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Team:Hong Kong HKUST/Project/module3
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Module Three
- Introduction
- Mechanism of MLS
- Parts Submission
- Characterization
Protein Trafficking
Introduction
In our project ,we will introduce 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).
Mechanism of Mitochondrial Leader Sequence
MLS will be attached to the N-terminal of enzyme, and bind to the receptor protein on mitochondrial membrane, and diffuse to contact site where inner membrane & outer membrane fuse, then bring the ACE enzyme into mitochondria. Afterward, it will be cleaved , leaving the enzyme in mitochondria.
Part Submission
The MLS was cloned from a commercial plasmid, pCMV/myc/mito (Invitrogen) by PCR. For the MLS biobrick, we are going to submit the MLS biobrick RFC 10 and RFC 25 , the Freiburg format which allow protein fusion , to facilitate other team to fuse the MLS with other protein for purpose of introducing other protein into mitochondria. The MLS is sequenced and is confirmed to be free from illegal site. It is ligated with pSB1C3 in biobrick.
