Red-emitting luciferases for bioluminescence reporter and imaging applications

Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

“…We constructed yeast reporters for a variety of beetle luciferases to evaluate their in vivo luminescence and spectral separation when expressed from the strong constitutive yeast promoter TEF1. A strain of S. cerevisiae (CEN.PK113-7D) was stably transformed with plasmids that expressed CBG99 and CBR click beetle luciferases (https://www.promega.com/ϳ/media/files/resources/ promega%20notes/85/introducing%20chroma-luc%20technolo gy.pdf?laϭen) and Ppy RE8 and Ppy RE9 red-shifted firefly luciferases (26). These yeasts were patched onto a solid medium containing beetle luciferin and allowed to grow overnight at 30°C before being imaged with a charge-coupled device (CCD) camera.…”

“…Redshifted firefly luciferase reporters pRS306-Ppy RE8 and pRS306-Ppy RE9 were created by replacing the CBR coding sequence of pRS306-CBR with that of Ppy RE8 or Ppy RE9 (26). To do this, Ppy RE8 and Ppy RE9 coding Padh1-3(BglII) ctacatAGATCTGGAGTTGATTGTATGCTTGG sequences were PCR amplified using primers 4 and 5 (Table 1) and introduced into pRS306-CBR using BglII and NheI, replacing CBR.…”

In Vivo Cell Tracking with Bioluminescence Imaging

“…Also, Kitamaya et al [30] reported on the thermal instability and pH-sensitive spectral property of firefly luciferase which can affect its use as a sensitive multicolor luminescence label or bioluminescence resonance energy transfer donor. In addition, the isolation of bright, red-shifted variants of Photinus pyralis luciferase in order to minimize light absorption and scattering by tissue for in vivo animal studies has also been reported [31][32][33].…”

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

“…Between l = 600-800 nm, the absorption of light by Hb decreases by a factor of approximately 50, resulting in less attenuation of red light. This wavelength range is within what is termed the "bio-optical window" and there has been much focus on engineering red-shifted Fluc enzymes that have maximum emission wavelengths in this range, [10][11][12][13][14][15] but these have peaked at wavelengths less than l = 645 nm.The most red-shifted luciferin analogues to date [16] are based upon amino derivatives (Figure 1 b), for example cyclic aminoluciferin (3 a: l max = 599 nm; 3 b: l max = 607 nm), [17] seleno-d-aminoluciferin (4: l max = 600 nm), [18] and a rationally designed 4-(dimethylamino)phenyl derivative conjugated to a thiazoline group (5: l max = 675 nm). [19] In particular cyclic aminoluciferin derivative 3 a has been shown to give improved bioluminescence imaging compared to luciferin (LH 2 ; 1) at dilute concentrations where the intracellular concentration of the luciferin or analogue is limiting.…”

“…Between l = 600-800 nm, the absorption of light by Hb decreases by a factor of approximately 50, resulting in less attenuation of red light. This wavelength range is within what is termed the "bio-optical window" and there has been much focus on engineering red-shifted Fluc enzymes that have maximum emission wavelengths in this range, [10][11][12][13][14][15] but these have peaked at wavelengths less than l = 645 nm.…”