1) Design of protein Caging:

In our design, we had two copies of Dronpa Fluorescent Protein with two BsaI cutting site in between to allow the insertion of our genes of interest .

Figure 3: construct design for 2 Dronpa domains

2) Molecular mechanism:

The enzyme of interest is placed between 2 copies of Dronpa fluorescent protein via a linker. By switching on Dronpa (violet light) the protein of interest is caged as the two copies would dimerize. And by switching off Dronpa (cyan light), the two copies would dissociate which will result in activation of the protein of interest.

Figure 4: Shining cyan and violet light using LEDS.

3) MutDronpa:

Using error prone PCR we have obtained several versions of Dronpa with several mutations from which we picked one version that interested us the most, as it had an R149H mutation which is located in the dimer interface. We proceeded in our experiments with 2 variants of Dronpa. The original version was annotated as wtDronpa and the mutated version was annotated as mutDronpa which has 2 mutations I4V and R149H in the first dronpa domain and an F78S mutation in the second domain.

Repressors bind DNA and setback transcription. In our project we developed a logic gate at the promoter level by creating dually repressed promoters using different combinations of the operators for TetR, P22 c2 and HK CI and it was interesting for us to test if these repressors can be controlled by light thus creating a light-inducible library of transcription factors. Dronpa hasn’t been used to control transcription factors, so we developed the following constructs to test if Dronpa can control proteins that function by dimerization.

Figure5: A diagram of the design of Dronpa-Repressors constructs.

By running a structure prediction test [figure 6], we could obtain some information regarding the expected behavior of the output of our design. In our design, the two Dronpa domains are connected to the repressors via long flexible linkers, which indicates that homodimers of repressors are likely to be formed even if caged by dronpa. The folding of the repressors that we are testing will be fast due to their small size and simple structure, which will make the dimerization of 2 copies of the repressors -before the complete folding of the second dronpa domains and caging the repressor- very likely. We expected in our design that the repressor homodimer will bind to its operator and once the second domain of Dronpa is fully folded, it will cage the already bound repressor to the DNA thus further stabilizing the repression when the violet light is shone.

Figure 6: Structure prediction of A) P22C2-dronpa fusion B)TetR-dronpa fusion C)HKCI-dronpa Fusion

To validate our design, we tested the three constructs in [figure 5] for both wtDronpa and mutDronpa with the logic gate of the dually repressed promoters in a cell free system giving us 96 combination.

Figure 7: In Vitro characterization of the activity of the repressors using cell free system

By illuminating our system with violet light, caging increase the repression (indicated by low mRFP levels) while cyan light decreases the repression (indicated by high mRFP levels) [Figure 7].