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

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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Team" class="navText">About Us</a></td>
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           <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/Project" class="navText">Our project</a></td>
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          <td width="165" bgcolor="#5C743D"><a href="https://2008.igem.org/Team:LCG-UNAM-Mexico/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>
-
<b><p>Session with our advisor Miguel A.<br /></b>
+
<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>
-
<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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-
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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 />
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   • 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>
+
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       </tr>  
       </tr>  
<tr>
<tr>
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         <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>

Latest revision as of 01:14, 29 October 2008

LCG-UNAM-Mexico:Notebook/June

Header image
iGEM 2008 TEAM
line decor
  
line decor

 
 
 
 
 
 
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.