Click beetle luciferase

Generate Constructs for Click Beetle Luciferase Complementation Fusion Proteins... 

Plate cells at a density of 1 X 10 —2 X 10 cells per well in 100 pi complete growth medium with serum in black walled, clear bottom 96-well plates for tissue culture. Since complementation of click beetle luciferase fragments produces less bioluminescence than intact CBGN or red, we typically use 96-weIl rather than 384-weU plates for cell-based assays. This approach allows us to use shorter acquisition times in each emission channel and improves resolution for kinetics of signahng. For 384-weU plate assays, we use 3 x 10 —5 X 10 cells perweU. We typically culture cells for two days in 96-weU plates before assays. 

Villalobos, V., Naik, S., Bruinsma, M., Dothaget, R., Pan, M., Samrakindi, M., etal. (2010). Dual-color click beetle luciferase heteroprotein fiagment complementation assays. Chemistry Biology, 17, 1018—1029. 

Firefly luciferase catalyzes a reaction that produces visible light in the 550-575 nm range. A click-beetle luciferase is also available that produces light at a peak closer to 595 nm. Both luciferases require the addition of an exogenous substrate (luciferin) for the light reaction to occur. Numerous luc-hased bioreporters have been constmcted for the detection of a wide array of inorganic and organic compounds of environmental concern. 

The bioluminescence systems of Phengodidae (railroad worms) and Elateroidae (click beetles) are basically identical to that of Lampyridae (fireflies), requiring firefly luciferin, ATP, Mg2+ and a luciferase for light emission. However, there seem to be some differences. Viviani and Bechara (1995) reported that the spectra of the luminescence reactions measured with the luciferases of Brazilian fireflies (6 species) shift from the yellow-green range to the red range with lowering of the pH of the medium, like in the case of the Photinus pyralis luciferase (see Section 1.1.5), whereas the spectra... 

The elaterid Pyrophorus is of special importance in the history of bioluminescence, because it was used by Dubois in his first demonstration of the luciferin-luciferase reaction in 1885. The Jamaican click beetle (Pyrophorus noctilucus) is commonly found in the West Indies. The beetle possesses two kinds of luminous organs. A... 

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