Team:Groningen/Project/secretion

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<h1>Introduction</h1>
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Silk production
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To distinguish extracellular proteins from cytoplasmic proteins, extracellular proteins are provided with a cleavable signal peptide. In general proteins are translocated in an unfolded state through a channel composed of integral membrane proteins. The precursor proteins are synthesized at the ribosome and are prevented from tight folding or aggregation, by chaperones. At this stage to possible pathways can be followed. The ribosome can synthesize the precursor protein directly at the translocation channel, called co-translation translocation. The other possibility is to synthesize the precursor protein prior to the translocation (post-translational translocation). After the translocation of the precursor protein, the signal peptide is cleaved off by signal peptidases during or shortly after the translocation [van Wely et al. 2001].
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<p>In our design we are employing the Gram-positive model bacterium Bacillus subtilis to secrete spider silk. B. subtilis is generally regarded as safe (GRAS) and is often used in industry for the commercial production of extracellular proteins because they only need to traverse a single membrane. In order to secrete the spider silk protein by the Sec-system of B. subtilis, we constructed various signal peptites in front of our silk protein that will be recognized by the signal recognition particle (SRP) and will be traversed over the membrane.
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Figure 2: schematic drawing of natural silk
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The spider silk we used is the so called dragline silk from the Argiope aurantia (MaSp2)  from Brookes et al (2008).  It is the strongest silk produced inside the spider. The spider silk consists out of a big repetitive domain (around 2500 base pairs) with an N and a C terminus (figure 2 b). (write something about figure 2a as well of crop the image).
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our spider silk
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We developed 12 different types of spider silk (Link towards biobricks and check the number) (figure 3). 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 4).
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<h3><i>B. subtilis</i> protein translocation machinery.</h3>
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Figure 3, our spider silks
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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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Figure 1. Schematic overview of the <i>B. subtilis</i> 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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<BR>KH van Wely <i>et al.</i> 2006 (FEMS Micro. Rev.)
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Figure 4: Not sure if this image needs to be on our site, if so please explain thoroughly.  
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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 5). The strep tag will be used for our coating mechanism.
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<h3>MotB</h3>
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<h3>FliZ</h3>
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Figure 5: the secretion pathway of silk�
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<h3>EstA</h3>
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<h3>LytB</h3>
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Revision as of 10:47, 10 September 2013

Silk production Figure 2: schematic drawing of natural silk The spider silk we used is the so called dragline silk from the Argiope aurantia (MaSp2) from Brookes et al (2008). It is the strongest silk produced inside the spider. The spider silk consists out of a big repetitive domain (around 2500 base pairs) with an N and a C terminus (figure 2 b). (write something about figure 2a as well of crop the image). our spider silk We developed 12 different types of spider silk (Link towards biobricks and check the number) (figure 3). 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 4). Figure 3, our spider silks Figure 4: Not sure if this image needs to be on our site, if so please explain thoroughly. 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 5). The strep tag will be used for our coating mechanism. Figure 5: the secretion pathway of silk�

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