Oct 05, 2025
Add Coelenterazine-h
- igemnthuth 1
- 1iGEM_NTHU_Taiwan
- iGEM NTHU
Protocol Citation: igemnthuth 2025. Add Coelenterazine-h. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmbzzog3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2025
Last Modified: October 05, 2025
Protocol Integer ID: 228984
Keywords: length reng fusion protein, optimal pairing of the spytag, maximum luminescence reference, length reng luciferase, luminescence intensity, spycatcher system, subsequent slca interaction signal, maximum luminescence value, relative standard for subsequent slca interaction signal, coelenterazine, reng fragment, positioning of the reng fragment, spycatcher, spytag, slca, assay, slca construct, reng
Abstract
Add Coelenterazine-h as the substrate (light-sensitive; prepare fresh on the day of use) to activate the full-length RenG luciferase and measure its maximum luminescence value. This serves as the relative standard for subsequent SLCA interaction signals. Since three configurations will be tested, record the strains and culture conditions to ensure consistency.
Next, measure the luminescence intensity of the three SLCA constructs to compare their complementation efficiency and background signals, thereby establishing the optimal pairing of the SpyTag/SpyCatcher system and the positioning of the RenG fragments.
- SLCA assay 1: Full-length RenG fusion protein, serving as the maximum luminescence reference.
- SLCA assay 2: SpyTag + RenG (N-terminal) with SpyCatcher + RenG (C-terminal).
- SLCA assay 3: SpyTag + RenG (C-terminal) with SpyCatcher + RenG (N-terminal).
Materials
- Strains harboring expression plasmids with the following constructs (designed plasmids already introduced by electroporation):
Full-length RenG
SpyTag–RenG (NTD, aa 1–155) and SpyCatcher–RenG (CTD, aa 156–314)
SpyTag–RenG (CTD) and SpyCatcher–RenG (NTD)
- THYE liquid medium (with appropriate antibiotic)
- Coelenterazine-h (working range 2–10 µM, pre-dissolved in DMSO; optimize experimentally to identify an appropriate concentration for downstream use)
- Sterile white 96-well luminometer plate (luminescence-compatible)
- ddH₂O or PBS
Troubleshooting
Strain Pre-culture and Preparation
For each strain, pick a single colony and inoculate into THYE medium containing the appropriate antibiotic. Incubate overnight at 37 °C, 220 rpm.
The next day, dilute 1:50 into fresh THYE medium with antibiotic and continue incubation until OD₆₀₀ ≈ 0.4–0.6 (mid-log phase).
Dispensing into a 96-well Plate
Dispense 100 μL of log-phase culture into each well of a white 96-well luminometer plate, preparing triplicates per condition.
In a separate row, dispense blank controls (medium only, no cells).
Adding Coelenterazine-h Substrate
Prepare the Coelenterazine-h working solution at [ ] μM in DMSO (light-protected).
Quickly add 20 μL of Coelenterazine-h to each well (final concentration [ ] μM) and mix immediately (gentle pipetting or plate tapping).
Luminescence Measurement
Within 3–5 minutes after substrate addition, measure luminescence for each well using a plate reader.
Record the raw RLU (Relative Light Units) for each well and subtract the blank (medium-only) background.
In SLCA assay 1, the luminescence of full-length RenG is defined as the maximum (Max RLU); report other assay signals as Relative Activity = (sample RLU) / (Max RLU).