Difference between revisions of "Team:Sydney Australia/Improve"

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<div class="para_container">
 
<div class="para_container">
 
<h2>Old Parts</h2>
 
<h2>Old Parts</h2>
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<div class = "divider1"></div>
 
<br>
 
<br>
 
<h4>Parts <a href="http://parts.igem.org/Part:BBa_M39904">BBa_M39904</a> and <a href="http://parts.igem.org/Part:BBa_M1877">BBa_M1877</a> were pre-existing registry parts described as ‘Human Insulin cDNA’ and ‘human insulin’ respectively. </h4>
 
<h4>Parts <a href="http://parts.igem.org/Part:BBa_M39904">BBa_M39904</a> and <a href="http://parts.igem.org/Part:BBa_M1877">BBa_M1877</a> were pre-existing registry parts described as ‘Human Insulin cDNA’ and ‘human insulin’ respectively. </h4>
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</div>
  
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<div class="para_container">
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<h2>Our Improved Parts</h2>
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<h2>What we Improved</h2>
 
<div class = "divider3"></div>
 
<div class = "divider3"></div>
 
<br>
 
<br>
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<div class="col-xs-4">
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<h4>Part BBa_M39904 (Figure 1) consisted of 345 base pairs and was submitted as a basic part, and showed no differentiation between A and B chains, or if there was a C-peptide present.</h4>
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<br>
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<h4>Part BBa_M1877 (Figure 2) consisted of only 12bp and so was clearly unfinished.</h4>
 
</div>
 
</div>
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<div class="col-xs-8">
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<figure><img class="crispy img-responsive" src="https://static.igem.org/mediawiki/2017/5/58/T--Sydney_Australia--improve_fig1.png" style="width:100%;">
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<figcaption>Figure 1: BBa_M39904 sequence as displayed in registry. The part is not compatible with many biobricks, and has no differentiation between A and B peptides.</figcaption>
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</figure>
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<br>
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<figure><img class="crispy img-responsive" src="https://static.igem.org/mediawiki/2017/3/36/T--Sydney_Australia--improve_fig2.png" style="width:100%;">
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<figcaption>Figure 2: BBa_M1877 sequence as displayed in registry. As human insulin is clearly more than 12bp this sequence was deemed incomplete.</figcaption>
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</figure>
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<div class="para_container">
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<h2>Our Improved Parts</h2>
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<div class = "divider2"></div>
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<br>
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<h4> We submitted <a href="http://parts.igem.org/Part:BBa_K2417000">BBa_K2417000</a>, <a href="http://parts.igem.org/Part:BBa_K2417001">BBa_K2417001</a> and <a href="http://parts.igem.org/Part:BBa_K2417006">BBa_K2417006</a> as improvements upon these (Figure 3).</h4>
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<figure><img class="crispy img-responsive img-center center-block" src="https://static.igem.org/mediawiki/2017/b/be/T--Sydney_Australia--improve_fig3.png" style="width:80%;">
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<figcaption>Figure 3: BBa_K2417000 – Ecotin-Proinsulin, BBa_K2417001 – Cytoplasmic-Proinsulin, BBa_K241006 - Proinsulin – are all improvement parts because they are much more clearly characterized and contains features for more efficient production and purification.</figcaption>
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</div>
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<div class="row">
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<h2>Summary</h2>
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<br>
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<div style="overflow-x:auto;">
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<table style="width:80%;margin:auto;">
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  <tr>
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    <th >Previous Part</th>
 +
    <th align="left">Characteristic</th>
 +
    <th align="left">How did we improve this?</th>
 +
    <th align="left">Which parts did we submit with this improvement?</th> 
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</tr>
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  <tr>
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    <td style="padding:1%">BBa_M399<a style="color:#94bdc1"04
 +
BBa_M1877</td>
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    <td>Basic part</td>
 +
    <td>We submitted composite parts that clearly distinguish between A and B chains, and the presence of a C peptide. The use of a composite part submission system allows users to access the basic parts they consist of for more detailed information.</td>
 +
    <td>BBa_K2417000
 +
BBa_K2417001
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</td>
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 +
  </tr>
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  <tr>
 +
    <td></td>
 +
    <td>Lacking complete sequence</td>
 +
    <td >Our parts are sequence verified, confirmed as complete and are based off NCIB sequences of human proinsulin.</td>
 +
        <td>BBa_K2417000
 +
BBa_K2417001
 +
BBa_K2417006
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</td>
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  </tr>
 +
    <tr>
 +
    <td></td>
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    <td>Lacking ribosomal binding site</td>
 +
    <td >Our parts contain an efficient extended ribosomal binding site (part BBa_K2417009) which aids successful expression.</td>
 +
    <td>BBa_K2417000
 +
BBa_K2417001
 +
BBa_K2417006
 +
 +
</td>
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  </tr>
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    </tr>
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    <tr>
 +
    <td></td>
 +
    <td>Lack of purification tags</td>
 +
    <td >We added an N-terminal His-tag (BBa_K2417008) to all of our proinsulin constructs to aid ease of purification. A His-tag allows purification using an affinity chromatography column.</td>
 +
        <td>BBa_K2417000
 +
BBa_K2417001
 +
BBa_K2417006
 +
 +
</td>
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  </tr>
 +
 +
  </tr>
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    <tr>
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    <td></td>
 +
    <td>No secretion tags</td>
 +
    <td >For some of our constructs we added tags to induce secretion of the protein to different parts of the cell. The addition of Ecotin to the N-terminus of proinsulin has been shown to induce its transport into the periplasm of E. coli, an oxidative environment favourable for the folding of disulphide bonds.</td>
 +
        <td>BBa_K2417000
 +
 +
</td>
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  </tr>
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 +
  </tr>
 +
    <tr>
 +
    <td>BBa_M39904</td>
 +
    <td>Biobrick incompatible</td>
 +
    <td >The construct entered into the database is only compatible with a single biobrick. All of our parts are compatible with all biobrick constructs.</td>
 +
    <td>BBa_K2417000
 +
BBa_K2417001
 +
BBa_K2417006
 +
 +
</td>
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  </tr>
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Revision as of 06:10, 31 October 2017

