Difference between revisions of "Team:UMaryland/Model"

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        <a href="https://2016.igem.org/Team:UMaryland">
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<img src="https://static.igem.org/mediawiki/2016/2/26/T--UMaryland--newlogo.jpeg" id="img-logo" width="128px">
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<ul>
 
<ul>
 
<li><a href="https://2016.igem.org/Team:UMaryland/projects">Projects</a></li>
 
<li><a href="https://2016.igem.org/Team:UMaryland/projects">Projects</a></li>
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</ul>
 
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<h11>Parts Collection</h11></br>
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<h21>BioBrick Devices Submitted to the Registry</h21></br>
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<h31>Furthering collaboration and standardization of genetic parts</h31></br>
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<h11>Modeling</h11></br>
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</div>
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<h21>Predicting Data and Optimizing Results</h21></br>
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        </div>
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<h31>Applying engineering principals to biological systems</h31></br>
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<div class="longText">
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<p>UMaryland iGEM submitted various basic and composite parts to the BioBrick Registry, which aims to increase standardization in synthetic biology by allowing genes to be added together easily. We synthesized the genes, put them inside standard BioBrick plasmids, and then characterized our parts.</p>
 +
<p>View:</p>
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<p>
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Basic Parts <input type="checkbox" class="filter" checked id="input-basic" />
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Composite Parts <input type="checkbox" class="filter" checked id="input-composite" />
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<div id="div-slideshow">
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<div class="profiles composite">
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<strong><a target="_blank" href="http://parts.igem.org/Part:BBa_K2032001">Fructose Pathway (BBa_K2032001)</strong></a>
 +
<img src="https://static.igem.org/mediawiki/2016/e/e7/T--UMaryland--BBa_K2032001_linear.jpg" class="linear" />
 +
<img src="https://static.igem.org/mediawiki/2016/1/14/T--UMaryland--BBa_K2032001_plasmid.jpg" class="plasmid" />
 +
<p>This composite biobrick part is a combination of the coding regions for three separate enzymes involved in the metabolization of methanol. Each coding region is preempted by a ribosome binding site in order to help counteract some of the translational issues associated with polycistronic mRNA. A lacI regulated promoter that allows for induction with IPTG was included in this construct to allow for selective gene expression along with the standard iGEM double terminator.</p>
 +
<p>The three enzymes encoded by this part are methanol dehydrogenase 2 (MDH), 3-hexulose-6-phosphate synthase (HPS), and 6-Phospho-3-hexuloisomerase (PHI). These enzymes serve to function as a three step pathway in which methanol is metabolized. MDH converts methanol to formaldehyde, producing a molecule of NADH in the process. Formaldehyde is then combined with a molecule of D-ribulose-5-phosphate taken from the pentose phosphate pathway to form one molecule of D-arabino-3-hexulose-6-phosphate via the usage of HPS. PHI then converts D-arabino-3-hexulose-6-phosphate to D-fructose-6-phosphate which can then undergo glycolysis.</p>
 +
</div>
 +
<div class="profiles basic">
 +
<strong><a target="_blank" href="http://parts.igem.org/Part:BBa_K2032002">Formate Pathway (BBa_K2032002)</a></strong>
 +
<img src="https://static.igem.org/mediawiki/2016/9/9b/T--UMaryland--BBa_K2032002_linear.jpg" class="linear" style="width: 944px; height: 323px;"/>
 +
<img src="https://static.igem.org/mediawiki/2016/a/a6/T--UMaryland--BBa_K2032002.png" class="plasmid" />
 +
<p>The Formate Pathway is a three enzyme pathway that begins with methanol and NAD+ as substrates, and culminates in the production of NADH molecules and carbon dioxide. This pathway is used to detoxify alcohols in the cellular environment. The pathway consists of a series of oxidations: methanol oxidized to formaldehyde by Methanol Dehydrogenase 2 (MDH2); formaldehyde oxidized to formate by Formaldehyde Dehydrogenase (FALDH); and finally formate oxidized to carbon dioxide by Formate Dehydrogenase (FDH). Each of these catalyzed reactions results in lower energy products than reactants, so every reaction is coupled to the production of one NADH molecule, which contributes to energy for the cell and biomass production.</p>
 +
<p>The first enzyme of the pathway, MDH2, has a low binding specificity and will oxidize many primary alcohols. The original use of this plasmid was to detoxify methanol as part of a larger pathway that consisted of eliminating methane gas from the atmosphere. The pathway has potential uses for detoxifying alcohols in the environment as well. As methanol becomes an increasingly popular liquid fuel source, one could imagine methanol spills in the future that require detoxification. This part contains one promoter that is IPTG inducible. The genes will be transcribed as a polycistronic mRNA strand. Each of the genes has a medium strength ribosome binding site. Modeling of this pathway has revealed that no toxic substrates should be produced when this pathway is expressed. The kinetics for each enzyme exist in such a way that there will be no buildup of formaldehyde of formic acid in the cell. Theoretically, this pathway should increase methanol resistance to cells that express it.</p>
 +
</p>
 +
</div>
 +
<div class="profiles basic">
 +
<strong><a target="_blank" href="http://parts.igem.org/Part:BBa_K2032003">Codon optimized MDH2 with Lac/pL promoter (BBa_K2032003)</strong></a>
 +
<img src="https://static.igem.org/mediawiki/2016/8/8c/T--UMaryland--BBa_K2032003_plasmid.jpg" class="plasmid" />
 +
<p>This is an intermediate used in the construction of BBa_K2032002. It contains the coding sequence for MDH2 which oxidizes methane to formaldehyde. It also contains the lac + pL promoter (BBa_R0011) which is repressed by LacI and induced by IPTG.</p>
 +
</div>
 +
<div class="profiles basic">
 +
<strong><a target="_blank" href="http://parts.igem.org/Part:BBa_K2032004">GroESL (BBa_K2032004)</strong></a>
 +
<p>Chaperone complex</p>
 +
<div class="profiles composite">
 +
<strong><a target="_blank" href="http://parts.igem.org/Part:BBa_K2032005">GroESL Composite (BBa_K2032005)</strong></a>
 +
<img src="https://static.igem.org/mediawiki/2016/5/59/T--UMaryland--BBa_K2032005_linear.jpg" class="linear" />
 +
<img src="https://static.igem.org/mediawiki/2016/3/30/T--UMaryland--BBa_K2032005_plasmid.jpg" class="plasmid" />
 +
<p>Contains promoter, rbs, and terminator along with GroESL coding region
 +
</div>
 +
<div class="profiles composite">
 +
<strong><a target="_blank" href="http://parts.igem.org/Part:BBa_K2032006">Fructose with RFP (BBa_K2032006)</strong></a>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b3/T--UMaryland--BBa_K2032006_linear.jpg" class="linear" />
 +
<img src="https://static.igem.org/mediawiki/2016/f/f9/T--UMaryland--BBa_K2032006_plasmid.jpg" class="plasmid" />
 +
<p>Fructose construct with RFP reporter</p>
 +
</div>
 +
<div class="profiles composite">
 +
<strong><a target="_blank" href="http://parts.igem.org/Part:BBa_K2032007">Formate with RFP (BBa_K2032007)</strong></a>
 +
<img src="https://static.igem.org/mediawiki/2016/f/fb/T--UMaryland--BBa_K2032007_linear.jpg" class="linear" />
 +
<img src="https://static.igem.org/mediawiki/2016/9/90/T--UMaryland--BBa_K2032007_plasmid.jpg" class="plasmid" />
 +
<p>This is the fructose methanol degradation pathway (BBa_K2032001) with an RFP marker (BBa_K801100) attached</p>
 +
</div>
 +
</div>
 +
</div>
 +
<!-- <table id="table-selector"> Table of navigational links
 
