Difference between revisions of "Team:UofC Calgary/Composite Part"

 
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<a href="https://2016.igem.org/Team:UofC_Calgary/Parts">Basic Parts</a>
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<a href="https://2016.igem.org/Team:UofC_Calgary/Parts">Parts Registry</a>
 
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             <img alt="" src="https://static.igem.org/mediawiki/2016/f/f5/T--UofC_Calgary--compositepartsbanner.jpg" data-bgposition="center center" data-bgfit="cover" data-bgrepeat="no-repeat" style="width: 100%; height: 100%;">
 
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                     <p style="color:#FFFFFF;">The backbone of the U of Calgary project are the composite parts. The composite parts are an amalgamation of different registry parts which act together to provide a novel function. The composite parts designed for this project are based around the Bowman-Birk Protease inhibitor (BBI) and the <a href="http://parts.igem.org/Part:BBa_K1444018">ComK part</a> that was submitted to the registry by the 2014 Calgary team.</p>
+
                     <p style="color:#FFFFFF;">The backbone of the U of Calgary project are the composite parts. Our composite parts are an amalgamation of different registry parts which act together to provide a novel function. The composite parts designed for this project are based around the modified Bowman-Birk Protease Inhibitor (mBBI) and the competency-inducing <a href="http://parts.igem.org/Part:BBa_K1444018"><i>ComK</i> part</a> that was submitted to the registry by the <a href="https://2014.igem.org/Team:Calgary/Notebook/Parts">2014 UofC_Calgary</a> team.</p><br>
  
<p style="color:#FFFFFF;">A variety of different parts were used across all of the BBI composite constructs. We used Pveg, a constitutive promoter, and double terminators to book end the rest of the internal genes. To improve secretion of BBI from <i>B. subtilis</i>, we added a secretion tag as well as a transdermal (TD1) tag. The secretion tag is a localization tag for the Sec pathway in <i>B. subtilis</i> <Ruan et al., 2013). TD1 is a part developed by a previous iGEM team <a href="http://parts.igem.org/Part:BBa_E0040:Experience> (USTC_CHINA 2013)</a> which is designed to allow for the trafficking of peptides across the skin.</p>
+
<p style="color:#FFFFFF;">A variety of different parts were used across all of the mBBI composite constructs. We used Pveg, a constitutive <i>E. coli</i> and <i>B. subtilis</i> promoter, and double terminators to book-end internal genes upon chromosomal integration. To improve secretion and transdermal delivery of mBBI from <i>B. subtilis</i>, we added a secretion (sec) tag as well as a transdermal (TD1) tag. The secretion tag is a localization tag for the Sec pathway in <i>B. subtilis</i> (Fu <i>et al.,</i> 2007). <a href="http://parts.igem.org/Part:BBa_E0040:Experience">TD1</a> is a part previously developed by the <a href="https://2013.igem.org/Team:USTC_CHINA">USTC_China 2013</a> iGEM team, and it is designed to allow for the trafficking of peptides across the skin.</p><br>
 
   
 
