Team:Groningen/Project/Construct

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<h1>Constructs</h1>
 
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<h1>Backbone</h1>
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<h2>An <i>amyE</i> locus integration backbone</h2>
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<h2>An <i>amyE</i> intergrational backbone</h2>
 
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To transform <i>B. subtilis</i> with our developed biobricks we have improved the <i>amyE</i> intergrational backbone from Munich's iGEM team 2012 (<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>) that had no inducible promoter. We've added the HyperSpank IPTG inducible promoter from Rudner's Lab 2004. This promoter is placed right in front of the prefix, so any biobrick can be inserted in this backbone (<a href="http://parts.igem.org/Part:BBa_K1085014">BBa_K1085014</a>) and can be induced with IPTG.<p>
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One of the big challenge of the iGEM Groningen project is the production of the silk protein in <i>Bacillus subtilis</i>. In order to face such a challenge the so called "<b>production backbone</b>" has been created.
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The backbone has been developed as an improved version of the <i>amyE</i> locus intergration backbone from Munich's iGEM team 2012 (<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>). Thus didn't contain any promoter, therefore the HyperSpank IPTG inducible promoter (Rudner's Lab 2004) has been added. The production backbone (Fig 2) (<a href="http://parts.igem.org/Part:BBa_K1085014">BBa_K1085014</a>) can be then used for the production of the desired protein upon IPTG induction.
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To transform <i>B. subtilis</i> with our developed silk biobricks we have improved the <i>amyE</i> integrational backbone from Munich's iGEM team 2012 (<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>) that had no inducible promoter. We have added the HyperSpank IPTG inducible promoter from Rudner's Lab 2004. This promoter is placed right in front of the prefix, so any biobrick can be inserted in this backbone (fig 2) (<a href="http://parts.igem.org/Part:BBa_K1085014">BBa_K1085014</a>) and can be induced with IPTG.</p>
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In figure 1 a close-up of the promoter is shown.  
In figure 1 a close-up of the promoter is shown.  
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<p>The HyperSpank promoter has a single nucleotide change (G->T) at the -1 position. This increases the expression levels but also causes leaky expression when IPTG is absence[1].
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To improve repression, a second lacO operator site has been inserted 71 bp upstream of the first. (David Rudner, Harvard Medical School)
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<p>The HyperSpank promoter has a single nucleotide change (G->T) at the -1 position. This increases the expression levels but also causes leaky expression when IPTG is absent [1].
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To improve repression, a second lacO operator site has been inserted 71 bp upstream of the first (David Rudner, Harvard Medical School).
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<font size="1">Figure 1: The HyperSpank promoter with in orange the lacO operators, in red the -35, -10, +1 signals and in green the prefix.</font>
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<font size="1"><p>Figure 1: The HyperSpank promoter with in orange the lacO operators, in red the -35, -10, +1 signals and in green the prefix.</p></font>
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<img src="https://static.igem.org/mediawiki/2013/d/d8/Backbone_fancy.png" width="100%"> <!--only insert the link, do not change the percentage!-->
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<font size="1">Figure 2: BBa_K1085014 with: The <i>amp</i> gene for ampicillin resistance in <i>E. coli</i>; The <i>cat</i> gene for chloramphenicol resistance in <i>B. subtilis</i>; The HyperSpank promoter with its repressor <i>lacI</i>; The <i>amyE</i> up- and downstream fragments for intergration in the <i>amyE</i> locus. </font>
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<font size="1"><p>Figure 2: BBa_K1085014 with: The <i>amp</i> gene for ampicillin resistance in <i>E. coli</i> (not shown); The <i>cat</i> gene for chloramphenicol resistance in <i>B. subtilis</i>; The HyperSpank promoter with its repressor <i>lacI</i>; The <i>amyE</i> up- and downstream fragments for intergration in the <i>amyE</i> locus.</p> </font>
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For the validation of this backbone, click <a href="https://2013.igem.org/Team:Groningen/Lab/experiments/Backbone">here</a>.
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<h3>Promoter activity</h3>
 
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<a href="http://parts.igem.org/Part:BBa_K1085014">BBa_K1085014</a> was constructed with two variants of GFP (one form our group and one from the biobrick system, <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840</a>). BBa_K1085014-GFPmg and BBa_K1085014-BBa_E0840 were transformed to <i>B. subtilis</i>. Overnight cultivated strains were diluted 1:100 in fresh medium and grown till OD~0.45 and induced with 1mM IPTG. A 1,2 and 3 hour sample was analysed under the microscope, results are shown in figure 3.
 
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<b>Figure 3<br> A:</b>
 
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<br><b>B:</b>
 
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<br><b>C:</b>
 
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<img src="https://static.igem.org/mediawiki/2013/d/dd/GFPdsm_expression.png" width="150%"></img>
 
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<font size="1">Figure 3: In (a) An overlay picture is shown from the phase-contrast and GFP-channel picture. In (b,c) the intensity in of ~40 cells in the GFP-channel, were analyzed from two pictures. The average intensity (AU) from the cells are plotted above with the standard deviation. In (b) a GFP from our group is shown and in (c)a biobricked GFP (BBa_E0840) is shown in a plot. wt=wildtype, T0= before induction, T1,2,3=#h of induction with IPTG.</font>
 
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<Br><Br><Br><Br><Br>
 
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<h2><i>Bacillus subtilis</i> silk genes collection derived from <i>Argiope aurantia</i> MaSp2</h2>
 
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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 “<b>The <i>Bacillus subtilis</i> silk assembly shop</b>”.
 
