Team:Imperial College London/Software Tool

From 2010.igem.org

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{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;width:300px;"|Select Protease
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;width:300px;"|Select Protease
-
|style="width:120px;"|
 
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Description
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Description
|-
|-
|align="center"|<html>
|align="center"|<html>
 +
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script>
 +
<script type="text/javascript">
 +
$(document).ready(function(){
 +
  var det = new Array();
 +
  det[1] = "This was our primary target. Read our wiki to find out more!";
 +
  det[2] = "Detecting HIV Protease with our system could be a quick, simple and cheap alternative way to diagnose HIV.";
 +
  det[3] = "Currently, a lumbar puncture is necessary to diagnose the devastating Chagas' disease. However, a simple blood test could test for a specific protease called cruzipain which is produced by the Trypanasoma cruzi parasite.";
 +
  det[4] = "Detecting this coagulation factor could be a way of determining if patients should be given warfarin or a low molecular weight heparin (LMWH) if they are displaying atrial fibrilation following a stroke. Factor X is the first member of the thrombin pathway which essentially leads to blood clotting.";
 +
  det[5] = "The complement system is part of the innate immune response to an acute infection. Detecting C3 convertase could give medical professionals a rapid method of seeing if a patient is fighting an acute infection.";
 +
  det[6] = "This protease is produced by the ''leishmania'' parasites which cause leishmaniasis, an NTD. Diagnosis currently takes at least 20 minutes and requires microscopy. Our kit would allow rapid detection with very simple apparatus. ";
 +
  det[7] = "This cysteine protease is made by the Tobacco Etch Virus (TEV). It is often used as a molecular biology tool as it is very well characterised and has a high degree of activity and specificity.";
 +
det[8] = "Caspases are used extensively in ''in vitro'' studies. They play a crucial role in apoptosis, including degrading genomic DNA and breaking down the cytoskeleton. Caspase 3 is an effector caspase and also plays a key role in the development of various human tissues.";
 +
det[9] = "Caspase 5 is an inflammatory caspase and is involved in cytokine maturation.";
 +
det[10] = "Caspase 9 is an initiator caspase and cleaves inactive pro-forms of effector caspases, thereby activating them";
 +
  var prefix = "GAATTCGCGGCCGCTTCTAG";
 +
  var promoter = "AATTTTGTCAAAATAATTTTATTGACAACGTCTTATTAACGTTGATATAATTTAAATTTTATTTGACAAAAATGGGCTCGTGTTGTACAATAAATGT";
 +
  var rbs = "TACTAGAGTCTTCAGAAAGGAGT";
 +
  var scar = "TACTAG";
 +
  var cwb = "ATGCGTTCTTATATAAAAGTCCTAACAATGTGTTTTCTGGGGCTCATACTTTTTGTGCCAACAGCTTTGGCCGATAACTCAGTGAAAAGAGTTGGGGGAAGCAATAGATACGGCACTGCTGTACAAATATCAAAGCAAATGTATTCAACAGCAAGTACAGCTGTAATTGTTGGTGGGAGTTCCTATGCAGATGCTATTTCAGCAGCACCTCTTGCTTACCAGAAGAATGCGCCATTGCTTTACACTAATTCTGATAAGCTTTCATATGAAACGAAAACAAGATTGAAAGAGATGCAGACTAAAAATGTAATTATTGTAGGCGGAACACCTGCTGTTTCTTCTAACACTGCTAACCAGATTAAAAGCTTGGGGATAAGTATTAAACGAATTGCAGGAAGCAACCGTTATGATACGGCTGCACGGGTGGCAAAAGCGATGGGTGCGACTTCAAAAGCTGTTATTTTGAACGGCTTCTTATATGCAGACGCTCCGGCCGTCATCCCTTATGCAGCGAAAAACGGGTATCCAATTCTTTTTACAAATAAAACATCTATAAATAGTGCGACTACGTCTGTGATAAAAGATAAGGGAATTTCGAGTACCGTTGTTGTAGGAGGCACTGGAAGTATCAGCAATACGGTATACAACAAGTTACCTTCTCCTACAAGAATTAGCGGTTCAAACAGATATGAGCTTGCTGCAAATATCGTACAAAAACTTAATTTATCAACAAGCACCGTATATGTAAGCAATGGATTCAGCTACCCTGACTCTATTGCAGGAGCTACACTGGCAGCTAAGAAGAAGCAATCTCTTATTCTTACAAATGGTGAAAATTTATCTACAGGAGCCCGTAAAATTATTGGAAGTAAAAACATGTCAAACTTTATGATTATCGGAAACACTCCTGCCGTAAGCACAAAGGTTGCTAATCAGCTAAAGAATCCAGTTGTA";
 +
  var linker = "AGCAGGGGTAGCAGGGCTGGTGGCGGTGGCGGTGGC";
 +
  var cleavage = new Array();
 +
  cleavage[1] = "AGCTGGCCTCTT";
