Sensitive Assay for Mycoplasma Detection in Mammalian Cell Culture

Mycoplasma contamination in mammalian cell cultures is often overlooked yet is a serious issue which can induce a myriad of cellular changes leading to false interpretation of experimental results. Here, we present a simple and sensitive assay to monitor mycoplasma contamination (mycosensor) based on degradation of the <i>Gaussia</i> luciferase reporter in the conditioned medium of cells. This assay proved to be more sensitive as compared to a commercially available bioluminescent assay in detecting mycoplasma contamination in seven different cell lines. The <i>Gaussia</i> luciferase mycosensor assay provides an easy tool to monitor mammalian cell contaminants in a high-throughput fashion

Directed Molecular Evolution Reveals Gaussia Luciferase Variants with Enhanced Light Output Stability

Gaussia Luciferase (Gluc) has proven to be a powerful mammalian cell reporter for monitoring numerous biological processes in immunology, virology, oncology, and neuroscience. Current limitations of Gluc as a reporter include its emission of blue light, which is absorbed by mammalian tissues, limiting its use in vivo, and a flash-type bioluminescence reaction, making it unsuited for high-throughput applications. To overcome these limitations, a library of Gluc variants was generated using directed molecular evolution and screened for relative light output, a shift in emission spectrum, and glow-type light emission kinetics. Several variants with a 10–15 nm shift in their light emission peak were found. Further, a Gluc variant that catalyzes a glow-type bioluminescence reaction, suited for high-throughput applications, was also identified. These results indicate that molecular evolution could be used to modulate Gluc bioluminescence reaction characteristics