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<th class="borderRight navigator borderTop" id="th-background" data-select="background"><p>Background</p></th>
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<th class="borderBoth navigator borderTop" id="th-pathway" data-select="pathway"><p>Pathway</p></th>
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<p>Three different constructs were synthesized by UMaryland iGEM in hopes of being co-cultured. They were:</p>
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<h4>Background</h4>
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<ul>
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<p>The Modeling portion of the UMaryland iGEM project proposes specific adjustments to the expression levels of enzymes in the methane digestion pathways. Our modeling efforts focus on ensuring the viability of our engineered organism, and optimizing the efficiencies of our pathways. Along the way, our team encountered stumbling blocks that we would like to illuminate for iGEM teams in the future. Incorporated into our modeling page is a concise guide on getting started with the Matlab applet, Simbiology, to model simple metabolic pathways. If you have no interest in this guide, please feel free to optimize your time by skipping over the purple text.</p>
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<li>sMMO Construct: oxidizing methane into methanol using oxygen</li>
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</div>
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<li>Formate Construct: further oxidizing methanol into carbon dioxide using NADH</li>
 +
<li>Fructose Construct: further oxidizing methanol into cellular metabolites</li>
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</ul>
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</div>
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<div class="longText" id="div-protocol">
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<h4>Protocol</h4>
 