Old Parts


Parts BBa_M39904 and BBa_M1877 were pre-existing registry parts described as ‘Human Insulin cDNA’ and ‘human insulin’ respectively.

What we Improved


Part BBa_M39904 (Figure 1) consisted of 345 base pairs and was submitted as a basic part, and showed no differentiation between A and B chains, or if there was a C-peptide present.


Part BBa_M1877 (Figure 2) consisted of only 12bp and so was clearly unfinished.

Figure 1: BBa_M39904 sequence as displayed in registry. The part is not compatible with many biobricks, and has no differentiation between A and B peptides.

Figure 2: BBa_M1877 sequence as displayed in registry. As human insulin is clearly more than 12bp this sequence was deemed incomplete.

Our Improved Parts


We submitted BBa_K2417000, BBa_K2417001 and BBa_K2417006 as improvements upon these (Figure 3).

Figure 3: BBa_K2417000 – Ecotin-Proinsulin, BBa_K2417001 – Cytoplasmic-Proinsulin, BBa_K241006 - Proinsulin – are all improvement parts because they are much more clearly characterized and contains features for more efficient production and purification.

Summary


Previous Part Characteristic How did we improve this? Which parts did we submit with this improvement?
BBa_M399 Basic part We submitted composite parts that clearly distinguish between A and B chains, and the presence of a C peptide. The use of a composite part submission system allows users to access the basic parts they consist of for more detailed information. BBa_K2417000 BBa_K2417001
Lacking complete sequence Our parts are sequence verified, confirmed as complete and are based off NCIB sequences of human proinsulin. BBa_K2417000 BBa_K2417001 BBa_K2417006
Lacking ribosomal binding site Our parts contain an efficient extended ribosomal binding site (part BBa_K2417009) which aids successful expression. BBa_K2417000 BBa_K2417001 BBa_K2417006
Lack of purification tags We added an N-terminal His-tag (BBa_K2417008) to all of our proinsulin constructs to aid ease of purification. A His-tag allows purification using an affinity chromatography column. BBa_K2417000 BBa_K2417001 BBa_K2417006
No secretion tags For some of our constructs we added tags to induce secretion of the protein to different parts of the cell. The addition of Ecotin to the N-terminus of proinsulin has been shown to induce its transport into the periplasm of E. coli, an oxidative environment favourable for the folding of disulphide bonds. BBa_K2417000
BBa_M39904 Biobrick incompatible The construct entered into the database is only compatible with a single biobrick. All of our parts are compatible with all biobrick constructs. BBa_K2417000 BBa_K2417001 BBa_K2417006