Difference between revisions of "Part:BBa K2039002"

Revision as of 17:21, 23 October 2016

Dimerization system FRB/FKBP linked to two subunit of tripartite GFP (GFP 10 & GFP 11)

This part is the association of part K2039000 and K2039001.

The FRB/FKBP12 system is an inducible system. Originally found in mammals, these two proteins form an heterodimer when rapamycin is added in the middle, it is particularly used in protein interaction studies. The FRB sequence has been modified in order to dimerize with a non toxic rapamycin analog (rapalog). The mutations introduced are : T2098L, K2095P, W2101F. It has also been codon optimized with Jcat plateforme to be expressed in bacteria.

FKBP protein is linked to GFP 10 and FRB* protein is linked to GFP 11.

The tripartite split-GFP is composed of two times of 20 amino-acids long GFP tags (GFP 10 and GFP 11) and a third complementary subsection (GFP 1-9).

Characterization

Test of the dimerization system with a tripartite GFP

We transformed E.coli with pSB1C3 coding for FKBP-GFP10 and FRB-GFP11 and pUC19 coding for the third part of GFP as we could not clone GFP1.9 in pSB1C3. Transformed cells where then incubated with the dimerization agent, rapalog. We try several concentration of rapalog but we did not see any GFP fluorescence with flow cytometer.

We did not obtain the fluorescent signal that we expected with the dimerization agent. We thought that maybe the rapalog does not penetrate into the cell. We made a second test with cell lysate in order to address this issue. Unfortunately we did not observe significant fluorescence either ( mean fluorescenece control : 72,33 +/- 6,29, mean fluorescence with 150nM rapalog : 85,33 +/- 2,36).

We did not have time to investigate further why the system did not work, but we have several ideas. We may have some expression issues with FRB*-GFP11 and FKBP-GFP10 as the western blot done with monoclonal antibody anti-GFP allow the revelation of GFP1.9 only. Another possibility is that there is some physical constraint that prevent GFP assembly, the linker length for example can generate this kind of constraint. Finally, the system can by well-constructed and expressed but we may not have found the optimal conditions to induce the dimerization (concentration of rapalog, timing, temperature).

Sequence and Features