Measurement
Being the first iGEM team at Shandong Experimental High School to work with bioluminescence imaging when we started this project, we have gained a lot of knowledge about bioluminescence over the past few months. We gathered information on safety, organic synthesis, necessary instruments, cell culture, storage, transfection, animal experiment, measuring, and other related topics that we wanted to share with others.
We performed a number of biological examinations to improve our measurement procedure after preparing our cyL molecules. Using a microplate reader, we first determined the bioluminescence intensity of our bioassay. However, the bioluminescence emission from these compounds is too weak in vitro to be detected by the microplate reader. Because of its great sensitivity, we decided to measure bioluminescence in vitro and in cellulo using an IVIS Spectrum imaging system.
Note that there is a problem with in vitro and cellulo imaging in 96-well microplates; the edge effect may disrupt imaging data in rows A and H. We then customized a pipette tip rack by removing the two outermost rolls of the plate, as shown in Scheme 1, in order to increase the precision of the imaging results. Please visit https://2024.igem.wiki/sehs/hardware for further information.
Scheme 1. Tailor-made pipette tip rack
We successfully completed the in vitro imaging of cyL and determined the kinetic parameters of each compound with the use of a customized pipette tip rack. When compared to D-luciferin and aminoluciferin, the low kinetic constants Km and Vmax of 5-cyL, 6-cyL, 7-cyL, and 8-cyL indicate that the reaction rate and affinity have clear potential (Table 1). These findings suggest that these cyL compounds may be more sensitive to firefly luciferase when used as novel substrates. We also evaluated the correlation between the concentration of ATP and the intensity of bioluminescence (Figure 1). Analogous to substrates D-luciferin and aminoluciferin, the bioluminescence intensity of cyL compounds exhibits a significant positive correlation with an increase in ATP concentration, suggesting potential applications for the cyL compounds as in vivo ATP sensors.
Table 1.The bioluminescence properties of 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin and aminoluciferin
| Substrates | λmax(nm) | Vmax (s-1) | Km (mol/L) |
|---|---|---|---|
| dLuc | 560 | 9.68×1010 | 10.9 |
| aLuc | 588 | 1.89×1010 | 6.63 |
| 5-cyL | 598 | 4.67×109 | 0.344 |
| 6-cyL | 610 | 3.69×109 | 0.274 |
| 7-cyL | 605 | 5.90×109 | 0.281 |
| 8-cyL | 613 | 4.367×109 | 0.313 |
Figure 1. In Vitro Bioluminescence Assay. Substrate dose-response analysis: left: (1) Total flux (p/s) of substrates at different substrate concentrations (μM). (2) The bioluminescence intensity of six luciferases at different substrate concentrations (μM); right: (1) Total flux (p/s) of substrates at different ATP concentrations (μM). (2) The bioluminescence intensity of substrates at different ATP concentrations(μM).
Our group successfully measured bioluminescence imaging in living U87-Luc cells to determine the bioluminescence intensity of various cyL compounds. Following the incubation period, the medium was withdrawn, and various concentrations of cyL compounds were added. As a result, these cyL compounds have excellent sensitivity, strong cell permeability, and bioluminescence activity. Under the same substrate concentration conditions, there is a clear positive association between the bioluminescence intensity and the concentration of U87-Luc cells (Figure 2).
Figure 2. Cell Bioluminescence imaging in U87-Luc cells. (left) Total flux(p/s) of five different luciferases at different cell concentrations (μM). (right) The light intensity of five luciferases at different cell concentrations (μM).
Even though we had to use this method to quantify bioluminescence, we intend to use optimized settings to file a patent after the jamboree. In the future, we would love to provide details on how to handle and examine plate data for bioluminescence cells.