Team:Groningen/Project/secretion

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<h1>Silk production</h1>
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<br>For the silk to be secreted the sec pathway is used. In using this pathway Signal Sequences are needed. this allows the bacillus to recognize the protein as something that needs to be moved to the outside of the cell.
 
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<br>The first Signal sequences that will be atempted are MotB FliZ EstA and LytB.
 
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We developed 12 different types of  <a href="https://2013.igem.org/Team:Groningen/Navigation/Parts">spider silks</a> (figure 1). We have one spider silk protein with a N and C terminus, a spider silk without a N and C terminus and a spidersilk without one block (explain the difference). A codon optimization script was developed to codon optimize the spider silk for <i>b. subtilis</i>.
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Codon optimization (state why we did codon optimization and link towards the codon  optimazation page on the wiki for all the details). Every silk gene was codon optimized twice with different results. All the codon optimized silk genes were ordered and made synthetically (figure 2).
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<h2>Protein targeting to the Sec translocase.</h2>
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<font size="1">Figure 1, Major ampullate Spidroin 2 (MaSp2) from <i>Argiope aurantia</i></font>
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<center><img src="https://static.igem.org/mediawiki/igem.org/8/82/Sec_bsub.gif" width="401" height="596"></img></center>
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<img src="https://static.igem.org/mediawiki/2013/7/78/Our_Silks.png" width="100%" >
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<font size="1">Figure 2, Our constructed silk. All <a href="https://2013.igem.org/Team:Groningen/Navigation/Parts">parts</a> are made into biobricks and can be combined together. </font>
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Figure 1. Schematic overview of the B. subtilis protein translocation machinery. The signal recognition particle (SRP) <Br>consists of scRNA, Ffh and HBsu. See text for details. The dashed line arrow indicates an SRP-independent pathway, <Br>possibly mediated by SecA that shuttles between the SecYEG-bound and free cytosolic state. Proteases that degrade <br>the secreted proteins are located near the membrane surface, in the cell wall and free in the suspending medium.
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The silk genes are provided with an signal peptide and a strep tag. The signal peptide will ensure the secretion of silk by the natural pathways of b. subtilis (figure 3). The strep tag will be used for our coating mechanism.
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<BR>K. H.M. van Wely <i>et al.</i> 2006 (FEMS Micro. Rev.)
 
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<font size="1">Figure 3: the secretion pathway of silk </font>
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Latest revision as of 11:05, 18 September 2013

Silk production

We developed 12 different types of spider silks (figure 1). We have one spider silk protein with a N and C terminus, a spider silk without a N and C terminus and a spidersilk without one block (explain the difference). A codon optimization script was developed to codon optimize the spider silk for b. subtilis. Codon optimization (state why we did codon optimization and link towards the codon optimazation page on the wiki for all the details). Every silk gene was codon optimized twice with different results. All the codon optimized silk genes were ordered and made synthetically (figure 2).



Figure 1, Major ampullate Spidroin 2 (MaSp2) from Argiope aurantia

Figure 2, Our constructed silk. All parts are made into biobricks and can be combined together.

The silk genes are provided with an signal peptide and a strep tag. The signal peptide will ensure the secretion of silk by the natural pathways of b. subtilis (figure 3). The strep tag will be used for our coating mechanism.
Figure 3: the secretion pathway of silk

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