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<h4>Results</h4>
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<h4>Creating the Pathway Architecture</h4>
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<div id="div-results">
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<img src="https://static.igem.org/mediawiki/2016/7/75/T--UMaryland--formateModel.jpg" width="100%" />
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<p>The models for our formate and fructose pathways each consist of two compartments, four reactions and 14 or 15 species, respectively. The initial goal for our project was to produce a co-culture of cells consisting of “sMMO Cells” that produce methanol and “Formate/Fructose Cells” that detoxify methanol, all in an effort to completely biodegrade methane. The first compartment in our model shown below is the “sMMO Cell” that converts methane to methanol, which travels to the second compartment of our model, either the “Formate” or “Fructose Cell.” Each species represents a different molecule involved in our pathway. The architecture for our Formate pathway displays a progression of compounds starting at methane and ending at carbon dioxide. In all, this pathway is facilitated by four enzymes.</p>
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<img src="https://static.igem.org/mediawiki/2016/e/eb/T--UMaryland--fructoseModel.jpg" width="100%" />
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<p>The first half of each pathway is identical. The first enzyme used in each pathway is sMMO, which catalyses the oxidation of methane to methanol while simultaneously oxidizing NADH to NAD+. Both pathways then proceed to oxidize methanol to formaldehyde, which is a reaction catalyzed by the MDH2 enzyme.</p>
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−
<p>Once formaldehyde is produced, the two pathways begin to differ. In the Formate Pathway, formaldehyde is first oxidized to formate, which is then oxidized to carbon dioxide by FALDH and FDH enzymes respectively. In the Fructose Pathway, formaldehyde is incorporated into a 5 carbon sugar by HPS to make D-arabinose 6-phosphate, a 6 carbon sugar. PHI then converts D-arabinose 6-phosphate into D-fructose-6-phosphate, which is a substrate of glycolysis.</p>
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<img class="figure" src="https://static.igem.org/mediawiki/2016/a/ae/T--UMaryland--absorbance.JPG">
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<small class="caption">Figure 1. Cell growth versus absorbance.</small>
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$('.titleText').css('left','15px');
 +
$('.titleText').css('margin-left','0px');
 +
} else {
 +
$('.titleText').css('left','50%');
 +
$('.titleText').css('margin-left','-40%');
 +
}
 
})
 
})
 
 
Line 769: Line 911:
 
});
 
});
 
</script>
 
</script>
−
         <script> $('#iGEM').css('display','block');
+
         <script> $('#iGEM').css('display','block'); </script>
−
+
−
$('body').css('display', 'block');</script>
+
 
<script>
 
<script>
 
$(document).ready(function(){
 
$(document).ready(function(){
−
   
+
  $('#div-figures').slick({
−
+
  });
 +
  $('body').css('display', 'block')
 
});
 
});
 
 
Line 786: Line 927:
 
}
 
}
 
});
 
});
 +
 
 
 
var toggle = 1;
 
var toggle = 1;
  
−
+
−
+
</script>
−
</script>
+
−
+
 
<script>
 
<script>
 
var clicked = '';
 
var clicked = '';
−
var current = 'background'; // sets initial display to be purpose
+
var current = 'purpose'; // sets initial display to be purpose
 
  $('.navigator').click( function() { // when any element with .class is clicked, trigger function
 
  $('.navigator').click( function() { // when any element with .class is clicked, trigger function
 
var selected = $(this).attr('data-select'); // grabs the name of the navigational element clicked
 
var selected = $(this).attr('data-select'); // grabs the name of the navigational element clicked
Line 808: Line 948:
 
dir = dir.replace('#','');
 
dir = dir.replace('#','');
 
if (dir == '') {
 
if (dir == '') {
−
dir = 'background';
+
dir = 'purpose';
 
}
 
}
 
changeText(dir)
 
changeText(dir)
 +
switch (dir) {
 +
case 'purpose':
 +
        break;
 +
    case 'background':
 +
        break;
 +
case 'experiment':
 +
        displayTable(dir)
 +
        break;
 +
case 'results':
 +
        displayTable(dir)
 +
        break;
 +
    default:
 +
        break;
 +
}
  
 
$(".subNav").click(function() {
 
$(".subNav").click(function() {
Line 819: Line 973:
 
});
 
});
  
 +
function displayTable(selected) {
 +
$('#ul-'+selected).css('display','block');
 +
}
  
 
function changeText(selected) {
 
function changeText(selected) {
 
$('.navigator').css('border-bottom','1px solid #A8A8A8'); // make all navigational element have no bottom border
 
