Team:SJTU-BioX-Shanghai/Parts2

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Membrane Anchor System

Membrane Anchor Rudder System

12SJTU-MA1-MA6.png



Membrane Achor 1 (BBa_K771001)
Membrane Achor 2 (BBa_K771002)
Membrane Achor 3 (BBa_K771003)
Membrane Achor 4 (BBa_K771004)
Membrane Achor 5 (BBa_K771005)
Membrane Achor 6 (BBa_K771006)

A novel device which could be used to switch direction of reactions through VVD and RNA signal. For more data, see http://partsregistry.org/wiki/index.php?title=Part:BBa_K771001.

Membrane Anchor Complex System

12SJTU-COMPLEX.png


Membrane Achor 2 without MS2(BBa_K771007)
Membrane Achor 3 (BBa_K771003)
Membrane Achor 4 (BBa_K771004)
Membrane Achor 5 without VVD(BBa_K771008)

A device designed to assemble enzymes on E.coli membrane to accelerate reactions and export products more efficiently.

Membrane Protein with A Selection Marker

Membrane Achor 0 (BBa_K771000)

12SJTU-MA0.png

Membrane Achor with GFP


Membrane Achor 0 -GFP(BBa_K77401)

12SJTU-project1-1.png

To justify the localization ability of fusion membrane proteins mentioned above, we constructed a novel fusion protein called BlaLG. β-lactamase is fused to the N-terminus of Lgt and GFP is fused to the C-terminus of Lgt. The fusion protein in under control of araBAD promoter .




split EGFP 1 with Membrane Achor 2 (BBa_K771407)
split EGFP 2 with Membrane Achor 3 (BBa_K771403)
split EGFP 1 with Membrane Achor 4 (BBa_K771404)
split EGFP 2 with Membrane Anchor 5(BBa_K771405)

To prove whether our membrane protein equipped with those interacting protein could assemble with each other, we conducted fluorescence complementation assay. In fluorescence complementation assay, proteins that are postulated to interact are fused to unfolded complementary fragments of EGFP and expressed in E.coli. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. EGFP was split into two halves, named 1EGFP and 2EGFP respectively. If there is interaction between two proteins which were fused with 1EGFP and 2EGFP, it is expected that fluorescence should be observed. Otherwise, no fluorescence could be observed.

Group 1, 2, 3 are designed to test the interaction of SH3domain & ligand, PDZdomain & ligand and GBDdomain & ligand respectively. Proteins within each group were expressed in E.coli. If there exists interaction within membrane proteins of each group, we expected to observe fluorescence on E.coli membrane. Control group is supposed to show much weaker fluorescence