Team:Groningen/Project/SilkAssemblyShop

From 2013.igem.org

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<ol><li><i>Design</i> your own silk protein</li>
<ol><li><i>Design</i> your own silk protein</li>
<li><i>Browse</i> the iGEM Groningen silk subunits collection:</li>
<li><i>Browse</i> the iGEM Groningen silk subunits collection:</li>
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<br>
<div align="center">  <!--you can use left/right or center to align the image-->
<div align="center">  <!--you can use left/right or center to align the image-->
<table  id="normal" width=60%> <!--change the percentage to determine the size of your image-->
<table  id="normal" width=60%> <!--change the percentage to determine the size of your image-->
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<th rowspan="2">Spider silk <br> subunit</th>
<th rowspan="2">Spider silk <br> subunit</th>
<th rowspan="2">Biobrick id</hd>
<th rowspan="2">Biobrick id</hd>
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<th colspan ="3">Construct Features</th>
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<th colspan="3">Construct Features</th>
</tr>
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</table>
</table>
</div>
</div>
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<br>
<li><i>Select</i> the subunits you need</li>
<li><i>Select</i> the subunits you need</li>
<li><i>Assemble</i> them together</li></ol>
<li><i>Assemble</i> them together</li></ol>
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</ul>
</ul>
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<h4>Assembly shop in action</h4>
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<p>
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Using the silk assembly shop idea the Groningen team developed its own personal silk gene version (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085018">BBa_K1085018</a>), which is composed by three subunits: <b>E1</b>, <b>E2</b> and <b>Tail</b> (Figure 1).
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<div align="left">
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<table id="layout" width=90%>
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<tr>
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<td>
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<img src="https://static.igem.org/mediawiki/2013/c/cf/Awesome.jpg" width="100%">
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</td>
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</tr>
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<tr>
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<td>
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<font size="1">Figure 1: This is the molecular schematic representation of BBa_K1085018 where the molecular features (ribosome binding site, strep-tag and stop codon) are shown.</font>
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</td>
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</tr>
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</table>
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</div>
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<br>The final construct contains all the features necessary in order to start the translation process, moreover the final protein shows the Strep-tag which can be used in order to detect the presence of the protein.
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<br>This proves that all the pieces can be assembled into any desired construct.
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</p>
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<br>
<h2>Secretion signal peptide accessory</h2>
<h2>Secretion signal peptide accessory</h2>
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Signal peptides sequences are introduced in the subunit design for the aim of, not only producing silk protein, but also secreting it. This has been done by embedding signal peptide particle (SRP) sequences, sourced from the <i>Bacillus subtilis</i> proteome, in front of the silk subunits with the purpose of directing the protein into the Sec secretion pathway (See Figure 1).
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Signal peptides sequences are introduced in the subunit design for the aim of, not only producing silk protein, but also secreting it. This has been done by embedding signal peptide particle (SRP) sequences, fished from the <i>Bacillus subtilis</i> genome, in front of the silk subunits with the purpose of directing the protein into the Sec secretion pathway (Figure 2).
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<br>
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<br>
<div align="center">
<div align="center">
<table id="layout" width=40%>
<table id="layout" width=40%>
<tr>
<tr>
<td>
<td>
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<img src="https://static.igem.org/mediawiki/igem.org/8/82/Sec_bsub.gif" width="100%">
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<img src="https://static.igem.org/mediawiki/2013/a/a0/Sec.jpg" width="100%">
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td>
<td>
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<font size="1">Figure 1: The schematic representation of the <i>Bacillus subtilis</i> Sec secretion pathway (van Wely et al. 2001). In the picture it is clearly visible how the SRP points the protein towards the extra cellular compartment.</font>
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<font size="1">Figure 2: The schematic representation (adapted from van Wely et al. 2001) shows how the signal recognition particle (SRP) helps in directing the silk protein towards the outer membrane compartment of <i>Bacillus subtilis</i>.  The Sec secretion machinery exports the silk protein. Proteases cleaves the SRP from the secreted protein.</font>
</td>
</td>
</tr>
</tr>
</table>
</table>
</div>
</div>
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<br>Ideally any E1 subunit can be fused to several signal sequences, therefore the produced protein is directed into the Sec pathway and end up in the extra-cellular compartment.
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<br>A selection of SRP sequences is made and those have been cloned in front of the E1 subunit in order to directs the produced silk protein towards the Sec pathway track.
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<br>The following signal sequences are fused together with silk subunit:
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<br>The following signal sequences are fused together with the E1 silk subunit:
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<br>
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<br>
<div align="center">  <!--you can use left/right or center to align the image-->
<div align="center">  <!--you can use left/right or center to align the image-->
<table  id="normal" width=60%> <!--change the percentage to determine the size of your image-->
<table  id="normal" width=60%> <!--change the percentage to determine the size of your image-->
<tr>
<tr>
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<th rowspan="2">Spider silk <br> subunit</th>
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<th rowspan="2">SRP source</th>
<th rowspan="2">Biobrick id</hd>
<th rowspan="2">Biobrick id</hd>
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<th colspan ="3">Construct Features</th>
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<th colspan ="2">Construct Features</th>
</tr>
</tr>
<tr>
<tr>
<th>RBS</th>
<th>RBS</th>
<th>Strep-tag</th>
<th>Strep-tag</th>
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<th>Stop codon</th>
 
