Composite

Part:BBa_K2039002

Designed by: Coline Sivelle   Group: iGEM16_Paris_Saclay   (2016-10-13)


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.

T--Paris Saclay--Testbiobrick1.png
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. Results are displayed on the plot below, each measurement was made in triplicate. We observe a slight difference between the mean fluorescence of the control (72,33 +/- 6,29) and the mean fluorescence with 150nM rapalog (85,33 +/- 2,36).

Paris-Saclay-Characterization.png

We observed a weak difference between the control and the induced samples. This difference may be more important if we consider a possible degradation of GFP parts during the lysate preparation. This experiment should be repeated for a more complete characterization.

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
    Illegal NheI site found at 716
    Illegal NheI site found at 739
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1255
    Illegal BamHI site found at 361
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 866
    Illegal AgeI site found at 1040
  • 1000
    COMPATIBLE WITH RFC[1000]


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