Difference between revisions of "Part:BBa K2039001"

Revision as of 17:21, 23 October 2016

Part expressing the dimerization protein FRB* and GFP11 (subunit of tripartite GFP)

This part is the complementary part of Bba_K2039000.

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.

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.

Figure 5: : No GFP florescence is observe with rapalog All the rapalog concentrations tested (5nM, 50nM, 500nM) display a signal inferior compared to the negative control.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 361
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 20: / Line 20: @@
 All the rapalog concentrations tested (5nM, 50nM, 500nM) display a signal inferior compared to the negative control. </center>
-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).
+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).
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