Team:TU-Eindhoven/Read-out/Tetramer

iGEM TU Eindhoven

Experiments Tetramer

To test if the newly created scaffold proteins are working as expected, we have designed several read-out systems for the heterodimer and for the tetramer.

Tetramer

The most important aspects of testing the tetramer are the activity of the homotetramer compared to the homodimer and the efficiency of our heterotetramer, for this we designed a caspase-9 activity assay and a 3 component BRET system assay respectively.

Caspase-9 Activity Assay

The caspase-9 activity assay consists of CT52 fused to caspase-9 proteins, these will dimerize on the scaffold after adding fusicoccin. This dimerization on the scaffold leads to an active caspase-9 dimer whose activity was examined using the fluorogenic substrate Ac-LEHD-AFC. When cut by caspase-9, this substrate releases a fluorescent group which can be measured. The difference in specific activity between the tetramer and dimer will be compared. We initially expected the tetramer to result in a higher activity, because it resembles the naturally occurring apoptosome more than a dimer, however the Protein-Protein interaction models predicted that the specific activity of caspase-9 on the tetramer is actually lower than that of the dimer.

The level of light measured is an indicator for the amount of activated caspase-9. This assay will also be conducted with a homodimeric T14-3-3 scaffold, in order to compare the result of the tetrameric scaffold assay to the results of the dimeric scaffold assay.

Figure 1: the tetrameric scaffold increases the local concentration of caspase-9 more than a dimer, inducing the apoptosis pathway more efficiently.

BRET Assay

The second assay will be conducted in order to verify if a tetramer with four different binding sites has successfully been created. The second assay is based on the

BRET System,

BRET stands for Bioluminescence Resonance Energy Transfer. It involves resonance energy transfer between a bioluminescent donor and a fluorescent acceptor.

the BRET system consists of three parts.

Figure 2: The BRET system is activated by the binding of NanoBiT and mNeonGreen on our scaffold.

Each of these parts is linked to a different form of CT52. The two CT52 split NanoLuc fragments will bind next to each other and lead to a functional NanoLuc, emitting light at a wavelength of 460 nm. On the other free binding sites CT52 with linked mNeonGreen will bind. In the BRET system, NanoLuc will be the donor and mNeonGreen will be the acceptor that changes the wavelength of the light emitted by NanoLuc to a wavelength of 517 nm (Figure 2). So if the tetramer scaffold is functional, light with a wavelength of 517 nm and 460 nm should be measured.