"><p align="center">J23100 </p></td>
+
-
    <td width="129" valign="bottom"><p align="center">BBa_J23113 </p></td>
+
-
    <td width="215" valign="bottom"><p align="center">Yes</p></td>
+
-
    <td width="135" valign="bottom"><p align="center">2547</p></td>
+
-
    <td width="246">&nbsp;</td>
+
-
  </tr>
+
-
</table>
+
-
<p><strong>Facts about  kinetics &amp; other things...</strong></p>
+
-
<p>
+
-
  Investigate a little  more about the parties involved in the system to begin with an outline  of modeling and defining how the design is theoretically feasible. <br />
+
-
  <br />
+
-
  <em>Note: </em>To join HSLwith  LuxR and enable the transcription of cI, HLS should be at a concentration of micromolar order.<br />
+
-
  <em>Note (2)</em>: Not all bioparts have been used previously, most DNA is  available but still there is no record of it working. We need to check  the quality of DNA to ensure that there will be no problems.</p>
+
       </tr>  
       </tr>  
-
<tr>
 
-
          <td class="subHeader" bgcolor="#99CC66" id="24">2008-06-24</td>
 
-
        </tr>
 
-
        <tr>
 
-
<td class="bodyText"><p align="center"><strong>Modeling</strong><br>
 
-
  <strong id="j4px695"><img src="http://docs.google.com/File?id=dntmktb_59hmv4brc3_b" alt="" name="graphics3" width="255" height="289" hspace="13" border="0" align="left" id="j4px696" /></strong><br><strong>Variables</strong> <br>
 
-
    Concentrations of:</p>
 
-
<ul>
 
-
  <li>LuxR  (constant). </li>
 
-
  <li>aiiA  (constant). </li>
 
-
  <li>AHL  (arbitrary). </li>
 
-
  <li>cI* (according to aiiA, AHL &amp; LuxR). </li>
 
-
  <li>RcnA (according to cI*). </li>
 
-
</ul>
 
-
<p>Knowing the initial concentrations and lifetime of proteins involved, as well as the efficiency action of aiiA (kinetics in general). <br />
 
-
* The concentration of Nickel (NiCl2) in the medium which supports the  cell according to Rodrigue et al. (2005) before inhibiting growth is 4 μ M for the  strain lacking rcnA, 10 μ M in the wildtype and up to 100 times more in  a strain with a multicopy gene.</p><br /><br /><br />
 
-
<p>&nbsp;</p>
 
-
<table border="0" cellspacing="0" cellpadding="0">
 
-
  <tr>
 
-
    <td width="50" height="40" valign="bottom"><p align="center">&nbsp;</p></td>
 
-
    <td width="149" valign="bottom"><p align="center"><strong>Concentration</strong></p></td>
 
-
    <td width="147" valign="bottom"><p align="center"><strong>Life span (half-life)</strong></p></td>
 
-
    <td width="155" valign="bottom"><p align="center"><strong>Substrate affinity</strong></p></td>
 
-
    <td width="159" valign="bottom"><p align="center"><strong>Notes</strong></p></td>
 
-
  </tr>
 
-
  <tr>
 
-
    <td width="50" valign="bottom"><p align="center"><strong>AHL</strong></p></td>
 
-
    <td width="149" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="147" valign="bottom"><p align="center">3 hrs.</p></td>
 
-
    <td width="155" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="159" valign="bottom"><p align="center">Conflictive information</p></td>
 
-
  </tr>
 
-
  <tr>
 
-
    <td width="50" rowspan="2"><p align="center"><strong>LuxR</strong></p></td>
 
-
    <td width="149" rowspan="2"><p align="center">?</p></td>
 
-
    <td width="147" valign="bottom"><p align="center">60 min- (~40-100)</p></td>
 
-
    <td width="155" rowspan="2"><p align="center">?</p></td>
 
-
    <td width="159" valign="bottom"><p align="center">&nbsp;</p></td>
 
-
  </tr>
 
-
  <tr>
 
-
    <td width="147" valign="bottom"><p align="center">2 min (35 min +AHL)</p></td>
 