</tr>
</tr>
<tr>
<tr>
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<th rowspan="2">E1</th>
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<th>EstA-E1</th>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085000">BBa_K1085000</a></td>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085035">BBa_K1085035</a></td>
<td></html>[[File:Green_tick.gif | 25px]]<html></td>
<td></html>[[File:Green_tick.gif | 25px]]<html></td>
<td></html>[[File:Green_tick.gif | 25px]]<html></td>
<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></td>
 
</tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085001">BBa_K1085001</a></td>
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<th>FliZ-E1</th>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085029">BBa_K1085029</a></td>
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<td></td>
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<td></td>
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</tr>
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<tr>
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<th rowspan="2">E2</th>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085002">BBa_K1085002</a></td>
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<td></td>
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<td></td>
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<td></td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085003">BBa_K1085003</a></td>
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<td></td>
<td></td>
<td></html>[[File:Green_tick.gif | 25px]]<html></td>
<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></td>
 
</tr>
</tr>
<tr>
<tr>
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<th rowspan="2">Tail</th>
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<th>MotB-E1</th>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085008">BBa_K1085008</a></td>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085033">BBa_K1085033</a></td>
<td></td>
<td></td>
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<td></td>
 
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
 
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</tr>
 
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<tr>
 
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085009">BBa_K1085009</a></td>
 
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<td></td>
 
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
 
<td></html>[[File:Green_tick.gif | 25px]]<html></td>
<td></html>[[File:Green_tick.gif | 25px]]<html></td>
</tr>
</tr>
<tr>
<tr>
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<td colspan="5"><font size="1">Table 1: ??.</font></td>
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<td colspan="4"><font size="1">Table 2: The signal recognition particle (SRP) sequences which were cloned in front of the E1 silk subunit are listed together with the features shown by the construct.</font></td>
</tr>
</tr>
</table>
</table>
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Why is it so important.
Why is it so important.
<br> The use of existing pathway so not lots of trouble
<br> The use of existing pathway so not lots of trouble
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<br>The signal sequences are nice addition to the registry
 
<br> how the pathway works and how the silk will be secreted
<br> how the pathway works and how the silk will be secreted
<br>
<br>

Latest revision as of 03:43, 5 October 2013

Silk Assembly Shop

Bacillus subtilis silk genes collection derived from Argiope aurantia MaSp2

One of the challenges that the project includes is the production of spider silk. In order to achieve this goal iGEM Groningen designed for you what we call “The Bacillus subtilis silk assembly shop”.
The 'shop' idea follows those conceptual steps:

  1. Design your own silk protein
  2. Browse the iGEM Groningen silk subunits collection:

    3. Spider silk
    subunit    		 Biobrick id Construct Features
    RBS Strep-tag Stop codon
    E1 BBa_K1085000 25px 25px
    BBa_K1085001 25px
    E2 BBa_K1085002
    BBa_K1085003 25px
    Tail BBa_K1085008 25px
    BBa_K1085009 25px 25px
    Table 1: The following silk subunits are listed: E1 whose sequence is codon-optimized to prevent the formation of secondary RNA structures in close proximity to the RBS, whereas E2 is more focused in reducing the presence of restriction sites and Tail whose sequence in order to match the translation rate between Bacillus subtilis and Argiope aurantia.