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<br>The 'shop' idea follows those conceptual steps:
 
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<ol><li>Design your own silk protein</li>
 
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<li>Browse the iGEM Groningen silk subunits collection:</li>
 
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<th rowspan="2">Spider silk <br> subunit</th>
 
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<th rowspan="2">Biobrick id</hd>
 
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<th colspan ="3">Construct Features</th>
 
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<th>RBS</th>
 
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<th>Strep-tag</th>
 
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<th>Stop codon</th>
 
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<th rowspan="2">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_K1085001">BBa_K1085001</a></td>
 
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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><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1085003">BBa_K1085003</a></td>
 
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<th rowspan="2">Tail</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_K1085009">BBa_K1085009</a></td>
 
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<td colspan="5"><font size="1">table 1: table description.</font></td>
 
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<li>Select the subunits you need</li>
 
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<li>Assemble them together</li></ol>
 
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At this point the silk product is ready to be produced (<a href="">See production backbone</a>) and employed for the desired purpose.
 
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The Groningen team enriched the iGEM database adding several new <i>Bacillus subtilis</i> 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.
 
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<br>It is the first time that silk-coding BioBricks suitable for <i>Bacillus subtilis</i> are introduced into the database. Therefore this work marks the starting point for further studies about silk within the <i>Bacillus subtilis</i> chassis inside the iGEM framework.
 
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The advantages shown by our collection are mainly two which can be summarized as follows:
 
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<ol><li><b>Less cloning steps</b>
 
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The iGEM database provides a lot of basic parts which can be employed for the development of the desired synthetic machine, however, the assembling protocols are often tedious and time consuming. For this reason all the silk subunits, produced by the Groningen team, have been created in different versions. The aim of such a design enables the end-user to reach quicker the desired silk protein without bothering too much about the molecular issues.</li>
 
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<li><b>Compatibility with BioBrick standards</b>
 
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We divided the several silk coding sequences in 3 big groups:
 
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<ul><li>Subunit E1</li>
 
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<li>Tail</li></ul>subunit E1, subunit E2 and Tail. Each of them has been produced and stored into the iGEM submission backbone (pSB1C3). Each of them shows certain features: signal sequences, strep-tag and Ribosome Binding Site (RBS).
 
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The aim of the molecular genetic design is to create a handy toolbox in order to produce any desired combination of silk-like genes which can be expressed in <i>Bacillus subtilis</i> for several purposes. Everything is designed within the RFC[23] standard framework. This enables the user to join together all the different parts creating in-frame fusion silk-like proteins.
 
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The following parts have been produced, sequenced and are available into the standard BioBrick submission backbone (pSB1C3):
 
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<br>What kind of spider silk gene is it
 
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<br>What are general problems with this gene
 
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<br>Where did we get it from.
 
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<br>Codon optimisation, solved the problems.
 
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<h2>Strep-tag</h2>
 
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<br>Why this is needed
 
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<br>For what purposes it comes in handy
 
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<h2>Signal peptide</h2>
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<h2>References</h2>
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<br>Why is it so important.
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<br> The use of existing pathway so not lots of trouble
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[1] &nbsp;&nbsp;&nbsp;&nbsp; Quisel, John D., William F. Burkholder, and Alan D. Grossman. "In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis." Journal of bacteriology 183.22 (2001): 6573-6578.
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<br>The signal sequences are nice addition to the registry
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<br> how the pathway works and how the silk will be secreted
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Latest revision as of 03:44, 5 October 2013

Backbone

An amyE locus integration backbone

To transform B. subtilis with our developed silk biobricks we have improved the amyE integrational backbone from Munich's iGEM team 2012 (BBa_K823023) that had no inducible promoter. We have added the HyperSpank IPTG inducible promoter from Rudner's Lab 2004. This promoter is placed right in front of the prefix, so any biobrick can be inserted in this backbone (fig 2) (BBa_K1085014) and can be induced with IPTG.

In figure 1 a close-up of the promoter is shown.

The HyperSpank promoter has a single nucleotide change (G->T) at the -1 position. This increases the expression levels but also causes leaky expression when IPTG is absent [1]. To improve repression, a second lacO operator site has been inserted 71 bp upstream of the first (David Rudner, Harvard Medical School).

Figure 1: The HyperSpank promoter with in orange the lacO operators, in red the -35, -10, +1 signals and in green the prefix.

Figure 2: BBa_K1085014 with: The amp gene for ampicillin resistance in E. coli (not shown); The cat gene for chloramphenicol resistance in B. subtilis; The HyperSpank promoter with its repressor lacI; The amyE up- and downstream fragments for intergration in the amyE locus.

For the validation of this backbone, click here.

References

[1]      Quisel, John D., William F. Burkholder, and Alan D. Grossman. "In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis." Journal of bacteriology 183.22 (2001): 6573-6578.

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