Team:Groningen/Project/Accumulation

From 2009.igem.org

(Difference between revisions)
(Arsenic: Support for our own ars sites.)
(Arsenic)
Line 117: Line 117:
&beta;1 = <input type="text" id="beta1" value="1"/> 1/second (???)<br/>
&beta;1 = <input type="text" id="beta1" value="1"/> 1/second (???)<br/>
&beta;2 = <input type="text" id="beta2" value="1"/> 1/second (???)<br/>
&beta;2 = <input type="text" id="beta2" value="1"/> 1/second (???)<br/>
 +
&beta;3 = <input type="text" id="beta3" value="1"/> 1/second (???)<br/>
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>-->
<!--As(III) = <input type="text" id="As3Concentration" value="10"/> &micro;M<br/>-->
OpN<sub>total</sub> = <input type="text" id="OpNTotalConcentration" value="0.00166054"/> &micro;M (??)<br/> <!-- 1/(avogadro's constant)/micrometer^3 in micromoles/liter -->
OpN<sub>total</sub> = <input type="text" id="OpNTotalConcentration" value="0.00166054"/> &micro;M (??)<br/> <!-- 1/(avogadro's constant)/micrometer^3 in micromoles/liter -->
-
OpG<sub>total</sub> = <input type="text" id="OpGTotalConcentration" value="0.0166054"/> &micro;M (??)<br/> <!-- 10/(avogadro's constant)/micrometer^3 in micromoles/liter -->
+
OpG<sub>total</sub> = <input type="text" id="OpGTotalConcentration" value="0"/> &micro;M (??)<br/>
 +
OpH<sub>total</sub> = <input type="text" id="OpHTotalConcentration" value="0.0166054"/> &micro;M (??)<br/> <!-- 10/(avogadro's constant)/micrometer^3 in micromoles/liter -->
<button onClick="computeArsenicEquilibrium()">Compute</button><br/>
<button onClick="computeArsenicEquilibrium()">Compute</button><br/>
Line 153: Line 155:
   var beta1Node = document.getElementById("beta1");
   var beta1Node = document.getElementById("beta1");
   var beta2Node = document.getElementById("beta2");
   var beta2Node = document.getElementById("beta2");
 +
  var beta3Node = document.getElementById("beta3");
   //var As3ConcentrationNode = document.getElementById("As3Concentration");
   //var As3ConcentrationNode = document.getElementById("As3Concentration");
   var OpNTotalConcentrationNode = document.getElementById("OpNTotalConcentration");
   var OpNTotalConcentrationNode = document.getElementById("OpNTotalConcentration");
   var OpGTotalConcentrationNode = document.getElementById("OpGTotalConcentration");
   var OpGTotalConcentrationNode = document.getElementById("OpGTotalConcentration");
 +
  var OpHTotalConcentrationNode = document.getElementById("OpHTotalConcentration");
   // Intermediates (mostly useful for debugging)
   // Intermediates (mostly useful for debugging)
Line 181: Line 185:
   var beta1 = Number(beta1Node.value); // 1/second
   var beta1 = Number(beta1Node.value); // 1/second
   var beta2 = Number(beta2Node.value); // 1/second
   var beta2 = Number(beta2Node.value); // 1/second
 +
  var beta3 = Number(beta3Node.value); // 1/second
   //var As3Concentration = Number(As3ConcentrationNode.value) * 1e-6; // micromolar -> molar
   //var As3Concentration = Number(As3ConcentrationNode.value) * 1e-6; // micromolar -> molar
   var OpNTotalConcentration = Number(OpNTotalConcentrationNode.value) * 1e-6; // micromolar -> molar
   var OpNTotalConcentration = Number(OpNTotalConcentrationNode.value) * 1e-6; // micromolar -> molar
   var OpGTotalConcentration = Number(OpGTotalConcentrationNode.value) * 1e-6; // micromolar -> molar
   var OpGTotalConcentration = Number(OpGTotalConcentrationNode.value) * 1e-6; // micromolar -> molar
 +
  var OpHTotalConcentration = Number(OpHTotalConcentrationNode.value) * 1e-6; // micromolar -> molar
   var OpTotalConcentration = OpNTotalConcentration + OpGTotalConcentration;
   var OpTotalConcentration = OpNTotalConcentration + OpGTotalConcentration;
Line 191: Line 197:
     var ArsRConcentration = 1;
     var ArsRConcentration = 1;
     var ArsDConcentration = 1;
     var ArsDConcentration = 1;
-
    var c1 = K3d * (tau1/Math.LN2) * beta1 * OpTotalConcentration;
 
