Team:Munich/Characterization

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Characterization

Characterization of the Firefly Luciferase (FLuc)

Aim

Determination of equilibrium parameters of the wild-type Firefly Luciferase (BBa_I712019) in vivo in mammalian cells using the cell-permeable substrate, D-Luciferin.

Design

As a first step, we ordered the sequence for the wild-type Firefly Luciferase (BBa_I712019) and ligated it into a backbone under the control of a CAG promoter optimised for HEK293T cells (BBa_K3113200) using the Gibson Assembly technique. After sequencing confirmation, we transfected HEK293T cells with the plasmid containing the sequence for Firefly Luciferase (pCAG_FLuc). Finally, we performed a luciferase assay with different D-Luciferin concentrations and measured the luminescence with a plate reader.

Materials and Measurement

HEK293T cells were transfected with pCAG_FLuc using our standardized protocol for transfection using Lipofectamine P3000. The cells were then grown for 48 hours at 37 °C and 5 % CO2. As negative control we included samples where water instead of substrate was added. A dilution series of D-Luciferin was prepared ranging from 0 μM up to 8 mM, which was then added 1:1 to the transfected cells, reaching final concentrations from 0 μM up to 4 mM. For the luciferase assay we used the GloMax® Discover Microplate Reader to measure the luminesence of our transfected cells. The assay was carried out at 37°C and no additional ATP or Mg2+ was added. For each substrate concentration we included four biological replicates, which were then averaged. D-Luciferin has a half-life of over 24 hours, allowing long-term measurements. Therefore, we measured our samples every five minutes over a period of approximately 8 hours, gaining more data for technical replicates.

Analysis

All the analyses were performed using Graphpad Prism (San Diego, CA).

Results

Time-resolved data of biological replicates for the Luciferin assay are summarized in Figure 1. We observed a concentration dependency of Firefly Luciferase in regard to D-Luciferin.
We determined the equilibrium binding constant, Kd(D-Luciferin), by plotting the averaged equilibrium values against the used D-Luciferin concentrations (Figure 2). The calculated equilibrium binding constant Kd(D-Luciferin) equals 0.9619 mM, representing the D-Luciferin concentration needed to achieve half-maximum binding at equilibrium. The solid line represents the fit used for the determination of the equilibrium binding constant.

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Figure 1: Luciferase Assay to determine equilibrium binding constant of the Firefly Luciferase and Luciferin. A dilution series of D-Luciferin was prepared reaching final concentrations from 0 μM up to 4 mM of D-Luciferin.
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Figure 2: The various D-Luciferin concentrations used throughout the luciferase assay were plotted against the averaged equilibrium values. The solid line represents a fit for specific binding (R2: 0.9892, n: 5).

Discussion

By following the luminescence signal over a longer time period and including four biological replicates, we obtained consistent data. We aimed to standardize this assay by establishig a standard curve with purified Firefly Luciferase from E.coli, but unfortunately we weren't able to successfully purify FLuc overexpressed from E.coli cells. However, we standardized our assay by seeding the same amount of cells into each well and transfecting them with the same concentration of DNA and included four biological replicates for each condition tested.