Difference between revisions of "Team:iTesla-SoundBio/design"

 
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<center>
 
<center>
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<h1>Engineer the Future<br>
 
 
<script>
 
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<br>That Prosperity and Environmentalism Coexist</h1>
 
 
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<div class="sidenav">
 
<div class="sidenav">
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<a href="#graphicsum">Graphic Summary</a>
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<a href="#Phase1"><img src="https://static.igem.org/mediawiki/2018/d/d8/T--itesla-soundbio--phase1.png" alt="Phase 1" style="width:100%"></a>
 
<br>
 
<br>
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<a href="#constructdesign">Construct Design</a>
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<a href="#Phase2"><img src="https://static.igem.org/mediawiki/2018/2/21/T--itesla-soundbio--phase2.png" alt="Phase 2" style="width:100%"></a>
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<br>
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<a href="#Biobrick">Biobrick: <br>Factor C gene in E. coli pSB1C3</a>
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<br>
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<a href="#Integration">Integration: <br>Factor C in B.Subtilis Chromosomal DNA</a>
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<br>
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<a href="#Expression">Expression: <br>Factor C protein verification</a>
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<br>
 
<br>
 
</div>
 
</div>
  
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<center><h2 style="margin-left: 200px;" id="lesserTitle">Introduction</h2></center>
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<center><p style="font-size: 25px; margin-left: 200px; margin-right: 40px;">The goal of our project was to recreate the first protein involved in the LAL cascade (Factor C) by using Bacillus subtilis, a gram-positive bacteria, to synthesize the protein of interest. Additionally, we wanted to find an alternative method to detect cleaved factor C. When detecting cleaved Factor C, our goal was to cut down on intermediate steps in the LAL cascade (factor B, clotting enzyme) and skip directly to an engineered reaction that is easily detectable, such as a visual assay. To briefly summarize our project design, it can be explained in two phases.</p></center>
  
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<div id="Phase1">
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<h2 style="margin-left: 200px;" id="lesserTitle">Phase 1</h2>
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<p><b>Overview:</b> Produce and purify Factor C from <em>Bacillus subtilis</em></p>
 +
<ol>
 +
<li>Acquire the amino acid sequence from factor C and translate it into codons. Codon optimizes it for <em>Bacillus subtilis</em></li>
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<ul style="font-size: 20px;">
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<li >After acquiring the full sequence for the Factor C gene, split into two fragments to meet IDT&rsquo;s limits on sequence length. The end of the first fragment and the end of the second fragment had a shared cut site of the BglII restriction enzyme.</li>
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</ul>
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</li>
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<li>Digest Fragment I and psb1C3 with EcoRI and PstI</li>
 +
<li>Ligate Fragment I and psb1C3</li>
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<li>Digest the Fragment I and psb1C3 construct with BglII and PstI</li>
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<li >Digest Fragment II with BglII and PstI </li>
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<li >Ligate Fragment II into the Fragment I and psb1C3 construct</li>
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<li >Transform into E. coli</li>
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<p>---------------- At this point we have a plasmid with the entire factor C gene in E. coli-----------------</p>
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<li>Miniprep E. coli for the Factor C gene</li>
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<li >Digest with BamHI and SacI to isolate factor C</li>
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<ul style="font-size: 20px;">
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<li >Gel extraction and purification of the factor C band &nbsp;</li>
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</ul>
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<li >Digest pAX01 with BamHI and SacII</li>
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<li>Ligate pAX01 and Factor C</li>
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<li>Transform into <em>B. subtilis 168</em></li>
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<li>His-tagged Protein Purification of factor C</li>
  
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</ol>
  
  
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<div class="main">
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<p>
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<div id="graphicsum">
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<b>Verification: </b>
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<center>
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western blot with Bacillus subtilis that has the pax0I Factor C insert. One will have Bacillus that has been induced by xylose and the other will not be induced.
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<img src="" alt="This is a graphic summary of our project", width=80%>
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</p>
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</center>
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<p><br /><br /></p>
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</div>
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<img style="width: 65%; margin-left: 330px" src="https://static.igem.org/mediawiki/2018/e/e5/T--iTesla-SoundBio--DesignP1.png">
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</br></br>
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<div class="horizontalline">
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<div id="Phase2">
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<h2 style="margin-left: 200px;" id="lesserTitle">Phase 2</id></h2>
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<div id="constructdesign">
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<p>
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<p class="text">
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<b>Objective:</b>  
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<u>Construct design: </u><br>
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Find an alternative procedure using the Factor C we produced to replace the LAL assay that is as effective, cheaper, and does not need horseshoe crab blood compared to industry options for endotoxin tests.
−
The goal for our project is to insert Factor C gene into the chromosomal DNA of B. Subtilis, and have it express this Factor C protein. At the same time, to fulfill iGEM competition requirements, we also need to insert Factor C gene into E.coli plasmid pSB1C3 and submit it as a biobrick for iGEM competition.
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−
<br>
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We decided to insert Factor C gene into E.coli plasmid pSB1C3 first; then, integrate Factor C gene into the chromosomal DNA of B. Subtilis using pAX01 integration factor (a B. Subtilis specific plasmid).
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<div id="Biobrick">
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<center><img src="https://static.igem.org/mediawiki/2018/c/c6/T--itesla-soundbio--twofragments.png", width=80%></center>
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<p class="text">
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<u>Biobrick: </u><br>
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Factor C in E.coli pSB1C3
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We couldn't order Factor C from factory because it is too large (about 3000 base pairs https://benchling.com/itesla-soundbio/f/gPUeHZXf-drafts/seq-Jq87odxl-psb1c3factor-c/edit), so we order it as two fragments, FC1 and FC2.
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First, insert FC1 into pSB1C3. Then, insert FC2 into the plasmid, too. This pSB1C3 with full Factor C will be submitted for biobrick.
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</p>
 