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  cleavage[2] = "TCTCAAAACTACCCTATCGTTCAA";
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  cleavage[3] = "AAAAAAGTTAAAGCTAAAAAA";
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  cleavage[4] = "ATCGACGGACGT";
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  cleavage[5] = "TTATTATCTCGTTCTGAAGAAGAC";
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  cleavage[6] = "CTGATTGCGTATCTGAAAAAAGCGACC";
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  cleavage[7] = "GAAAACTTATACTTCCAAGGA";
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  cleavage[8] = "GACATGCAAGACGGA";
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  cleavage[9] = "TTAGAACATGACGGA";
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  cleavage[10] = "TTAGAACATGACGGA";
 +
  var aip = "GAAATGCGCCTTAGCAAATTCTTCAGGGACTTCATTCTTCAAAGGAAAAAA";
 +
  var terminator = "TAATAA";
 +
  var suffix = "TACTAGTAGCGGCCGCTGCAG";
 +
 +
  $("#paraselect").change(function()
 +
  {
 +
    var message_index
 +
 +
    message_index = $("#paraselect").val();
 +
    $("#parades").empty();
 +
 +
    if (message_index > 0)
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  $("#parades").append(det[message_index]);
 +
  });
 +
 +
  $('#parago').click(function() {
 +
  var message_index
 +
  message_index = $("#paraselect").val();
 +
  $("#stem").empty();
 +
  $("#spre").text(prefix);
 +
  $("#spro").text(promoter);
 +
  $("#srbs").text(rbs);
 +
  $("#ssca").text(scar);
 +
  $("#scwb").text(cwb);
 +
  $("#slin").text(linker);
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  $("#scle").text(cleavage[message_index]);
 +
  $("#saip").text(aip);
 +
  $("#ster").text(terminator);
 +
  $("#ssuf").text(suffix);
 +
  });
 +
});
 +
</script>
<style type="text/css">
<style type="text/css">
  #paraselect {
  #paraselect {
   width:300px;
   width:300px;
-
   height:25px;
+
   height:28px;
   font-size:1.5em;
   font-size:1.5em;
   color:#555555;
   color:#555555;
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</style>
</style>
<select id="paraselect">
<select id="paraselect">
-
   <option value="schistosoma">Schistosoma Elastase</option>
+
   <option value="1">Schistosoma Elastase</option>
-
   <option value="hiv">HIV Protease</option>
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   <option value="2">HIV Protease</option>
-
   <option value="cruzipain">Cruzipain</option>
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   <option value="3">Cruzipain</option>
-
   <option value="factorxa">Factor Xa</option>
+
   <option value="4">Factor Xa</option>
-
   <option value="complementc1r">Complement - C1r</option>
+
   <option value="5">C3 Convertase</option>
 +
  <option value="6">Leishmanolysin</option>
 +
  <option value="7">TEV</option>
 +
  <option value="8">Caspase 3</option>
 +
  <option value="9">Caspase 5</option>
 +
  <option value="10">Caspase 9</option>
</select>
</select>
</html>
</html>
-
|<html>
+
|'''<html>
 +
<p id="parades">This was our primary target. Read our wiki to find out more!</p>
 +
</html>'''
 +
|-
 +
|align="center"|<html>
<style type="text/css">
<style type="text/css">
#parago {
#parago {
   width:120px;
   width:120px;
-
   height:25px;
+
   height:28px;
   font-size:1.5em;
   font-size:1.5em;
   color:#555555;
   color:#555555;
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<button type="button" id="parago">Generate!</button>
<button type="button" id="parago">Generate!</button>
</html>
</html>
-
|'''<html>
+
|
-
This was our primary target. Read our wiki to find out more!
+
-
</html>'''
+
|-
|-
|style="width:860px;background:#d5d5d5;border: solid 20px #d5d5d5;" colspan="3"|'''<html>
|style="width:860px;background:#d5d5d5;border: solid 20px #d5d5d5;" colspan="3"|'''<html>
-
Awaiting sequence generation...
+
<style type="text/css">
 +
#sequenceout {
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  width:820px;