-
    <td width="159" valign="bottom"><p align="center">&nbsp;</p></td>
 
-
  </tr>
 
-
  <tr>
 
-
    <td width="50" valign="bottom"><p align="center"><strong>aiiA</strong></p></td>
 
-
    <td width="149" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="147" valign="bottom"><p align="center">24 hrs</p></td>
 
-
    <td width="155" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="159" valign="bottom"><p align="center">&nbsp;</p></td>
 
-
  </tr>
 
-
  <tr>
 
-
    <td width="50" valign="bottom"><p align="center"><strong>cI*</strong></p></td>
 
-
    <td width="149" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="147" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="155" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="159" valign="bottom"><p align="center">&nbsp;</p></td>
 
-
  </tr>
 
-
  <tr>
 
-
    <td width="50" valign="bottom"><p align="center"><strong>RcnA</strong></p></td>
 
-
    <td width="149" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="147" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="155" valign="bottom"><p align="center">?</p></td>
 
-
    <td width="159" valign="bottom"><p align="center">&nbsp;</p></td>
 
-
  </tr>
 
-
</table>
 
-
<p><em>Assumption 1:</em> Once there is nickel in the medium, RcnR does not matter  for the pump regulation. This because there will be  large concentrations of metal,  so we can assume that RcnR will always be linked to a molecule and it  will therefore be unable to suppress the transcript of rcnA; the noise that  the few RcnR free molecules can cause,  will be  indistinguishable from normal behaviour of the pump. <br />
 
-
</p>
 
-
<p><em>Assumption  2:</em> Any decrease in the concentration of AHL is due to aiiA. It is  believed that the natural degradation of this is irrelevant in the time  scale analysis. Either way, a process will not be distinguishable from  the other and even when the first is estimated, it would not be very  informative for the analysis, so we intend to  take this assumption as  true. <br />
 
-
  </p>
 
-
<p><em>Assumption 3:</em> The transcription of cI * depends solely on the  concentration of AHL. LuxR is not a limiting step, ie, it is in  constant concentration and in sufficient quantity to be always  available to associate with AHL. Only to simplify the analysis, at  least as a first approximation.</p>
 
-
<p><strong>Initial outline:</strong></p>
 
-
<p>(v1) AHL0 <br />
 
-
    (v2) aiiA + AHL -&gt; aiiA <br />
 
-
    (v3) AHL + LuxR -&gt; cI* <br />
 
-
    (v4,v5) ρ + cI* &lt;--&gt; ρ.cI* <br />
 
-
    ρ -&gt; ρ +RcnA <br />
 
-
    RcnA + Ni -&gt; RcnA <br />
 
-
    RcnA -&gt; Ø </p>
 
-
<p align="center">&nbsp;</p>
 
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          <td class="subHeader" bgcolor="#99CC66" id="26">2008-06-26</td>
 
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<td class="bodyText"><p><p><strong>Experimental work</strong></p>
 
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<p>&nbsp;</p>
 
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<p>1. Take the sequences (fasta format) <br />
 
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  2. Once you have the sequence find appropriate reading frames <br />
 
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  3. Make the restriction map<br />
 
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  -- Nedcutter, check the page for NewEngland Biolabs (because we are going to use enzymes from that company) <br />
 
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  For rcnA and rcnR, the regulatory region that was among the two genes was not explained. <br />
 
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  We have to take the whole sequence in fasta format and use it in a program called Gene Construction Kit. This shows reading frames and restriction sites. </p>
 
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<p>&nbsp;</p>
 
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<p>In fruitfly.org: 9005/seq_tools/promoter.html we can look for primers and we can adjust parameters. We can also analyze the  stability energy, and seek the lowest point of stability. This point is generally the -10box. To find inverted repeats, we shall use the program StemLoop of the parcel of GCG (genetics computer group). This program calls in the sequence in a GCG format. To find direct repeats we will use the program &quot;repeat&quot;. For rcnR and rcnA we found three direct repeats between the -10 box and the translation start of rcnA.We suggest that this is a regulatory region. Based on this, we designed the primers, trying to preserve the regulatory region and changing its promoter.</p>
 