   
<p style="color:#FFFFFF;">The second major composite part was the ComK construct. We modified the original ComK sequence, which was first submitted by the 2014 Calgary Team, to remove unwanted restriction sites from within Comk itself. The ComK gene and xylose inducible promoters were then flanked with sequences homologous to AmyE in <i>B. subtilis</i>. The flanking sequences were added to allow for the integration of a controllable ComK into the <i>B. subtilis</i> genome. The integration of a xylose inducible ComK is to make the transformation of <i>B. subtilis</i> less time and resource intensive. </p>
+
<p style="color:#FFFFFF;">The second major composite part was the <i>ComK</i> construct. ComK is a transcription factor and master regulator for competency in <i>B. subtilis</i>. We modified the original <a href="http://parts.igem.org/Part:BBa_K1444018"><i>ComK</i></a> sequence, which was first submitted by the <a href="https://2014.igem.org/Team:Calgary/Notebook/Parts">2014 UofC_Calgary</a> team, to remove unwanted restriction sites from within the <i>ComK</i> gene itself. The <i>ComK</i> gene and xylose-inducible promoter were then flanked with sequences homologous to the <i>amyE</i> locus in <i>B. subtilis</i>, a non-essential gene responsible for the degradation of starch. These flanking sequences were added to allow for the integration of a controllable ComK into the <i>B. subtilis</i> genome. The integration of a xylose-inducible ComK allows for the transformation of <i>B. subtilis</i> to be more time-efficient and less resource-intensive. </p><br>
<p style="color:#FFFFFF;">Our last construct does not fall into either of the two previous categories. The construct is an integration cassette which is a mix of both the BBI and Comk constructs. The cassette contains Thrc integration sequences which flank the sec and TD1 tag. However the BBI sequence is replaced with the sequences for the BamHI and XmaI restriction enzymes. Addition of the restriction sites is to make a platform for future biotherapeutics that could be mass produced in bacteria, a mini-pharmacy.
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 +
<p style="color:#FFFFFF;">Our last construct does not fall into either of the two previous categories. The construct is an integration cassette, which is a mix of both the mBBI and <i>Comk</i> constructs. Our cassette contains <i>thrC</i> integration sequences which flank the sec and TD1 tag. However, the mBBI sequence is replaced with the restriction sites for BamHI and XmaI restriction enzymes. Addition of the restriction sites allows for directional cloning of any desired insert, allowing <i>B. subtilis</i> to be used as a platform for future biotherapeutics, creating a portable mini-pharmacy.
 
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               <div class="row">
 
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                             <div class="col-sm-3" style="padding-left:40px">
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                             <div class="col-sm-3" style="padding-left:0px">
 
                                   <h3 class="human-practices-div-2">BBa_K2008001
 
                                   <h3 class="human-practices-div-2">BBa_K2008001
 
   
 
   
 
  </h3>
 
  </h3>
  <p class="human-practices-div-2"> <em>E. coli</em> – GFP-BBI 5 <br>BBI with N-terminal KSCI solubility tag fused to GFP <br><a href="http://parts.igem.org/Part:BBa_K2008001">Registry Link</a>
+
  <p class="human-practices-div-2">BBI with N-terminal KSCI solubility tag fused to GFP for expression in <em>E. coli</em></p>
</p>
+
 
 +
<ul><li><p class="human-practices-div-2">Sequence-confirmed</p></li><li><p class="human-practices-div-2">Submitted to the registry</p></li><li><p class="human-practices-div-2"><a href="http://parts.igem.org/Part:BBa_K2008001">Registry Link</a></p></li></ul>
 
                             </div>
 
                             </div>
                             <div class="col-sm-3" style="padding-left:28px; padding-right:28px">  
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                             <div class="col-sm-3" style="padding-left:7px; padding-right:7px">  
 
                                   <h3 class="human-practices-div-2">BBa_K2008002
 
                                   <h3 class="human-practices-div-2">BBa_K2008002
 
</h3>
 
</h3>
  <p class="human-practices-div-2"> <em>B. subtilis</em>– pVeg -> sec-> TD1-> BBI-GFP (BBI 1 GFP w/o) <br><a href="http://parts.igem.org/Part:BBa_K2008002">Registry Link</a>
+
  <p class="human-practices-div-2">BBI with N-terminal sec and TD1 tags fused to GFP and under the pVeg promoter for expression in <em>B. subtilis</em></p><ul><li><p class="human-practices-div-2">Sequence-confirmed</p></li><li><p class="human-practices-div-2">Submitted to the registry</p></li><li><p class="human-practices-div-2"><a href="http://parts.igem.org/Part:BBa_K2008002">Registry Link</a></p></li></ul>           
</p>              
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                             </div>
<div class="col-sm-3" style="padding-left:28px; padding-right:28px">  
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<div class="col-sm-3" style="padding-left:7px; padding-right:7px">  
 
