Team:Uppsala/signal-peptide

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                                                                 <li><a href="https://2013.igem.org/Team:Uppsala/signal-peptide">Signal peptide</a></li>
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                                                 <li><a href="https://2013.igem.org/Team:Uppsala/metabolic-engineering">Metabolic engineering</a>
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href="https://2013.igem.org/Team:Uppsala/miraculin">Miraculin</a></li>
href="https://2013.igem.org/Team:Uppsala/miraculin">Miraculin</a></li>
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<li><a href="https://2013.igem.org/Team:Uppsala/carotenoid-group">Carotenoid group</a></li>
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<h1 class="main-title">  Signal peptides </h1>
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<h1 class="main-title-left">  Signal peptides </h1>
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<h3> How signal peptides work </h3>
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<img class="symbol-pic" src="https://static.igem.org/mediawiki/2013/4/40/Uppsala2013_Signal_peptide_symbol.png">
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A signal peptide, also called leader peptide, is the first part of extracellular proteins and can vary in length, its a short sequence fused to the N-terminus of a protein. The ones we work with are 25 amino acids and 50 amino acids in length. The signal peptides enables the protein to be translocated through the bacterial plasma membrane via the SecY complex. [1]
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<h3> Lactobacillus - A champion of protein secretion </h3>
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One of the specific advantageous traits of the lactobacillus genus is that they are great at secreting proteins. Therefore we decided to synthesize the signal peptide usp45 from lactococcus lactis according to the Freiburg fusion standard to enable fusion with any protein. This peptide has been shown to work in lactobacillus reuteri together with GFP and will probably work in other lactobacillus species. [2]
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Our goal is fuse this with a protein called miraculin [3]. This peptide acts as a sweetener by binding to taste receptors on the tongue and gives acidic or sour foods a sweet taste. Since it needs to bind to these receptors it is easier to reap its benefits if it is readily available in the probiotic product rather than contained within the bacteria.  
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<h1> How signal peptides work </h1>
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<p>A signal peptide, also called leader peptide, is the first part of extracellular proteins and can vary in length, its a short sequence fused to the N-terminus of a protein. The signal peptides we work with are 25 and 50 amino acids in length. The signal peptides enables the protein to be translocated through the bacterial plasma membrane via the SecY complex.<sup><a href="#l1">[1]</a></sup></p> 
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<h3> References: </h3>  
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<a id="b1"></a>
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[1] Rapoport T. (Nov. 2007). "Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.". Nature 450 (7170): 663–9.
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<h1> Lactobacillus - A champion of protein secretion </h1>
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[2] Chi-Ming Wu. (April 2006). “Green fluorescent protein is a reliable reporter for screening signal peptides functional in Lactobacillus reuteri” Journal of Microbiological Methods (2006) 181-186.
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<p>One of the specific advantageous traits of the lactobacillus genus is that they are great at secreting proteins. Therefore we decided to synthesize the signal peptide usp45 from lactococcus lactis according to the Freiburg fusion standard to enable fusion with any protein. This peptide has been shown to work in lactobacillus reuteri together with GFP and will probably work in other lactobacillus species.<sup><a href="#l2">[2]</a></sup></p>
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[3] https://2013.igem.org/Team:Uppsala/miraculin
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<a href="https://static.igem.org/mediawiki/2013/9/9d/Uppsala2013_signal-peptides.jpg" data-lightbox="roadtrip"><img class="method-plasmid" src="https://static.igem.org/mediawiki/2013/9/9d/Uppsala2013_signal-peptides.jpg"></a>
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<p>One of the goals in our project was to make a probiotic bacteria express the protein miraculin (Mir in picture) which would work as a sweetener. However because it works by binding to receptors on the tongue and the protein is too big to pass through the cell membrane of the bacteria it needs to be actively secreted to work optimally. Fusing a signal peptide to the N-terminus of miraculin can solve this. We have synthesised miraculin fused together with usp45. Because we received the synthesised miraculin gene late, we did not have time to put it in pSB1C3 and send it to the registry. We have also not yet had the chance to transform and characterise it in Lactobacillus.</p>
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<h1> References: </h1>  
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<a name="l1">[1]</a> Rapoport T. (Nov. 2007). "Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.". Nature 450 (7170): 663–9.
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<br><br>
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<a name="l2">[2]</a> Chi-Ming Wu. (April 2006). “Green fluorescent protein is a reliable reporter for screening signal peptides functional in Lactobacillus reuteri” Journal of Microbiological Methods (2006) 181-186.
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Latest revision as of 21:30, 28 October 2013

Signal peptides

How signal peptides work

A signal peptide, also called leader peptide, is the first part of extracellular proteins and can vary in length, its a short sequence fused to the N-terminus of a protein. The signal peptides we work with are 25 and 50 amino acids in length. The signal peptides enables the protein to be translocated through the bacterial plasma membrane via the SecY complex.[1]

Lactobacillus - A champion of protein secretion

One of the specific advantageous traits of the lactobacillus genus is that they are great at secreting proteins. Therefore we decided to synthesize the signal peptide usp45 from lactococcus lactis according to the Freiburg fusion standard to enable fusion with any protein. This peptide has been shown to work in lactobacillus reuteri together with GFP and will probably work in other lactobacillus species.[2]

One of the goals in our project was to make a probiotic bacteria express the protein miraculin (Mir in picture) which would work as a sweetener. However because it works by binding to receptors on the tongue and the protein is too big to pass through the cell membrane of the bacteria it needs to be actively secreted to work optimally. Fusing a signal peptide to the N-terminus of miraculin can solve this. We have synthesised miraculin fused together with usp45. Because we received the synthesised miraculin gene late, we did not have time to put it in pSB1C3 and send it to the registry. We have also not yet had the chance to transform and characterise it in Lactobacillus.

References:

[1] Rapoport T. (Nov. 2007). "Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.". Nature 450 (7170): 663–9.

[2] Chi-Ming Wu. (April 2006). “Green fluorescent protein is a reliable reporter for screening signal peptides functional in Lactobacillus reuteri” Journal of Microbiological Methods (2006) 181-186.