Team:Hong Kong HKUST/Project/module3

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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>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/experiment">Experiments</a></li>
 
<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/notebook">Notebook</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module4">Glyoxylate Shunt</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module2">FA Quantification & Cell Viability</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module2">FA Sensing Mechanism</a>
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<a href=#2>Mechanism of MLS</a>
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<a href=#2>Biology of Mitochondrial Leader Sequence (MLS)</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module4">Glyoxylate Shunt</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module2">FA Sensing Mechanism</a>
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<div class="nine columns"><p id="1"></p>
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<h3>Overview</h3>
<h3>Overview</h3>
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In vertebrates, the signal sequence coding region promotes translocation of precursor peptide. In our project, we introduced bacterial enzymes to mammalian cell to modify metabolic pathway. However, unlike bacteria, citric acid cycle in mammalian cells is compartmentalized in mitochondria. The glyoxylate enzymes (<a href="http://parts.igem.org/Part:BBa_K1119002">AceA</a> and <a href="http://parts.igem.org/Part:BBa_K1119003">AceB</a> proteins, denoted ACE enzymes) should be targeted to mitochondria for their functionality. To do so, we fused ACE enzymes with Mitochondrial Leader Sequence (MLS). We have constructed and qualitatively and quantitatively <a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization/mls">characterized</a> Mitochondrial Leader Sequence BioBrick <a href="http://parts.igem.org/Part:BBa_K1119001">BBa_K1119001</a>.
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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.
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<br>
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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.
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<br>
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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.
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<h3>Mechanism of MLS</h3>
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<h3>Biology of Mitochondrial Leader Sequence (MLS)</h3>
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<p>MLS is attached to the N-terminal of the protein in interest. The MLS will be recognized by receptor on outer mitochondrial membrane. The complex of receptor and attached protein will diffuse to contact site, where the outer and inner membrane come into contact. The protein will be translocated across mitochondrial membrane via a protein translocator. Preprotein is unfolded and bind to the TOM complex on mitochondrial outer membrane, then it will pass to TIM complex on inner membrane side. Mitochondrial chaperone will be involved in pulling preprotein into mitochondria and refold the protein.
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<p>In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.</p>
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<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>
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<img src="https://static.igem.org/mediawiki/2013/8/87/MLS_mechanism.png" >
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<h3>Reference</h3>
<h3>Reference</h3>
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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.<br><br>
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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>
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Bohnert, M., & Pfanner, N. (2007). A dynamic machinery for import of mitochondrial precursor proteins. FEBS Letters, 581, 2802-2810. Retrieved from http://ac.els-cdn.com/S0014579307002530/1-s2.0-S0014579307002530-main.pdf?_tid=95ba5a1a-27a7-11e3-b268-00000aab0f01&acdnat=1380308827_541781deb3d3577ca5de6f02f5152202<br><br>
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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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<br>
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Latest revision as of 12:42, 28 October 2013

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.