                                   <h3 class="human-practices-div-2">BBa_K2008003  
 
                                   <h3 class="human-practices-div-2">BBa_K2008003  
 
</h3>
 
</h3>
  <p class="human-practices-div-2"> <em>B. subtilis</em> – pVeg->sec-TD1-KSCI-BBI-GFP (BBI 5 GFP w/o)<br><a href="http://parts.igem.org/Part:BBa_K2008003">Registry Link</a>  
+
  <p class="human-practices-div-2">BBI with N-terminal KSCI solubility, sec, and TD1 tags fused to GFP and under the pVeg promoter for expression in <em>B. subtilis</em></p><ul><li><p class="human-practices-div-2">Sequence-confirmed</p></li><li><p class="human-practices-div-2">Submitted to the registry</p></li><li><p class="human-practices-div-2"><a href="http://parts.igem.org/Part:BBa_K2008003">Registry Link</a></p></li></ul>               
</p>               
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                             </div>
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                             <div class="col-sm-3" style="padding-right:0px">     
 
                                   <h3 class="human-practices-div-2">BBa_K2008004
 
                                   <h3 class="human-practices-div-2">BBa_K2008004
 
</h3>
 
</h3>
  <p class="human-practices-div-2"><em>B. subtilis</em> – secretion tag, transdermal tag, and BBI without internal restriction sites (BBI 1 w/o)<br><a href="http://parts.igem.org/Part:BBa_K2008004">Registry Link</a>
+
  <p class="human-practices-div-2">BBI with N-terminal sec and TD1 tags under the pVeg promoter for expression in <em>B. subtilis</em></p><ul><li><p class="human-practices-div-2">Sequence-confirmed</p></li><li><p class="human-practices-div-2">Submitted to the registry</p></li><li><p class="human-practices-div-2"><a href="http://parts.igem.org/Part:BBa_K2008004">Registry Link</a></p></li></ul>
 
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</p>          
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  <div class="row">
 
  <div class="row">
                             <div class="col-sm-3" style="padding-left:40px">
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          <div class="col-sm-3" style="padding-left:0px">
                                   <h3 class="human-practices-div-2">BBa_K2008005
+
            <img src="https://static.igem.org/mediawiki/2016/8/8d/T--UofC_Calgary--BBa_K2008001img.png" width="300px" height="88"px/>
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          </div>
 +
          <div class="col-sm-3" style="padding-left:7px; padding-right:7px">
 +
            <img src="https://static.igem.org/mediawiki/2016/4/48/T--UofC_Calgary--BBa_K2008002img.png" width="300px" height="88"px/>
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          </div>
 +
          <div class="col-sm-3" style="padding-left:7px; padding-right:7px">
 +
            <img src="https://static.igem.org/mediawiki/2016/0/08/T--UofC_Calgary--BBa_K2008003img.png" width="300px" height="88"px/>
 +
          </div>
 +
          <div class="col-sm-3" style="padding-right:0px">
 +
            <img src="https://static.igem.org/mediawiki/2016/e/e1/T--UofC_Calgary--BBa_K2008004img.png" width="300px" height="88"px/>     
 +
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<div class="row">
 +
                             <div class="col-sm-3" style="padding-left:0px">
 +
                                   <h3 class="human-practices-div-2"><font color=#A2310F>♥</font>BBa_K2008005
 
  </h3>
 
  </h3>
  <p class="human-practices-div-2"> <em>B. subtilis</em> – secretion tag, transdermal tag, solubility tag and BBI without internal restriction sites (BBI 5 w/o)<br><a href="http://parts.igem.org/Part:BBa_K2008005">Registry Link</a>
+
  <p class="human-practices-div-2">BBI with KSCI solubility, sec, and TD1 tags under the pVeg promoter for expression in <em>B. subtilis</em></p><ul><li><p class="human-practices-div-2">Sequence-confirmed</p></li><li><p class="human-practices-div-2">Characterized</p></li><li><p class="human-practices-div-2">Submitted to the registry</p></li><li><p class="human-practices-div-2"><a href="http://parts.igem.org/Part:BBa_K2008005">Registry Link</a></p></li></ul>
</p>
+
 
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                             </div>
                             <div class="col-sm-3" style="padding-left:28px; padding-right:28px">  
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                             <div class="col-sm-3" style="padding-left:7px; padding-right:7px">  
 