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<p>&nbsp;</p>
 
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<p><em>Primer design.</em> The region should be rich in GC, of about 20 nucleotides with a 50% GC content at least and it should finish in G. The program can also show the double chain to facilitate the design of oligo lower. If they are rich in AT, they can be longer primers to increase its Tm. </p>
 
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<p>&nbsp;</p>
 
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<p>The most popular program at the center is Oligo. Here we open a new window and paste the sequence. This will open two windows. The first one with the Tm, and the other one with the free energy. The program can calculate all oligos and show potential couples with its parameters. We can also specify were we want the oligo to be located. Once the program generates it, we can analyze its biochemical properties. </p>
 
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<p><em>Trying to k Delta G so it won't be lower than -10. </em></p>
 
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<p><br />
 
-
  The differences between the TMS should not be greater than 5 degrees. Enzymes used in PCR use magnesium chloride. The most reliable and processed use magnesium acetate. It is said that 10mM of dinucleotidos is an optimal concentration for PCR, .4 mM is used in the lab. Once we have the oligo, we add a site at the far restraining 5 '. And add nucleotides in the 5 'end to ensure that the enzyme is positioned correctly and efficiently cut. These nucleotides are different for each enzyme, and they also protect the  5' end. </p>
 
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<p>&nbsp;</p>
 
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<p>Two plasmids are used as a basis prK415 P. Our advisor, Miguel, already has isolated DNA and this DNA will be used to transform and to have a  the plasmid reserved. 2ul of the plasmid and competent cells treated with calcium chloride. The theory says that positive ions are attached to the membrane, so the membrane has a positive charge. As DNA has a negative charge, once they are mixed at 4 degrees Celsius for 20 minutes, we are going to take the tube and put it at 42 degrees centigrade. This stress produces holes in the membrane and many things will be capable or entering or exiting through the membrane, including DNA. Then it remains 2 more minutes at this temperature. The we return it to ice for 5 more minutes to recover. Later, the cells are placed in 1ml of rich medium (LB) were they are allowed to grow at 37 degrees for one hour at 300 revolutions per minute (this allows them to recover).</p>
 
-
<p>&nbsp;  </p>
 
-
<p>100ul are taken and used to plate in petri dishes with the antibiotic. It is left to grow for an entire day and at the end, isolated colonies should appear.<br />
 
-
  Bacteria with kanamycin 5ml of two strains, one with a deletion in rcnA and another one with any deletion except for rcnA . For 6 hours, the bacteria will have an exponential growth. Genomic DNA will be extracted. </p>
 
-
<p>&nbsp;</p>
 
-
<p>The contents of the tube will be put in an eppendorf, we centrifuge and then we withdraw the liquid medium with a syringe. Before we lyse de cells, we need to wash with TE 10 1 (Tris 10uM EDTA 1uM), with pH 8. Vortex, to separate and disintegrate. Again, we centrifuge and remove supernatant. To lyse, we add 400-450 ul TE5020pH8 and SDS 10% and K proteinase. We leave it at 37 degrees for 20 minutes. The medium goes from an opaque color to a light color when lysis happens. We add  ethanol 100% once we have lysed the cells and we vortex. In the presence of ethanol DNA is precipitated, so we add 1ml of ethanol. Then we centrifuge for a few minutes and we have pellet. We wash three times with ethanol 70%, which solubilised salts and the small molecules (including RNA). We remove all the ethanol, this tube is placed in a specific centrifuge. The vacuum from this centrifuge will remove the remain solvent. It is necessary to remove all the ethanol, because this affects the pH. TE 10 1 RNAs 10mg per ml, this Stock solution is divided 1000 times and  50ul approx are added. To check the quality of the DNA extracted, we use an agarose gel.</p>
 
