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Overview
Below you can find a description about the approaches that we used to proof that we indeed have made two constructs that will aggregate and form a large structure, similar to a gel.
We have investigated the following conditions:
- Scaffold Construct (14-3-3 with GFP) + Binding Partner (CT33 with Strep-tag and mCherry) + Inducer + Strep-tactin
- Scaffold Construct (14-3-3 with GFP) + Binding Partner (CT33 with Strep-tag and mCherry) + Inducer
- Scaffold Construct (14-3-3 with GFP) + Binding Partner (CT33 with Strep-tag and mCherry) + Strep-tactin
Table 1: Concentration of our constructs used to obtain the results
| Construct |
Proteins |
Concentration (µM) |
| Scaffold |
14-3-3 with GFP |
66 |
| Center Point |
Strep-tactin |
49.5 |
| Binding Partner |
CT33 with mCherry and strep-tag II |
198 |
| Inducer |
Fusicoccin |
330 |
FRET Measurements
The first approach was executed with a plate reader measurement. The first step is to excite the fluorophore GFP, which will then emit light with a higher wavelength. A part of this emitted light has a wavelength that can excite the fluorophore mCherry, which will then also emit light with an even higher wavelength. This principle is called Förster Resonance Energy Transfer (FRET)[1] and mostly used to indicate a close proximity between two proteins. If our system does not behave as expected, there would be no difference between the induced and non-induced state.
In Figure 1, fluorescence measurements are shown after 1 h and after 24 h. As can be seen for the results after 1 h, the systems behaves as expected as the FRET signal for the condition with all constructs has the highest signal. However, the condition without fusicoccin also shows a high signal, which is unexpected. The results after 24 h show that the FRET signal is relatively low for the condition without fusicoccin, compared to the other 2 conditions, indicating that fusicoccin is needed for network formation after a longer incubation time. For Strep-tactin, the FRET signal fof the condition overnight without Strep-tactin is higher than the condition with all constructs.
Microscopy
An even better indication of close proximity between the two constructs can be generated by using a microscope. With the microscope we can make two images, one where we visualize GFP and one where we visualize mCherry, and overlay them to see that the two different constructs are close to each other.
Additionally, we can even visualize large clusters by measuring one of the fluorophores and compare this with all the other states described above.
In Figure 2, fluorescence microscopy images are shown after 48 h of incubation. As can be seen from the overlay spectra, the presence of fusicoccin as well as the presence of Strep-tactin is crucial for network formation. Without fusicoccin, no network formation is visible after 48 h. Without the presence of Strep-tactin, the mCherry and GFP signals are in close proximity, but it is unclear whether network formation has occured. This indicates that our system works as expected: a network is formed in presence of an inducer, and multivalency is also key to network formation.
Conclusion
The FRET results show no clear difference between the different conditions, which is probably due to a too high background signal. The microscopy results show a clear difference between the different conditions. We see that all constructs together lead to clusters with sizes of approximately 10-20 µm. The absence of Strep-tactin results in very small clusters, probably presenting single scaffold constructs bound to binding partners. This interaction is due to the presence of Fusicoccin. If Fusicoccin is absent, we only see signals in the GFP microscopy results and not for the mCherry. Therefore the overlay picture also shows no signal leading us to conclude that Fusicoccin indeed acts as an inducer for our system.
[1] B.T. Bajar, E.S. Wang, S. Zhang, M.Z. Lin and J. Chu, "A Guide to Fluorescent Protein FRET Pairs", Sensors (Basel), vol 16, no. 9, pp 1488, (2016)
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