</p>
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</div>
 
  
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<p>
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<div class="horizontalline">
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<b>Option I:</b>  
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</div>
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We could synthesize all the relevant proteins for the natural coagulation cascade
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<div id="Integration">
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<p class="text">
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<u>Integration: </u><br>
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Factor C in B. Subtilis Chromosomal DNA.
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We then digest full Factor C from Biobrick, and insert it into pAX01, a B. Subtilis integration factor. Then, we transform B. Subtilis with pAX01. After plasmid pAX01 enters B. Subtilis, there is a chance that it will integrate into chromosomal DNA.
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</p>
 
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<div id="expression">
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<li>Here is how it could look in terms of design:</li>
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<p class="text">
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<ul>
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<u>Expression</u>
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<li>Since it is not Factor C we do not have to transfer it to Bacillus subtilis</li>
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Factor C protein extract: <br>
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<li>1) Get the DNA sequence and add a His-tag for Factor B,</li>
−
B. Subtilis are used to express Factor C because they are gram positive and will not damage the Factor C protein. In contrast, E. coli is gram negative and will cleave FC protein. After B. Subtilis express and produce Factor C, we can then extract Factor C protein for further experiment.   
+
<li>2) DIgest with enzymes so that we can ligate it into psb1C3</li>
 +
<li>3) Transform into E. coli</li>
 +
<li>4) His-tagged Protein Purification of factor C</li>
 +
<li>5) repeat with clotting enzyme, and substrate</li>
 +
<li>6) Once all the other compounds have been isolated, we can mix them with factor C</li>
 +
<ul>
 +
<li>Pros: Should work, in theory</li>
 +
<li>Cons: Almost certainly difficult/expensive</li>
 +
</ul>
 +
</ul>
 +
</ul>
 
</p>
 
</p>
 
</div>
 
</div>
  
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Latest revision as of 14:15, 16 October 2018

Safety Protocols
InterLab
Team Members Attributions
Human Practices Collaborations
Project Description Design Results Biobrick Parts Lab Notebook
iT-SB 2018 iT-SB 2017

Phase 1
Phase 2

Introduction

The goal of our project was to recreate the first protein involved in the LAL cascade (Factor C) by using Bacillus subtilis, a gram-positive bacteria, to synthesize the protein of interest. Additionally, we wanted to find an alternative method to detect cleaved factor C. When detecting cleaved Factor C, our goal was to cut down on intermediate steps in the LAL cascade (factor B, clotting enzyme) and skip directly to an engineered reaction that is easily detectable, such as a visual assay. To briefly summarize our project design, it can be explained in two phases.

Phase 1

Overview: Produce and purify Factor C from Bacillus subtilis

  1. Acquire the amino acid sequence from factor C and translate it into codons. Codon optimizes it for Bacillus subtilis
    • After acquiring the full sequence for the Factor C gene, split into two fragments to meet IDT’s limits on sequence length. The end of the first fragment and the end of the second fragment had a shared cut site of the BglII restriction enzyme.
  2. Digest Fragment I and psb1C3 with EcoRI and PstI
  3. Ligate Fragment I and psb1C3
  4. Digest the Fragment I and psb1C3 construct with BglII and PstI
  5. Digest Fragment II with BglII and PstI
  6. Ligate Fragment II into the Fragment I and psb1C3 construct
  7. Transform into E. coli

    8. ---------------- At this point we have a plasmid with the entire factor C gene in E. coli-----------------

  8. Miniprep E. coli for the Factor C gene
  9. Digest with BamHI and SacI to isolate factor C
    • Gel extraction and purification of the factor C band  
  10. Digest pAX01 with BamHI and SacII
  11. Ligate pAX01 and Factor C
  12. Transform into B. subtilis 168
  13. His-tagged Protein Purification of factor C

Verification: western blot with Bacillus subtilis that has the pax0I Factor C insert. One will have Bacillus that has been induced by xylose and the other will not be induced.





Phase 2

Objective: Find an alternative procedure using the Factor C we produced to replace the LAL assay that is as effective, cheaper, and does not need horseshoe crab blood compared to industry options for endotoxin tests.

Option I: We could synthesize all the relevant proteins for the natural coagulation cascade

  • Here is how it could look in terms of design:
    • Since it is not Factor C we do not have to transfer it to Bacillus subtilis
    • 1) Get the DNA sequence and add a His-tag for Factor B,
    • 2) DIgest with enzymes so that we can ligate it into psb1C3
    • 3) Transform into E. coli
    • 4) His-tagged Protein Purification of factor C
    • 5) repeat with clotting enzyme, and substrate
    • 6) Once all the other compounds have been isolated, we can mix them with factor C
      • Pros: Should work, in theory
      • Cons: Almost certainly difficult/expensive