  3. Select the subunits you need
  4. Assemble them together
At this point the silk product is ready to be produced (See production backbone) and employed for the desired purpose.

The Groningen team enriched the iGEM database adding several new Bacillus subtilis BioBricks containing spider silk gene subunits. This provides the iGEM community with a new bunch of parts; which, in the future, will help anyone in assembling its own silk protein and customize it with different features.
It is the first time that silk-coding BioBricks suitable for Bacillus subtilis are introduced into the database. Therefore this work marks the starting point for further studies about silk within the Bacillus subtilis chassis inside the iGEM framework.

The advantages shown by our collection are mainly three which can be summarized as follows:

  1. Accessible design The iGEM database provides a lot of basic parts (such as RBS and terminator), but often achieving the desired goal turns out to be really tedious and time consuming, since the creation of a working construct needs to go through a lot of steps. iGEM Groningen took into account this problematic aspect while designing the silk collection, therefore all the silk subunits (each of them coding for the same final protein product) have been created in different versions (see Table 1). Such a design enables the end-user to quickly reach the desired silk protein construct without bothering too much about the molecular issues. This kind of approach helps to make synthetic biology and specifically working with silk protein production in Bacilus subtilis more accessible.
  2. Compatibility with BioBrick standards We are aware that the BioBrick assembly standard is the foundation of the iGEM method, allowing all different laboratories to build on each others work. That is why the silk subunits are compatible with the following standard:
    • BBF RFC 10
    • BBF RFC 12
    • BBF RFC 21
    • BBF RFC 23
    • BBF RFC 25
    • BBF RFC 1000
  3. Dealing with repetitiveness One of the issue while working with the silk protein is the high repetitiveness of the coding sequence. The problem is that the molecular techniques, such as PCR, are less effective on this type of DNA template. The subunit design provides us with a solution, since the molecular steps do not depend anymore on the silk coding sequence itself, but on the universal flanking regions.

Strep-tag

The Strep-tag is there for two reasons:
  • mainly as a binding component to Streptavidin (See coating mechanism)
  • as well as a way of detecting the protein production

Assembly shop in action

Using the silk assembly shop idea the Groningen team developed its own personal silk gene version (BBa_K1085018), which is composed by three subunits: E1, E2 and Tail (Figure 1).

Figure 1: This is the molecular schematic representation of BBa_K1085018 where the molecular features (ribosome binding site, strep-tag and stop codon) are shown.

The final construct contains all the features necessary in order to start the translation process, moreover the final protein shows the Strep-tag which can be used in order to detect the presence of the protein.
This proves that all the pieces can be assembled into any desired construct.


Secretion signal peptide accessory

Signal peptides sequences are introduced in the subunit design for the aim of, not only producing silk protein, but also secreting it. This has been done by embedding signal peptide particle (SRP) sequences, fished from the Bacillus subtilis genome, in front of the silk subunits with the purpose of directing the protein into the Sec secretion pathway (Figure 2).

Figure 2: The schematic representation (adapted from van Wely et al. 2001) shows how the signal recognition particle (SRP) helps in directing the silk protein towards the outer membrane compartment of Bacillus subtilis. The Sec secretion machinery exports the silk protein. Proteases cleaves the SRP from the secreted protein.

A selection of SRP sequences is made and those have been cloned in front of the E1 subunit in order to directs the produced silk protein towards the Sec pathway track.
The following signal sequences are fused together with the E1 silk subunit:

SRP source Biobrick id Construct Features
RBS Strep-tag
EstA-E1 BBa_K1085035 25px 25px
FliZ-E1 BBa_K1085029 25px
MotB-E1 BBa_K1085033 25px
Table 2: The signal recognition particle (SRP) sequences which were cloned in front of the E1 silk subunit are listed together with the features shown by the construct.

Retrieved from "http://2013.igem.org/Team:Groningen/Project/SilkAssemblyShop"