Difference between revisions of "Part:BBa K2039000"

Revision as of 23:36, 19 October 2016

Part expressing the dimerization protein FKBP and GFP10 (subunit of tripartite GFP)

This part is the complementary part of Bba_K2039001.

The FRB/FKBP12 system is an inducible dimerization system. Originally found in mammals, these two proteins form an heterodimer when rapamycin is added in the medium. It is particularly useful for 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.

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 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