Team:Hong Kong HKUST/module3backup

Overview

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).

Mechanism of MLS

MLS is attached to the N-terminal of the protein in interest, and be recognized by receptor in the outer membrane of 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. Chaperone proteins will help to pull the protein across the membrane and help the refolding of protein. https://static.igem.org/mediawiki/2013/8/87/MLS_mechanism.png

Linkage to Project

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.

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. Click here to see our submitted parts.

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.