Team:Hong Kong HKUST/Project/module3
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+ | |||
+ | |||
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+ | |||
+ | <p id="back-top" style="display:none;"> | ||
+ | <a href="#top"><span><img src="http://515alive.com/theme/img/up-arrow.png" style="width:90%;"><br><br>BACK TO TOP</span></a> | ||
+ | </p> | ||
<a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a> | <a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a> | ||
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li> | ||
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li> | ||
+ | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/attribution">Attribution</a></li> | ||
+ | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/acknowledge">Acknowledgement</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/ | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project">Project</a> |
<ul> | <ul> | ||
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li> | ||
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/ | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modules">Modules Description</a></li> |
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/ | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/data">Data Page</a></li> |
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li> | ||
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/ | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li> |
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results"> | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Result</a></li> |
+ | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/future">Future Work</a></li> | ||
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a> | ||
<ul> | <ul> | ||
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook"> | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li> |
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols"> | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li> |
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety"> | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">Safety</a></li> |
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a> | ||
<ul> | <ul> | ||
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/ | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/cp">Country Profile</a></li> |
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/ | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/blog">Blog</a></li> |
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/ | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/interview">Interviews</a></li> |
- | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/ | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/article/genet">Article</a></li> |
+ | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/video">Videos</a></li> | ||
+ | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/presentation">Presentations</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
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<div class="two columns"> | <div class="two columns"> | ||
- | <ul class="side-nav"> | + | <br> |
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<li> | <li> | ||
- | <h6> | + | <h6>Modules</h6> |
</li> | </li> | ||
<li class="divider"></li> | <li class="divider"></li> | ||
- | + | <a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module4">Glyoxylate Shunt</a> | |
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</li> | </li> | ||
+ | <li> | ||
+ | Protein Trafficking | ||
+ | <ul><li> | ||
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- | <a href= | + | <a href=#1>Overview</a> |
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- | <a href= | + | <a href=#2>Biology of Mitochondrial Leader Sequence (MLS)</a> |
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<li> | <li> | ||
- | + | <a href=#3>Reference</a> | |
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+ | </li> | ||
+ | </ul> | ||
</li> | </li> | ||
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- | <a href="https:// | + | <a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module2">FA Sensing Mechanism</a> |
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</li> | </li> | ||
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</div> | </div> | ||
<div class="ten columns team-bios-container"> | <div class="ten columns team-bios-container"> | ||
<div class="row" id="ugd-members"> | <div class="row" id="ugd-members"> | ||
<div class="twelve columns"> | <div class="twelve columns"> | ||
- | <h2 class="centered"> | + | <h2 class="centered">Protein Trafficking</h2> |
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<div class="row"> | <div class="row"> | ||
- | <div class="nine columns"> | + | <div class="nine columns"><p id="1"></p> |
- | <h3> | + | <h3>Overview</h3> |
+ | In our project, we introduced bacterial glyoxylate enzymes into mammalian cells to create alternative metabolic pathway. However, unlike their native environment in bacteria, the two enzymes needed to find their way through the high compartmentalized system in order to reach the citric acid cycle where they could act on. | ||
<br> | <br> | ||
- | + | To guide the glyoxylate enzymes into the mitochondria where mammalian citric acid cycle resides, we produced recombinant glyoxylate enzymes by attaching the Mitochondrial Leader Sequence (MLS) to their N-termini. | |
- | <br> | + | <br> |
+ | We have also constructed the MLS in its own into standard BioBricks (<a href="http://parts.igem.org/Part:BBa_K1119000">BBa_K1119000</a> & <a href="http://parts.igem.org/Part:BBa_K1119001">BBa_K1119001</a>), and we quantitatively characterized their behavior using GFP reporter. | ||
</div> | </div> | ||
- | <div class=" | + | </div> |
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+ | <div class="nine columns"><p id="2"></p> | ||
+ | <h3>Biology of Mitochondrial Leader Sequence (MLS)</h3> | ||
+ | <p>In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.</p> | ||
+ | <p>The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. (Alberts, 2002) <br>For the MLS we used, four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.(Invitrogen, 2012)</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/8/87/MLS_mechanism.png" > | ||
</div> | </div> | ||
</div> | </div> | ||
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- | <div class=" | + | <div class="nine columns"><p id="3"></p> |
- | + | <div class="nine columns"><p id="5"></p> | |
- | <div class="nine columns"> | + | <h3>Reference</h3> |
- | < | + | Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2010). <i>Essential cell biology.</i> (3rd ed., p. 505). UK: Garland Science.<br><br> |
- | < | + | pCMV/<i>myc</i>/mito Invitrogen.(2012).pShooter™ Vector(pCMV/<i>myc</i> vectors).Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf <br><br> |
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- | + | Alberts, B. (2002). <i>Molecular biology of the cell.</i> (4th ed., pp. 1050-1061). New York:Garland Science. | |
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Latest revision as of 12:42, 28 October 2013
-
Modules
Glyoxylate Shunt
- Protein Trafficking
- FA Sensing Mechanism
Protein Trafficking
Overview
In our project, we introduced bacterial glyoxylate enzymes into mammalian cells to create alternative metabolic pathway. However, unlike their native environment in bacteria, the two enzymes needed to find their way through the high compartmentalized system in order to reach the citric acid cycle where they could act on.To guide the glyoxylate enzymes into the mitochondria where mammalian citric acid cycle resides, we produced recombinant glyoxylate enzymes by attaching the Mitochondrial Leader Sequence (MLS) to their N-termini.
We have also constructed the MLS in its own into standard BioBricks (BBa_K1119000 & BBa_K1119001), and we quantitatively characterized their behavior using GFP reporter.
Biology of Mitochondrial Leader Sequence (MLS)
In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.
The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. (Alberts, 2002)
For the MLS we used, four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.(Invitrogen, 2012)
Reference
Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2010). Essential cell biology. (3rd ed., p. 505). UK: Garland Science.pCMV/myc/mito Invitrogen.(2012).pShooter™ Vector(pCMV/myc vectors).Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf
Alberts, B. (2002). Molecular biology of the cell. (4th ed., pp. 1050-1061). New York:Garland Science.