 +
  word-wrap: break-word;}
 +
</style>
 +
<div id="sequenceout"><span id="stem">Awaiting sequence generation... </span><span id="spre" style="color:#F6CF39"></span><span id="spro" style="color:#FC8E42"></span><span id="srbs" style="color:#D34649"></span><span id="ssca" style="color:#A851AA"></span><span id="scwb" style="color:#AB6197"></span><span id="slin" style="color:#6273A8"></span><span id="scle" style="color:#6A9ECD"></span><span id="saip" style="color:#68904E"></span><span id="ster" style="color:#9DB742"></span><span id="ssuf" style="color:#F6CF39"></span></div>
</html>'''
</html>'''
|-
|-
-
|colspan="3"|
+
|
'''<span style="color:#F6CF39">Yellow</span> - Biobrick Prefix/Suffix'''
'''<span style="color:#F6CF39">Yellow</span> - Biobrick Prefix/Suffix'''
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'''<span style="color:#D34649">Red</span> - Ribosome Binding Site'''
'''<span style="color:#D34649">Red</span> - Ribosome Binding Site'''
 +
 +
'''<span style="color:#A851AA">Violet</span> - Scar'''
'''<span style="color:#AB6197">Purple</span> - Cell Wall Binding Domain'''
'''<span style="color:#AB6197">Purple</span> - Cell Wall Binding Domain'''
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'''<span style="color:#9DB742">Light Green</span> - Terminator'''
'''<span style="color:#9DB742">Light Green</span> - Terminator'''
 +
|[[Image:ICswticon.png|500px]]
 +
|}
 +
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|What else could we attach to a cell wall binding domain?
 +
|-
 +
|
 +
'''Attaching enzymes to the surface of ''B. subtilis'':'''
 +
 +
This could be used for a variety of applications including biofuel production. In order for ''B. subtilis'' to absorb lignocellulose material, and use it to produce biofuels, this material first needs to be broken down into monosaccharides. Lignocellulose breakdown enzymes, such as cellulase, hemicellulase, and ligninase. could therefore be attached to the surface of ''B. subtilis''.
 +
 +
 +
'''Flagellin: A Pathogen-associated molecular pattern (PAMP):'''
 +
 +
PAMPs are sensed by our innate immune system, specifically by pattern-recognition receptors (PRRs). PAMPs are often shared between many different classes of microbes, so they work well as a generic target for the immune system early on in an infection.
 +
 +
Flagellin is one such PAMP and is recognised by a PRR called Toll-like receptor 5 (TLR-5). Flagellin monomers make up flagella of many bacteria, giving them motility which often confers pathogenicity.
 +
 +
Engineering a flagellin monomer to be bound to the exterior of our organism could initiate an immune response, such as the production of the cytokine TNF (tumor necrosis factor) which could help inhibit viral replication in the body.
 +
 +
 +
'''Antigen presentation:'''
 +
 +
Antigen presentation is vital when initiating the adaptive immune response. There are many different proteins that we could engineer to be on the surface of our organism, in order for the organism to be phagocytosed by antigen presenting cells (APCs). These APCs would then display the antigen on their surface, held in place by a majorhistocompatability complex (MHC) Class II molecule.
 +
 +
Using this system one could initiate long-term immunity to a variety of pathogens.
 +
 +
 +
 +
'''DNA binding proteins:'''
 +
 +
This could be a very useful application for our system because it could be used to bind DNA in the extracellular environment. We could use DNA-binding domains (DBDs) from proteins like transcription factors to bind a specific DNA sequence.
 +
 +
 +
 +
'''Gene expression libraries:'''
 +
 +
By attaching each member of a protein library to the surface of a cell, it would be possible to screen for ligands to the proteins. This could be applied to the testing of ligand-binding of proteins after site-directed mutagenesis, allowing the identification of key amino acid residues for ligand binding.
|}
|}

