Achivements

We designed and successfully tested three light inducible split proteases: CIBN/CRY2PHR light inducible split TEV protease, CIBN/CRY2PHR light inducible split PPVp and CIBN/CRY2PHR light inducible split TEVpE.

Introduction

In the recent years, light has been extensively explored as a trigger signal for activation of different biological processes. Small molecules and other chemical signals lack spatial resolution and their temporal resolution is limited by the time required for the cell permeation. In comparison, induction by light as developed by the optogenetics offers many advantages. It is fast as well as inexpensive and allows for excellent spatial, temporal and dose-dependent control.

Initially we decided to test the LOVpep/ePDZ system. This system has been used previously at iGEM, by EPF_Lausanne 2009, Rutgers 2011 and Rutgers 2012 and in mammalian cells by Freiburg_2014. AsLOV2 is a small photosensory domain from Avena sativa phototropin 1 with a C-terminal Jα helix. The Jα helix is caged in darkness but unfolds upon blue light (< 500 nm) photoexcitation, which is crucial for phototropin signaling.

Results

For initial testing and characterization of the system, we fused the LOVpep and ePDZb Muller2014 to corresponding segments of the split firefly luciferase. We tested different positions of the split protein on the PDZ domain, while the split protein was kept at the N-terminus of the LOVpep domain due to the importance of the C-terminal peptide epitope (1).

We tested two orientations of ePDZ (ePDZ fused to split luciferase to N or C terminus) and ratios of the constructs ePDZ vs. cLuc:LOVpep were tested (2).

As the measured signal was the highest with split luciferase on the N-terminus of the ePDZ domain and a 1:3 ratio of LOVpep:ePDZ, all subsequent experiments were performed with this ratio. An important feature for real life applications is the ability of the system to be stimulated multiple times. Therefore, repeated association and dissociation was tested in the real time, by adding luciferin to the medium and measuring bioluminescence upon induction by light (Figure 4.9.1.C.). The system showed a delayed, but successful induction the first time, but the second induction was much weaker. The results indicated that the LOVpep/ePDZ system in this setup could not be induced more than once, so we decided to test another system.

As it has previously been shown on the example of split Cre recombinase Kennedy2010 the CRY2-CIB1 interaction seems to be a suitable tool for the reconstitution of split proteins, allowing for spatial, temporal and dose-dependent optical control of protein dimerization. CRY2 system has also been sued before at iGEM: Duke 2012, Marburg 2013, HNU_China 2014 and ANU_Canberra 2015

We adapted this system for the reconstitution of split luciferase to create a blue-light sensor, which enables easy characterization for further experiments. The N- and C-terminal split fragments of the firefly luciferase were fused to the C-terminus of the CRY2PHR and the CIBN proteins, since this topology has previously been shown to work with the Cre recombinase (3).

As the measured signal was the highest with a 1:3 ratio of CRY2PHR/CIBN (4A), all subsequent experiments were performed with this ratio. Next we tested if this system could be induced repeatedly in real time. The CRY2PHR/CIBN system shows maximum activity after 2 minutes of induction, returns to background in 10 minutes after the stimulus is removed, and can be induced repeatedly (4B).

To implement light as one of the input signals for protease-based signaling pathway or logic functions, we fused the CRY2PHR and the CIBN domains to the N- and C-terminal split domains of 3 different proteases (TEVp, TEVpE and PPVp) (5A) and tested their activity and orthogonality with bioluminescence assays (5B) and western blotting (5C). The bioluminescence assay was based on the split firefly luciferase with cleavage site for either TEVp, TEVpE or PPVp. Cleavage of this reporter results in the luciferase activity reconstitution and thus an increase in the luminescence.

The reporter used for the western blot analysis was the luciferase reporter with the appropriate cleavage substrate inserted at a permissible site and an AU1 tag at the N-terminal. Uncleaved luciferase appears as a single band on a western blot, while partial cleavage of luciferase results in two bands (uncleaved at 65 kDa and cleaved at 55 kDa) and complete cleavage results in only the smaller band (6). We showed that substrates carrying specific target peptide for proteases were cleaved after induction of split protease reporters.

Both methods showed a successful, fast and dose-dependent response. This is the first time split TEV protease has been shown to work as a light inducible system. Also this is the first time TEVpE and PPVp were prepared as split proteins and shown to function in an inducible system.