Development of a Destabilized Firefly Luciferase Enzyme for Measurement of Gene Expression

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

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Firefly luciferase as a reporter of regulated gene expression in higher plants

Plant Molecular Biology Reporter ◽

10.1007/bf02668909 ◽

1992 ◽

Vol 10 (4) ◽

pp. 324-337 ◽

Cited By ~ 90

Author(s):

Andrew J. Millar ◽

Sharla R. Short ◽

Kazuyuki Hiratsuka ◽

Nam-Hai Chua ◽

Steve A. Kay

Keyword(s):

Gene Expression ◽

Higher Plants ◽

Firefly Luciferase ◽

Regulated Gene Expression

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The inducible lac operator-repressor system is functional in zebrafish cells

10.1101/2020.02.10.939223 ◽

2020 ◽

Author(s):

Sierra S. Nishizaki ◽

Torrin L. McDonald ◽

Gregory A. Farnum ◽

Monica J. Holmes ◽

Melissa L. Drexel ◽

...

Keyword(s):

Gene Expression ◽

Model Organism ◽

Firefly Luciferase ◽

Regulatory Elements ◽

Regulatory Sequences ◽

Flexible Tool ◽

Lac Operator ◽

Promising Tool ◽

Reporter Assays ◽

Spatio Temporal

AbstractBackgroundZebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, a component of the E. coli lac operon which has been adapted for use in many other species and is a valuable, flexible tool for studying the inducible modulation of gene expression, has not previously been tested in zebrafish.ResultsHere we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish.ConclusionsOur results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe that this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.

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Evaluation of Luciferase Thermal Stability by Arginine Saturation in the Flexible Loops

Current Proteomics ◽

10.2174/1570164616666190320151005 ◽

2020 ◽

Vol 17 (1) ◽

pp. 30-39

Author(s):

Farzane Kargar ◽

Mojtaba Mortazavi ◽

Masoud Torkzadeh-Mahani ◽

Safa Lotfi ◽

Shahryar Shakeri

Keyword(s):

Thermal Stability ◽

Structural Changes ◽

Specific Activity ◽

Temperature Stability ◽

3D Models ◽

Firefly Luciferase ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Original Activity ◽

Luciferase Enzyme

Background: The firefly luciferase enzyme is widely used in protein engineering and diverse areas of biotechnology, but the main problem with this enzyme is low-temperature stability. Previous reports indicated that surface areas of thermostable proteins are rich in arginine, which increased their thermal stability. In this study, this aspect of thermophilic proteins evaluated by mutations of surface residues to Arg. Here, we report the construction, purification, and studying of these mutated luciferases. Methods: For mutagenesis, the QuikChange site-directed mutagenesis was used and the I108R, T156R, and N177R mutant luciferases were created. In the following, the expression and purification of wild-type and mutant luciferases were conducted and their kinetic and structural properties were analyzed. To analyze the role of these Arg in these loops, the 3D models of these mutants’ enzymes were constructed in the I-TASSER server and the exact situation of these mutants was studied by the SPDBV and PyMOL software. Results: Overall, the optimum temperature of these mutated enzymes was not changed. However, after 30 min incubation of these mutated enzymes at 30°C, the I108R, T156R, N177R, and wild-type kept the 80%, 50%, 20%, and 20% of their original activity, respectively. It should be noted that substitution of these residues by Arg preserved the specific activity of firefly luciferase. Conclusion: Based on these results, it can be concluded that T156R and N177R mutants by compacting local protein structure, increased the thermostability of luciferase. However, insertion of positively charged residues in these positions create the new hydrogen bonds that associated with a series of structural changes and confirmed by intrinsic and extrinsic fluorescence spectroscopy and homology modeling studies.