     var c2 = K4d * (tau2/Math.LN2) * beta2 * OpNTotalConcentration;
     var c2 = K4d * (tau2/Math.LN2) * beta2 * OpNTotalConcentration;
-
     if (!(c1>0)) throw new Error("c1<=0");
+
     if (!(c2>0)) throw new Error("c2<=0, c2="+c2);
-
    if (!(c2>0)) throw new Error("c2<=0");
+
     do {
     do {
Line 201: Line 205:
       var D2 = Math.sqrt(b2*b2 + c2);
       var D2 = Math.sqrt(b2*b2 + c2);
       ArsDConcentration = -b2 + D2;
       ArsDConcentration = -b2 + D2;
-
       var b1 = 0.5 * K3d * (ArsDConcentration/K4d + 1);
+
       var b1 = 0.5 * (K3d * (ArsDConcentration/K4d + 1)
 +
                      - (tau1/Math.LN2) * beta3 * OpHTotalConcentration);
 +
      var c1 = K3d * (tau1/Math.LN2) * (beta1 * OpTotalConcentration
 +
                                        + beta3 * OpHTotalConcentration * (ArsDConcentration/K4d + 1));
       var D1 = Math.sqrt(b1*b1 + c1);
       var D1 = Math.sqrt(b1*b1 + c1);
       ArsRConcentration = -b1 + D1;
       ArsRConcentration = -b1 + D1;
       // Some general assertions
       // Some general assertions
-
       if (!(b1>0)) throw new Error("b1<=0");
+
       if (!(b2>0)) throw new Error("b2<=0, b2="+b2);
-
       if (!(b2>0)) throw new Error("b2<=0");
+
       if (!(ArsDConcentration>0)) throw new Error("ArsD<=0, ArsD="+ArsDConcentration);
-
       if (!(ArsDConcentration>0)) throw new Error("ArsD<=0");
+
       //if (!(b1>0)) throw new Error("b1<=0, b1="+b1);
-
       if (!(ArsRConcentration>0)) throw new Error("ArsR<=0");
+
      if (!(c1>0)) throw new Error("c1<=0, c1="+c1);
 +
       if (!(ArsRConcentration>0)) throw new Error("ArsR<=0, ArsR="+ArsRConcentration);
       // Check that we don't encounter too steep a derivative
       // Check that we don't encounter too steep a derivative
       var b2d = 0.5 * (K4d/K3d);
       var b2d = 0.5 * (K4d/K3d);
       var ArsDConcentrationd = -b2d + (2*b2*b2d)/D2;
       var ArsDConcentrationd = -b2d + (2*b2*b2d)/D2;
 +
      var c1d = (tau1/Math.LN2) * beta3 * OpHTotalConcentration * (K3d/K4d) * ArsDConcentrationd;
       var b1d = 0.5 * (K3d/K4d) * ArsDConcentrationd;
       var b1d = 0.5 * (K3d/K4d) * ArsDConcentrationd;
-
       var fd = -b1d + (2*b1*b1d)/D1;
+
       var fd = -b1d + (2*b1*b1d+c1d)/D1;
-
       if (Math.abs(fd)>1) throw new Error("fd>1, no convergence?"); // According to Wikipedia
+
       if (Math.abs(fd)>1) throw new Error("|fd|>1, no convergence?"); // According to Wikipedia
     } while(Math.abs(oldArsR-ArsRConcentration)>1e-6*Math.abs(ArsRConcentration));
     } while(Math.abs(oldArsR-ArsRConcentration)>1e-6*Math.abs(ArsRConcentration));

Revision as of 14:06, 14 July 2009

[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]

Introduction

Once heavy metals have entered the cell it is key to keep them there. As these metals are toxic to cell survival in critical amounts evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variaty of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..

Metallothioneins

Metallothioneins are a class of low molecular-weight metal-binding proteins rich in cysteines residues. They are capable of binding a variety of heavy metals. And they have readily been used to create cell based systems for purification of contaminated water [http://www.ncbi.nlm.nih.gov/pubmed/9758654][http://www.ncbi.nlm.nih.gov/pubmed/18618649]. In addition to their wide application possibilities they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one[http://www.ncbi.nlm.nih.gov/pubmed/9579658]. Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium[http://www.ncbi.nlm.nih.gov/pubmed/16890348], arsenic [http://www.ncbi.nlm.nih.gov/pubmed/16984198][http://www.ncbi.nlm.nih.gov/pubmed/15294789][http://www.ncbi.nlm.nih.gov/pubmed/18326684], mercuryhttp://www.ncbi.nlm.nih.gov/pubmed/9758654 1[http://www.ncbi.nlm.nih.gov/pubmed/9342882][http://www.ncbi.nlm.nih.gov/pubmed/17920767], nickel[http://www.ncbi.nlm.nih.gov/pubmed/12727263] or a combination of metalshttp://www.ncbi.nlm.nih.gov/pubmed/9579658 3[http://www.ncbi.nlm.nih.gov/pubmed/18313216]. Metal-protein complexes can be quantified using a fluorescent molecule[http://www.ncbi.nlm.nih.gov/pubmed/19133293].

Alternatives

Inclusion bodies[http://www.ncbi.nlm.nih.gov/pubmed/3297654]
(Bacterio)Ferritins
Phytochelatins
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]

Inhibitory characteristics?