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<p>&nbsp;</p>
 
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<p><em>Transforming bioparts: </em>Bacteria needed to extract DNA plasmid. Centrifuge, wash and put solution 1. Glucose, TRIS, EDTA and sometimes RNAs 1. Sodium hydroxide and SDS in the solution 2, sodium hydroxide denatures the DNA. Solution 3 with sodium acetate neutralizes the base. Wash and dry every time.</p></p>
 
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Latest revision as of 01:14, 29 October 2008

LCG-UNAM-Mexico:Notebook/June

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iGEM 2008 TEAM
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June

2008-06-03

GROUP SESSION:

Session with Dr. Miguel

We did some brainstorming in regards to the project. How to measure the efflux, and how to build the system.
• Monitor changes in pH using colorimetry or electrodes, choose the electrodes wisely.
• If we use a nickel transporter in E. coli we can easily obtain the mutated strain (deleted trasporter).
• Introducing the gene into a multicopy plasmid and establish positive and negative controls
• An electrode is not really necessary, measurements can be done with conventional methods.

Researching Metal Efflux Pumps:
(The metal must be ionizable, maybe using a simple salt, the anion doesn't matter).

We decided to research how are metals transported by E. coli , each member of the team chose a specific metal:

• Cobalt - Mariana
• Zinc - Jimena
• Cadmium - Mariana GS
• Nickel - Libertad
• Iron - Atahualpa
• TeO3 (+2) - Isaac
• Cu (2 +) - Minerva
• CrO4 (2 -) - Daniela
• AsO4 (3 -) - Enrique
• Hg - Carlos
• Pb - Martin

Vectors we could use:

  • prk404 y7o prk415: resistant to tratracicline, maybe 4 - 9 copies.
  • pbbr1mcs5: resistant to kentamicine up to 10 copies.
  • puc: up to 20 copies resistant to ampicillin.
  • pjet: resistant to ampicillin up to 600 copies.
  • Plbb: clorma resistant, up to 12 copies with LacI in cis (it can be controlled with IPTG)
  • REMEMBER not to combine the vector with the same replication origin.

    Once the most suitable gene has been chosen, primers must be designed:
    • Tm = (2 (A + T)) + (4 * (C + G))
    • Choose the single cut site.
    • Take in account compatibility of restriction sites.
    • Read more to start writing.

    2008-06-05

    GROUP SESSION:
    Expositions: Choosing the efflux pump:

    Nickel and cobalt
    • Very specific
    • We don't know how to get the Co inside the cell.
    • We can keep the wild type entry, and regulate the efflux.
    • All this in E. coli.
    • Co is very toxic, it can damage the cell, we better only use Nickel.
    • We have two pumps we can test.
    • The cell hold up to 2 millimolar of Ni.
    • It has more than one system for Ni entry.
    • RcnA & RcnR (~200 & ~300 aa) are used during Ni efflux.
    • Pending: How does Co enter the cell and the mechanism of Ni entry.

    Zinc
    • Uses ZnuABC, Zupt and ZntB to enter the cell. To leave the cell it uses ZntA and ZitB.
    • The only specific entry for Zn is ZnuABC, the others also transport other metals.
    • Regulated by Zurt, which binds Zn.
    • To withdraw Zn: ZntA only works at high concentrations, but is not specific for Zn; ZitB is not specific either and it only operates at low concentrations.
    • Problem: It is difficult to regulate their extrusion.
    • Zn is essential.

    Cadmium
    • Its entry is mediated by a transportation system of divalent ions, it is cotransported with Manganese, which is essential for the cell, so the entry is not tightly regulated.
    • It is toxic to the cell, but it doesn't seem too serious.
    • The efflux can be mediated by multiple systems, all found in E. coli (CzcD, CzcCBA, CadA, ...).
    • Legatzki et al. (2003) made an experiment in which they use a mutant E coli GG48 ((delta) zntA & (delta) zitB) that accumulates both Zn and Cd, but when they transform it with a plasmid with zntA & cadA of R. metallidurans, it regained resistance. It is true that we will not regulate all systems involved, but according to their experiment, change is quite significant. It could be useful.
    • Genes are large, up to ~800aa.
    • Pending: What concentration can the cell hold?