                                   <h3 class="human-practices-div-2">BBa_K2008006
 
                                   <h3 class="human-practices-div-2">BBa_K2008006
 
   
 
   
 
</h3>
 
</h3>
  <p class="human-practices-div-2"> <em>B. subtilis</em> - Improved ComK: removal of internal restriction sites <br><a href="http://parts.igem.org/Part:BBa_K2008006">Registry Link</a>
+
  <p class="human-practices-div-2">Improved version of <i>comK</i> gene with removal of internal restriction sites</p><ul><li><p class="human-practices-div-2">Sequence-confirmed</p></li><li><p class="human-practices-div-2">Submitted to the registry</p></li><li><p class="human-practices-div-2"><a href="http://parts.igem.org/Part:BBa_K2008006">Registry Link</a></p></li></ul>               
</p>               
+
 
                             </div>
 
                             </div>
<div class="col-sm-3" style="padding-left:28px; padding-right:28px">  
+
<div class="col-sm-3" style="padding-left:7px; padding-right:7px">  
                                   <h3 class="human-practices-div-2">BBa_K2008007  
+
                                   <h3 class="human-practices-div-2"><font color=#A2310F>♥</font>BBa_K2008007  
 
</h3>
 
</h3>
  <p class="human-practices-div-2"> <em>B. subtilis</em> - Improved ComK: addition of AmyE homology <br><a href="http://parts.igem.org/Part:BBa_K2008007">Registry Link</a>  
+
  <p class="human-practices-div-2">Improved version of <i>comK</i> with addition of flanking regions of <i>amyE</i> homology</p><ul><li><p class="human-practices-div-2">Sequence-confirmed</p></li><li><p class="human-practices-div-2">Submitted to the registry</p></li><li><p class="human-practices-div-2"><a href="http://parts.igem.org/Part:BBa_K2008007">Registry Link</a></p></li></ul> 
</p>              
+
           
 
                             </div>
 
                             </div>
                             <div class="col-sm-3" style="padding-right:40px">     
+
                             <div class="col-sm-3" style="padding-right:0px">     
                                   <h3 class="human-practices-div-2">BBa_K2008008  
+
                                   <h3 class="human-practices-div-2"><font color=#A2310F>♥</font>BBa_K2008008  
 
</h3>
 
</h3>
  <p class="human-practices-div-2"> <em>B. subtilis</em> - Bacillus subtilis modular secretion platform with ThrC homology for chromosomal integration <br><a href="http://parts.igem.org/Part:BBa_K2008008">Registry Link</a>
+
  <p class="human-practices-div-2">Modular secretion platform containing sec and TD1 tags with flanking regions of <i>thrC</i> homology for chromosomal integration in <i>B. subtilis</i></p><ul><li><p class="human-practices-div-2">Sequence-confirmed</p></li><li><p class="human-practices-div-2">Submitted to the registry</p></li><li><p class="human-practices-div-2"><a href="http://parts.igem.org/Part:BBa_K2008008">Registry Link</a></p></li></ul>         
</p>          
+
 
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                             </div>
 
                 </div>
 
                 </div>
+
  <div class="row">
 +
          <div class="col-sm-3" style="padding-left:0px">
 +
            <img src="https://static.igem.org/mediawiki/2016/f/fb/T--UofC_Calgary--BBa_K2008005img.png" width="300px" height="88"px/>
 +
          </div>
 +
          <div class="col-sm-3" style="padding-left:7px; padding-right:7px">
 +
          <img src="https://static.igem.org/mediawiki/2016/b/b8/T--UofC_Calgary--BBa_K2008006img.png" width="300px" height="88"px/>
 +
          </div>
 +
          <div class="col-sm-3" style="padding-left:7px; padding-right:7px">
 +
            <img src="https://static.igem.org/mediawiki/2016/d/d1/T--UofC_Calgary--BBa_K2008007img.png" width="300px" height="88"px/>
 +
          </div>
 +
          <div class="col-sm-3" style="padding-right:0px">
 +
            <img src="https://static.igem.org/mediawiki/2016/e/e5/T--UofC_Calgary--BBa_K2008008img.png" width="300px" height="88"px/>     
 +
          </div>
 +
        </div>
 +
<br><br><p style="color:#FFFFFF;">References</p><br>
 +
<p style="color:#FFFFFF;">Fu, L.L., Xu, Z.R., Li, W.F., Shuai, J.B., Lu, P., and Hu, C.X. (2007). Protein secretion pathways in <i>Bacillus subtilis</i>: implication for optimization of heterlogous protein secretion. Biotechnology Advances <i>25</i>(1): 1-12.</p>
 +
 