Latest revision as of 03:57, 28 October 2010

Software Tool
We realised early on that our detection module could be designed with a sensitivity to different proteases. By changing the cleavage site the system can accept a wide variety of inputs. This tool is designed to facilitate a quick custom sequence generation of the entire surface protein construct.
Select Protease Description

This was our primary target. Read our wiki to find out more!
Awaiting sequence generation...

Yellow - Biobrick Prefix/Suffix

Orange - Promoter

Red - Ribosome Binding Site

Violet - Scar

Purple - Cell Wall Binding Domain

Dark Blue - Adjustable Linker

Light Blue - Protease Cleavage Site

Dark Green - Autoinducing Peptide

Light Green - Terminator

ICswticon.png
What else could we attach to a cell wall binding domain?

Attaching enzymes to the surface of B. subtilis:

This could be used for a variety of applications including biofuel production. In order for B. subtilis to absorb lignocellulose material, and use it to produce biofuels, this material first needs to be broken down into monosaccharides. Lignocellulose breakdown enzymes, such as cellulase, hemicellulase, and ligninase. could therefore be attached to the surface of B. subtilis.


Flagellin: A Pathogen-associated molecular pattern (PAMP):

PAMPs are sensed by our innate immune system, specifically by pattern-recognition receptors (PRRs). PAMPs are often shared between many different classes of microbes, so they work well as a generic target for the immune system early on in an infection.

Flagellin is one such PAMP and is recognised by a PRR called Toll-like receptor 5 (TLR-5). Flagellin monomers make up flagella of many bacteria, giving them motility which often confers pathogenicity.

Engineering a flagellin monomer to be bound to the exterior of our organism could initiate an immune response, such as the production of the cytokine TNF (tumor necrosis factor) which could help inhibit viral replication in the body.


Antigen presentation:

Antigen presentation is vital when initiating the adaptive immune response. There are many different proteins that we could engineer to be on the surface of our organism, in order for the organism to be phagocytosed by antigen presenting cells (APCs). These APCs would then display the antigen on their surface, held in place by a majorhistocompatability complex (MHC) Class II molecule.

Using this system one could initiate long-term immunity to a variety of pathogens.


DNA binding proteins:

This could be a very useful application for our system because it could be used to bind DNA in the extracellular environment. We could use DNA-binding domains (DBDs) from proteins like transcription factors to bind a specific DNA sequence.


Gene expression libraries:

By attaching each member of a protein library to the surface of a cell, it would be possible to screen for ligands to the proteins. This could be applied to the testing of ligand-binding of proteins after site-directed mutagenesis, allowing the identification of key amino acid residues for ligand binding.

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