    Iron
    • There are many ways to get iron, including through siderophores.
    • Problem: On entering the cell, it forms a complex with a global regulator (fur) involved in many important functions. Essential.
    • The pump Fief (~920kb) is ok, highly specific and the only way to remove the iron. .

    Tellurium
    • Not so much an extrusion pump, because there is a transformation by means of an enzyme, which is not well known.
    • All resistance genes are in two plasmids.
    • Entry to the cell is based on a ionic potential in membranes.
    • It is not necessary, rather toxic.
    • We can not regulate the entry, and when the Tellurium enters, unless there is resistance, the cell dies immediately.
    • The role of the genes involved in resistance is not well understood.

    Copper
    • When it enters, it is reduced from 2+ to 1+, because the extrusion systems only recognize Cu1+.
    • Cells hold up to 3.5 miniMolar.
    • CusCFAB operon is responsible for efflux, regulated transcriptionally by cusRS. There is probably a biopart. Known in E. coli.
    • CusRS is ~1000 aa. Cus CFAB ~2000aa.
    • Problem: Its size!
    • Admission is ATPase dependent... by pumps? Described in yeast and animals, it is known to enter to E. coli, but how can we regulate it?
    • It is not essential, it is highly toxic.
    • Pending: How is the entry mediated?

    Arsenic
    • Resistance is in a plasmid in E. coli. Five genes (Ars [RABCD] ~ 1.4Kb), the plasmid is in total ~ 4.4kb. Some genes on the chromosome are also involved; they are not necessary but the resistance is reduced from 10 to 100 times if they are absent.
    • Do they have a translational control?
    • The pump works with ATP.
    • The entry is not specific, active transport.
    • Toxic.
    • This is the most studied bomb.
    • This operon is cloned into a vector.
    • Pending: What concentration can it hold?

    Mercury
    • Free diffusion, and three carriers are known.
    • It is highly toxic, to reduce its toxicity it is reduced.
    • Ther is no well-known system of entry.
    • The pumps are quite specific.
    • Also toxic in the cells environment, therefore cells absorb it, for processing.
    • Pending: Getting Hg out of the cells?

    Lead
    • It enters together with manganese, Zn or Co.
    • It is highly toxic to E. coli because it affects membranes.
    • Calcium pumps that transport it into the cells are known, but they are animals.
    • To remove it, it uses the Cd detoxification systems, there are no specific system.
    • Pending: Finding a target, Concentration that cells can endures?


    Not useful for our purposes

    • Iron
    • Lead
    • Mercury
    • Tellurium

    Probably useful
    • Zinc (Cons: It is essential.)
    • Copper (Cons: It is very big.)

    Favourites
    • Cobalt & Nickel
    • Cadmium
    • Arsenic

    2008-06-17

    GROUP SESSION:
    Project Design

    Experimental

    Pump we will use: Nickel.

    Articles:

  • Complex Transcriptional Control Links NikABCDE-HYDROGEN with Dependent Nickel Transport Expression in E. coli (2005).
  • Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function (2006).
  • Identification of rcnA (yohM), Nickel and Cobalt Resistance Gene in Escherichia coli (2005).
  • Pending:

  • Check bioparts
  • Design vectors
  • Design primers
  • Strain with deletion of rcnA.

  • Preliminary design:
    • The mechanism of entry of Nickel will remain wildtype.
    • In the absence of Nickel, RcnR (whose gene will remain in the chromosome with its normal regulation) will repress rcnA (which will be deleted from the chromosome and inserted into a plasmid).
    • By adding (*) to the system we will repress transcription of rcnA, even in the presence of Nickel, so this will be will be our signal to retain the metal inside the cell and modify the concentration of the medium (if it is not enough to turn off the pump, it will be necessary to find a new level of regulation).
    • How will we turn off the signal (*)?

    Task: Suggest a molecule for (*)!

    Modeling

    Pending:

  • Response vs. Concentration (experimental part).
  • Set thresholds & limitations.
  • Efficiency of interactions?
  • Defining variables:
  • - Metal concentration.
    - Repressor concentration.
    - (*) concentration.