 
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Latest revision as of 01:12, 20 October 2016

iGEM Calgary 2016

Composite Parts

The backbone of the U of Calgary project are the composite parts. Our composite parts are an amalgamation of different registry parts which act together to provide a novel function. The composite parts designed for this project are based around the modified Bowman-Birk Protease Inhibitor (mBBI) and the competency-inducing ComK part that was submitted to the registry by the 2014 UofC_Calgary team.


A variety of different parts were used across all of the mBBI composite constructs. We used Pveg, a constitutive E. coli and B. subtilis promoter, and double terminators to book-end internal genes upon chromosomal integration. To improve secretion and transdermal delivery of mBBI from B. subtilis, we added a secretion (sec) tag as well as a transdermal (TD1) tag. The secretion tag is a localization tag for the Sec pathway in B. subtilis (Fu et al., 2007). TD1 is a part previously developed by the USTC_China 2013 iGEM team, and it is designed to allow for the trafficking of peptides across the skin.


The second major composite part was the ComK construct. ComK is a transcription factor and master regulator for competency in B. subtilis. We modified the original ComK sequence, which was first submitted by the 2014 UofC_Calgary team, to remove unwanted restriction sites from within the ComK gene itself. The ComK gene and xylose-inducible promoter were then flanked with sequences homologous to the amyE locus in B. subtilis, a non-essential gene responsible for the degradation of starch. These flanking sequences were added to allow for the integration of a controllable ComK into the B. subtilis genome. The integration of a xylose-inducible ComK allows for the transformation of B. subtilis to be more time-efficient and less resource-intensive.


Our last construct does not fall into either of the two previous categories. The construct is an integration cassette, which is a mix of both the mBBI and Comk constructs. Our cassette contains thrC integration sequences which flank the sec and TD1 tag. However, the mBBI sequence is replaced with the restriction sites for BamHI and XmaI restriction enzymes. Addition of the restriction sites allows for directional cloning of any desired insert, allowing B. subtilis to be used as a platform for future biotherapeutics, creating a portable mini-pharmacy.

Composite Parts

BBa_K2008001

BBI with N-terminal KSCI solubility tag fused to GFP for expression in E. coli

BBa_K2008002

BBI with N-terminal sec and TD1 tags fused to GFP and under the pVeg promoter for expression in B. subtilis

BBa_K2008003

BBI with N-terminal KSCI solubility, sec, and TD1 tags fused to GFP and under the pVeg promoter for expression in B. subtilis

BBa_K2008004

BBI with N-terminal sec and TD1 tags under the pVeg promoter for expression in B. subtilis

BBa_K2008005

BBI with KSCI solubility, sec, and TD1 tags under the pVeg promoter for expression in B. subtilis

  • Sequence-confirmed

  • Characterized

  • Submitted to the registry

  • Registry Link

BBa_K2008006

Improved version of comK gene with removal of internal restriction sites

BBa_K2008007

Improved version of comK with addition of flanking regions of amyE homology

BBa_K2008008

Modular secretion platform containing sec and TD1 tags with flanking regions of thrC homology for chromosomal integration in B. subtilis



References


Fu, L.L., Xu, Z.R., Li, W.F., Shuai, J.B., Lu, P., and Hu, C.X. (2007). Protein secretion pathways in Bacillus subtilis: implication for optimization of heterlogous protein secretion. Biotechnology Advances 25(1): 1-12.

iGEM

iGEM is an international competition promoting synthetic biology as a means to solve social, economic and humanitarian problems around the globe. The iGEM Jamboree is held in Boston annually. In 2016, over 300 teams are competing against each other.

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