Team:Hong Kong HKUST/module3backup

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<h3>Overview</h3>
<h3>Overview</h3>
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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 ACE proteins should be targeted to mitochondria for their functionality. To do so, we fused ACE enzymes with Mitochondrial Leader Sequence (MLS).
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In nature, eukaryotic cell mitochondria usually have their protein encoded by gene in nucleus and produced from cytosol. In this process, MLS will act as signal peptide to target the protein into mitochondria.
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In our module, we will construct the MLS BioBrick <a href="http://parts.igem.org/Part:BBa_K1119001">K1119001</a>, and characterize it quantitatively[link].
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<h3>Mechanism of MLS</h3>
<h3>Mechanism of MLS</h3>
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MLS is attached to the N-terminal of enzyme, and bind to the receptor protein on mitochondrial membrane, and diffuse to contact site where inner and outer membrane fuse, then bring the ACE enzyme into mitochondria. Afterward, it is be cleaved, leaving the enzyme in mitochondria.
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MLS is attached to the N-terminal of the protein in interest, and be recognized by receptor in the outer mitochondria membrane. The complex of receptor and attached protein diffuse to a contact site, where protein is translocated across both the outer and inner membrane by a protein translocator. The MLS will be cleaved off by signal peptidase inside the mitochondria afterward. Chaperone proteins will help to pull the protein across the membrane and help the refolding of protein.
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<img src="https://static.igem.org/mediawiki/2013/8/87/MLS_mechanism.png" >
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<h3>Linkage to Project</h3>
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<h3>Linkage to project</h3>
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The MLS was cloned from a commercial plasmid, pCMV/myc/mito (Invitrogen) by PCR. For the MLS BioBrick, we have submitted the MLS BioBrick in RFC 10 and RFC 25, the Freiburg format which allows protein fusion, to facilitate other team to fuse the MLS with other protein for purpose of introducing other protein into mitochondria.  
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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 ACE proteins should be targeted to mitochondria for their functionality. To do so, we fused ACE enzymes with Mitochondrial Leader Sequence (MLS).  
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For characterization, MLS and green fluorescence protein was fused with constitutive mammalian CMV promoter. The promoter was cloned from pEGFP-N1 (Clonetech) in RFC10 format, since such part could not be found in partsregistry. The CMV cloned for our characterization construct was also submitted. The two construct for characterization, the CMV promoter – green fluorescent protein – polyadenylation sequence – pSB1C3 and CMV promoter – mitochondria leader sequence – green fluorescence protein – polyadenylation sequence – pSB1C3 composite parts are also submitted. <a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Click here to see our submitted parts.</a>
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<h3>Reference</h3>
<h3>Reference</h3>
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.
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.
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Latest revision as of 15:13, 27 September 2013



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Protein Trafficking

Overview

In nature, eukaryotic cell mitochondria usually have their protein encoded by gene in nucleus and produced from cytosol. In this process, MLS will act as signal peptide to target the protein into mitochondria.
In our module, we will construct the MLS BioBrick K1119001, and characterize it quantitatively[link].

Mechanism of MLS

MLS is attached to the N-terminal of the protein in interest, and be recognized by receptor in the outer mitochondria membrane. The complex of receptor and attached protein diffuse to a contact site, where protein is translocated across both the outer and inner membrane by a protein translocator. The MLS will be cleaved off by signal peptidase inside the mitochondria afterward. Chaperone proteins will help to pull the protein across the membrane and help the refolding of protein.

Linkage to project

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 ACE proteins should be targeted to mitochondria for their functionality. To do so, we fused ACE enzymes with Mitochondrial Leader Sequence (MLS).

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.

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