Photoreceptors are valuable optogenetic tools which, upon coupling with other proteins, activate certain functions in a controlled spatial and temporal manner when exposed to the appropriate wavelength of light. However, the usage of photoreceptors suffers from many drawbacks including the toxicity of the light to the cells, photobleaching of the receptors and the delay in the response i.e. the time needed for transcription and translation of the target protein to be controlled-. The emergence of Fluorescent light-inducible proteins is an attractive alternative that doesn’t suffer from these drawbacks.

Dronpa is one of the reversible photoswitchable fluorescent proteins (RSFPs), these are proteins that are switched on and off reversibly by specific wavelengths. Dronpa is switched on by default “fluorescent” and is switched off when illuminated by cyan light (~500nm). Dronpa Fluorescence is recovered by shining violet light (~400nm)

Figure 2:A fluorescent light-inducible protein design based on Dronpa Lys145Asn- From Zhou, X.X. and Lin, M.Z., 2013.

In Our project we wanted to explore other possible design strategies to control protein activity by light.

1) Our general design:

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 interst.

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.

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 to us to test if these repressors can be controlled by light thus creating a light inducible library of transcription factors. TetR, HK CI and P22 C2 function as homodimers. Our original hypothesis was that caging with Dronpa will prevent them from dimerization. To test this hypothesis we created the following constructs:

Figure5:

The three constructs have been tested with the logic gate in a cell free system, Results:

Figure 6:

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

Figure 7: Structure prediction of A) P22C2-dronpa fusion B)TetR-dronpa fusion C)HKCI-dronpa Fusion

Figure 8: Results of microscopic photos to test for the aggregation of the Repressors

The transcription factors that we have tested bind DNA as Homodimers, In our original hypothesis we wanted to create repressors that are caged by violet light and uncaged by cyan light, following the design that was created and validated by The Lin lab. We had two scenarios in mind for caging the repressors by Dronpa:

a) Binding of 2 dronpa domains to the repressors might prevent them from dimerization.

b) Since the repressors are very small in size, they might still be able to form a dimer. But in this case, this will result in the concentration of 4 copies of Dronpa which in turn will lead to an aggregation that will render the repressors nonfunctional.

However, the results we have obtained from testing our parts in a cell free system were very surprising. By illuminating our system with Violet light, the repressors are activated (indicated by low mRFP levels) while cyan light decreases their activity (indicated by high mRFP levels) Figure 7, which is the exact opposite of what we have expected.

As an interpretation of these data, we came up with the following scenario. The three repressors that we have tested are very small in size, and sin

T7 RNA polymerase (RNAP) transcribes only DNA downstream of a T7 promoter and it has a low error rate. Putting into account that T7 RNAP can undergo dramatic conformational changes during the transition from an initiation complex to an elongation complex. We hypothesized that adding two copies of Dronpa might control the activity of T7 RNAP by altering its conformation. To test this hypothesis we made the following construct:

Results:

L: Left, the structure of T7 RNAP binding DNA (PDB: 1t7p). Right:The prediction of the structure of Dronpa caging of T7 RNAP which suggests that the addition of the two dronpa copies is causing drastic changes in the the structure of the polymerase.

The inactivation of T7RNAP was confirmed using the following construct which is considered a self reporter as upon its activation mrfp should be generated. By cloning the part and plating the cells overnight in the dark, no RFP was generated

1) image with GFP filter, 2) image with RFP filter, 3) Bright field images.

Using error prone PCR we have obtained a version of Dronpa with 2 mutations I4V and R149H in the first dronpa domain and F78S in the second domain.

• Using the standard assembly, we added the pt7 mrfp reporter (BBa_K1758105) to our constructs of T7RNAP-Dronpa wt and T7RNAP-Dronpa mut.
• prepare overnight cultures of the colonies containing the 2 constructs ( T7RNAP-Dronpa wt + pt7 mrfp reporter) and ( T7RNAP-Dronpa mut + pt7 mrfp reporter) in LB with ampicillin
• Using a loop, streak colonies of each cultures on 2 plates (LB agar + ampicillin)
• Incubate the plates overnight in 37 degree with 2 conditions, dark and cyan
• Visualize the plates using bright field, gfp and rfp filters

In our construct for Dronpa we have used a T7 terminator, which is known to have a high readthrough depending on its contextuality. This suggests that the red colonies observed in figure () are more likely to be due to a transcription readthrough rather than T7RNAP activation.

Dronpa has been used successfully to cage proteases (Ref) and kinases (Ref). However it has been never used for optical control of enzymes with small substrates, as even if the enzyme is caged by 2 dronpa domains, that shouldn’t stop small molecules from reaching the enzyme’s active site.

β-galactosidase activity is measured by X-Gal (5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside) which is a chromogenic substrate that produces a blue color that can easily be detected visually. We made the following construct and we put it under testing.

Results:

Legend Native PAGE A) image with gfp filter B) Coomassie stain for Dilutions of wild type dronpa protein(wells 1-5) and Mut dronpa protein (wells 6-10) to confirm the previous results aren’t due to different protein concentration

Grow an overnight culture 16 h in LB with Ampicillin added

- Protein extraction using the bugbusters reagent and following the bugbuster protocol

Shining light :

Legend

The proteins extracted from the 3 different versions of pdDronpa were divided in 2 pcr tubes for each were light were shown for 30 seconds and for 30 minutes ( 2 different conditions ) light intensity is 20 mW/cm2

For each of the 3 different versions of pdDronpa they were placed in a pcr tubes and UV Led were shone for 30 seconds 8

- After light illumination, X-Gal dissolved in DMSO and diluted in PBS was added to to reach the final concentration of 5x

Dilution factor	1	1.25	1.66	2.5	5
Ul of Proteins	25	20	15	10	5
Ul ofPBS	-	5	10	15	20

Zhou, X.X., Chung, H.K., Lam, A.J. and Lin, M.Z., 2012. Optical control of protein activity by fluorescent protein domains. Science, 338(6108), pp.810-814.

Zhou, X.X. and Lin, M.Z., 2013. Photoswitchable fluorescent proteins: ten years of colorful chemistry and exciting applications. Current opinion in chemical biology, 17(4), pp.682-690.

Zhou, X.X., Fan, L.Z., Li, P., Shen, K. and Lin, M.Z., 2017. Optical control of cell signaling by single-chain photoswitchable kinases. Science, 355(6327), pp.836-842.