Modelling

Arsenic

Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its buoyancy capacity. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add upto a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).

At this moment we use four different variables:

  1. Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]
  2. Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: Koster et al
  3. The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]
  4. Cell density. Source: see our gas vesicle page.

awAs(III) = g/mol
nAs(III) / Mcell(dry) = millimole/kg
Mcell(dry) / Mcell(wet) =
ρcell = kg/m3

As(III) intake per volume of cells
= g/m3
= µmol/liter (TODO: check)

The following tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell. See our Modelling page for information on the constants/variables used. Note that the computations currently involve more variables/constants than strictly necessary.

K1d = µM (?)
K2d = µM
K3d = µM
K4d = µM
τ1 = min (??)
τ2 = min (??)
β1 = 1/second (???)
β2 = 1/second (???)
β3 = 1/second (???)
OpNtotal = µM (??)
OpGtotal = µM (??)
OpHtotal = µM (??)

Operator = µM
ArsR = µM
ArsD = µM
ArsRop = µM
ArsDop = µM

As(III)total/As(III) =

Planning and requirements:

  • Modelling
    • Speed
    • Metaliotheines concentration
  • Lab
    • Measurements
      • Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.
    • Biobrick Bba_K129004
    • Rest

Literature

  1. Brady et al.:[http://www.ncbi.nlm.nih.gov/pubmed/18618649 The use of hollow fiber cross-flow microfiltration in bioaccumulation and continuous removal of heavy metals from solution by Saccharomyces cerevisiae], Biotechnology and bioengineering (1994) 44(11);1362-1366
  2. Cadosch et al.: [http://www.ncbi.nlm.nih.gov/pubmed/19133293 Uptake and intracellular distribution of various metal ions in human monocyte-derived dendritic cells detected by Newport Green DCF diacetate ester] Journal of Neuroscience Methods (2009) 178(1);182-187
  3. Chang et al.:[http://www.ncbi.nlm.nih.gov/pubmed/9579658 Cysteine contributions to metal binding preference for Zn/Cd in the b-domain of metallothionein], Protein Engineering 1998 11(1);41–46
  4. Chen et al.: [http://www.ncbi.nlm.nih.gov/pubmed/9758654 Hg2+ removal by genetically engineered Escherichia coli in a hollow fiber bioreactor], Biotechnology progress (1998) 14(5);667-71
  5. Chen & Wilson: [http://www.ncbi.nlm.nih.gov/pubmed/9342882 Genetic engineering of bacteria and their potential for Hg2+ bioremediation], Biodegradation (1997) 8(2);97-103
  6. Deng et al.:[http://www.ncbi.nlm.nih.gov/pubmed/16890348 Cadmium removal from aqueous solution by gene-modified Escherichia coli JM109] Journal of hazardous materials (2007) 139(2);340-4
  7. Deng et al.: [http://www.ncbi.nlm.nih.gov/pubmed/17920767 Continuous treatment process of mercury removal from aqueous solution by growing recombinant E. coli cells and modeling study], Journal of hazardous materials (2008) 153(1-2);487-92
  8. Deng et al.: [http://www.ncbi.nlm.nih.gov/pubmed/12727263 Bioaccumulation of nickel from aqueous solutions by genetically engineered Escherichia coli], Water research (2003) 37(10);2505-11.
  9. Fowler: [http://www.ncbi.nlm.nih.gov/pubmed/3297654 Intracellular Compartmentation of Metals in Aquatic Organisms: Roles in Mechanisms of Cell injury], Environmental Health Perspectives (1987) 71;121-128
  10. Kao et al.: [http://www.ncbi.nlm.nih.gov/pubmed/18313216 Biosorption of nickel, chromium and zinc by MerP-expressing recombinant Escherichia coli], Journal of hazardous materials (2008) 158(1);100-106
  11. Kostal et al.: [http://www.ncbi.nlm.nih.gov/pubmed/15294789 Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR], Applied and environmental microbiology (2004) 70(8);4582–4587
  12. Ngu & Stillman: [http://www.ncbi.nlm.nih.gov/pubmed/16984198 Arsenic binding to human metallothionein], Journal of the American Chemical Society (2006) 128(38);12473-83.
  13. Singh et al.: [http://www.ncbi.nlm.nih.gov/pubmed/18326684 Highly Selective and Rapid Arsenic Removal by Metabolically Engineered Escherichia coli Cells Expressing Fucus vesiculosus Metallothionein] Applied and environmental microbiology (2008) 74(9);2924-7
  14. Yung-Fen et al.: [http://www.jbc.org/cgi/content/full/282/23/16783?view=long&pmid=17439954 ArsD Residues Cys12, Cys13, and Cys18 Form an As(III)-binding Site Required for Arsenic Metallochaperone Activity], The Journal of Biological Chemistry (2007) VOL 282(23); 16783–16791
  15. Yung-Fen et al.: [http://www.springerlink.com/content/684280r475276335/ ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase], Journal of Bioenergetics and Biomembranes (2007) 39;453-458