$('.navigator').css('border-bottom','1px solid #A8A8A8'); // make all navigational element have no bottom border
−
$('#th-' + selected).css('border-bottom', '4px solid #A8A8A8'); // the clicked navigational element given border // hides all text
+
$('#th-' + selected).css('border-bottom', '4px solid #A8A8A8'); // the clicked navigational element given border
−
var widthText = $('.longText').outerWidth(false);
+
$('.blockText').css('display','none');
−
$('.textSection').css('width', widthText);
+
$('.subNavList').css('display','none'); // hides all text
−
$('.textSection').css('display','none');
+
$('.blockText').css('margin-left', '60px') // slighly shifts right all text to set up for animation
−
$('.subNavList').css('display','none'); // slighly shifts right all text to set up for animation
+
$('#div-' + selected).css('display','block');
−
$('.textSection').css('margin-left', '60px') // slighly shifts right all text to set up for animation
+
displayTable(selected) // the div containing the text is displayed
−
$('#div-' + selected).css('display','block'); // the div containing the text is displayed
+
$('#div-' + selected).animate({ marginLeft: '0px'}, 200); // the text is animated to quickly move left
−
$('#div-' + selected).animate({ marginLeft: '0px'}, 200);
+
−
$('.textSection').css('width', '100%');
+
 
current = selected; // sets the current display
 
current = selected; // sets the current display
 
location.hash = current;
 
location.hash = current;

Revision as of 07:42, 19 October 2016

</div> </div> Modeling

Parts Collection
BioBrick Devices Submitted to the Registry
Furthering collaboration and standardization of genetic parts

UMaryland iGEM submitted various basic and composite parts to the BioBrick Registry, which aims to increase standardization in synthetic biology by allowing genes to be added together easily. We synthesized the genes, put them inside standard BioBrick plasmids, and then characterized our parts.

View:

Basic Parts Composite Parts

Fructose Pathway (BBa_K2032001)

This composite biobrick part is a combination of the coding regions for three separate enzymes involved in the metabolization of methanol. Each coding region is preempted by a ribosome binding site in order to help counteract some of the translational issues associated with polycistronic mRNA. A lacI regulated promoter that allows for induction with IPTG was included in this construct to allow for selective gene expression along with the standard iGEM double terminator.

The three enzymes encoded by this part are methanol dehydrogenase 2 (MDH), 3-hexulose-6-phosphate synthase (HPS), and 6-Phospho-3-hexuloisomerase (PHI). These enzymes serve to function as a three step pathway in which methanol is metabolized. MDH converts methanol to formaldehyde, producing a molecule of NADH in the process. Formaldehyde is then combined with a molecule of D-ribulose-5-phosphate taken from the pentose phosphate pathway to form one molecule of D-arabino-3-hexulose-6-phosphate via the usage of HPS. PHI then converts D-arabino-3-hexulose-6-phosphate to D-fructose-6-phosphate which can then undergo glycolysis.

Formate Pathway (BBa_K2032002)

The Formate Pathway is a three enzyme pathway that begins with methanol and NAD+ as substrates, and culminates in the production of NADH molecules and carbon dioxide. This pathway is used to detoxify alcohols in the cellular environment. The pathway consists of a series of oxidations: methanol oxidized to formaldehyde by Methanol Dehydrogenase 2 (MDH2); formaldehyde oxidized to formate by Formaldehyde Dehydrogenase (FALDH); and finally formate oxidized to carbon dioxide by Formate Dehydrogenase (FDH). Each of these catalyzed reactions results in lower energy products than reactants, so every reaction is coupled to the production of one NADH molecule, which contributes to energy for the cell and biomass production.

The first enzyme of the pathway, MDH2, has a low binding specificity and will oxidize many primary alcohols. The original use of this plasmid was to detoxify methanol as part of a larger pathway that consisted of eliminating methane gas from the atmosphere. The pathway has potential uses for detoxifying alcohols in the environment as well. As methanol becomes an increasingly popular liquid fuel source, one could imagine methanol spills in the future that require detoxification. This part contains one promoter that is IPTG inducible. The genes will be transcribed as a polycistronic mRNA strand. Each of the genes has a medium strength ribosome binding site. Modeling of this pathway has revealed that no toxic substrates should be produced when this pathway is expressed. The kinetics for each enzyme exist in such a way that there will be no buildup of formaldehyde of formic acid in the cell. Theoretically, this pathway should increase methanol resistance to cells that express it.

Codon optimized MDH2 with Lac/pL promoter (BBa_K2032003)

This is an intermediate used in the construction of BBa_K2032002. It contains the coding sequence for MDH2 which oxidizes methane to formaldehyde. It also contains the lac + pL promoter (BBa_R0011) which is repressed by LacI and induced by IPTG.

GroESL (BBa_K2032004)

Chaperone complex

GroESL Composite (BBa_K2032005)

Contains promoter, rbs, and terminator along with GroESL coding region

Fructose with RFP (BBa_K2032006)

Fructose construct with RFP reporter

Formate with RFP (BBa_K2032007)

This is the fructose methanol degradation pathway (BBa_K2032001) with an RFP marker (BBa_K801100) attached