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Use of the firefly luciferase gene as a reporter of gene expression in plants

Plant Molecular Biology Manual ◽

10.1007/978-94-009-0951-9_18 ◽

1989 ◽

pp. 359-369 ◽

Cited By ~ 3

Author(s):

Stephen H. Howell ◽

David W. Ow ◽

Michel Schneider

Keyword(s):

Gene Expression ◽

Firefly Luciferase ◽

Luciferase Gene ◽

Firefly Luciferase Gene

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A synthetic luciferin improves in vivo bioluminescence imaging of gene expression in cardiovascular brain regions

Physiological Genomics ◽

10.1152/physiolgenomics.00055.2016 ◽

2016 ◽

Vol 48 (10) ◽

pp. 762-770 ◽

Cited By ~ 3

Author(s):

Hayk Simonyan ◽

Chansol Hurr ◽

Colin N. Young

Keyword(s):

Gene Expression ◽

Targeted Delivery ◽

Bioluminescence Imaging ◽

Firefly Luciferase ◽

Brain Regions ◽

Cmv Promoter ◽

Brain Nuclei ◽

Molecular Events ◽

In Vivo Bioluminescence Imaging

Bioluminescence imaging is an effective tool for in vivo investigation of molecular processes. We have demonstrated the applicability of bioluminescence imaging to spatiotemporally monitor gene expression in cardioregulatory brain nuclei during the development of cardiovascular disease, via incorporation of firefly luciferase into living animals, combined with exogenous d-luciferin substrate administration. Nevertheless, d-luciferin uptake into the brain tissue is low, which decreases the sensitivity of bioluminescence detection, particularly when considering small changes in gene expression in tiny central areas. Here, we tested the hypothesis that a synthetic luciferin, cyclic alkylaminoluciferin (CycLuc1), would be superior to d-luciferin for in vivo bioluminescence imaging in cardiovascular brain regions. Male C57B1/6 mice underwent targeted delivery of an adenovirus encoding the luciferase gene downstream of the CMV promoter to the subfornical organ (SFO) or paraventricular nucleus of hypothalamus (PVN), two crucial cardioregulatory neural regions. While bioluminescent signals could be obtained following d-luciferin injection (150 mg/kg), CycLuc1 administration resulted in a three- to fourfold greater bioluminescent emission from the SFO and PVN, at 10- to 20-fold lower substrate concentrations (7.5–15 mg/kg). This CycLuc1-mediated enhancement in bioluminescent emission was evident early following substrate administration (i.e., 6–10 min) and persisted for up to 1 h. When the exposure time was reduced from 60 s to 1,500 ms, minimal signal in the PVN was detectable with d-luciferin, whereas bioluminescent images could be reliably captured with CycLuc1. These findings demonstrate that bioluminescent imaging with the synthetic luciferin CycLuc1 provides an improved physiological genomics tool to investigate molecular events in discrete cardioregulatory brain nuclei.

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Firefly Luciferase as a Reporter to Study Gene Expression in Streptococcus mutans,

Plasmid ◽

10.1006/plas.2000.1451 ◽

2000 ◽

Vol 43 (2) ◽

pp. 184

Author(s):

Steven D. Goodman ◽

Qian Gao

Keyword(s):

Gene Expression ◽

Streptococcus Mutans ◽

Firefly Luciferase ◽

Study Gene Expression

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Real-Time Monitoring of Chloroplast Gene Expression by a Luciferase Reporter: Evidence for Nuclear Regulation of Chloroplast Circadian Period

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.863-870.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 863-870 ◽

Cited By ~ 43

Author(s):

Takuya Matsuo ◽

Kiyoshi Onai ◽

Kazuhisa Okamoto ◽

Jun Minagawa ◽

Masahiro Ishiura

Keyword(s):

Gene Expression ◽

Circadian Clock ◽

Real Time ◽

Nuclear Genome ◽

Firefly Luciferase ◽

Luciferase Reporter ◽

Chloroplast Gene ◽

Chloroplast Gene Expression ◽

Period Gene ◽

Circadian Expression

ABSTRACT Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.

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