Firefly Luciferase Luc

Firefly luciferase (Luc) catalyzes a reaction that produces visible light in die 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. Niunerous /wc-based bioreporters have been constructed for the detection of a wide array of inorganic and organic compounds of environmental concern. 

This section describes the bacterial luciferase (Lux) in the first part and the firefly luciferase (Luc) in the second part. 

For each well, prepare transfection mix B, containing 45 ng of the control Firefly luciferase (F-luc) plasmid pGL3 (Promega), 1 fd Lipofec-tamine 2000 (Invitrogen), and 100 fd of optiMEM. 

Figure 6.2 Critical parameters of the miR/mRNA co-transfection method. (A) Titration of mRNA amount. HeLa cells were transfected with increasing amounts of cap tail R-luc-4 sites mRNA and a fixed amount of firefly (F-luc) mRNA. R-luc expression (luciferase activity) was measured 5 h after transfection. (B) Titration of miCXCR4 concentration. HeLa cells were transfected with cap tail R-luc-4 sites mRNA, F-luc mRNA, and varying concentrations of miCXCR4. Luciferase activity was measured 16 h after transfection and fold-repression by the miR was calculated as in Fig. 6.1D (C) Time-course of miR-mediated repression. HeLa cells were co-transfected with cap tail R-luc-4 sites and F-luc (control) mRNAs, either with or without miCXCR4, and harvested at different time points. Repression was calculated as detailed in Fig. 6.1 and plotted against time (mRNA transfection data series depicted by the circles). Analogous plasmid DNA transfections are shown for reference (pDNA, diamonds). Averaged results from several experiments are shown with standard deviation. Data were previously published (Humphreys etal., 2005). Copyright PNAS, reprinted with permission.

Plasmid DNA encoding firefly luciferase gene under the cytomegalovirus promoter (pCMV-luc) was obtained from Stratagene (pCMV-Tag4, CA). 

DNA solution Luciferase gene plasmid p55pCMV-lVS-luc containing the firefly luciferase cDNA under the control of the cytomegalovirus (CMV) promoter (Plasmid Factory, Bielefeld, Germany) (pBLuc 5 mg/ml) see also Note 3. 

Luciferase reporter plasmid p55pCMV-lVS-luc +containing the firefly luciferase cDNA under the control of the cytomegalovirus (CMV) promoter. 

Fig. 16 Panel for ligand-attached PEG-siRNA conjugate through the acid-labile linkage system, a Chemical structure of Lac-PEG-siRNA conjugate, which is readily cleaved at the pH corresponding to that of the endosome (pH 5.5). b Evaluahon of RNAi activities against the firefly luciferase coded gene in cultured HuH-7 cells under various conditions. Normalized ratios between the firefly luciferase activity (firefly luc.) and the renilla luciferase activity (renilla luc.) are shown as ordinate ( = 3, SD)...

Reagents. Recombinant aequorin (Aq) with a free cys residue was from Chisso Corporation (Yokohama, Japan). Thermostable biotinylated firefly luciferase (b-Luc) was obtained from Kikkoman Corporation (Chiba, Japan). Aq labeled anti-Dig Fab fragment and b-Luc/streptavidin complex were produced by previously reported.1 HRP-labeled anti-DNP was purchased from LSL Co. (Tokyo). 

Simultaneous luminescent measurement of aequorin, biotinylated firefly luciferase and HRP. Aq, b-Luc and HRP solutions (5 pL of each) diluted with 0.05 mol/L HEPES-KOH buffer (pH 7.0) containing 5 mmol/L EGTA and 10% Block Ace (Dainippon SumitomoPharma Co., Ltd., Osaka, Japan) were introduced... 

An interesting variation on imaging caspase-3 activity in cells undergoing apoptosis was recently reported by Laxman et al. (41). In this chapter, the authors created a construct in which the firefly luciferase gene (Luc) was inserted between two estrogen receptor-DEVD sequences (i.e., ER-DEVD-Luc-DEVD-ER), which is the peptide sequence recognized and cleaved by caspase-3. 

The specificity for ATP makes it possible to perform Luc assays in the presence of other NTPs and NDPs. Indeed, this specificity allows the firefly luciferase to be used as a sensitive reagent for the ATP assay (64). In an elegant in vivo application, the ATP assay is exploited as a rapid method for distinguishing drug-sensitive and drug-resistant mycobacteria following transformation of the bacteria (clinical isolates) with luciferase reporter phages (65). 

Firefly luciferase from P. pyralis is a 50-kDa single polypeptide (550 amino acids). A C-terminal tripeptide sequence, —Ser—Lys—Leu, serves as the peroxisomal translocation signal, and its removal abolishes import of the enzyme into peroxisomes. Luc is hardly soluble in water, and its solubilization requires some salt. In solvents of relatively low ionic strengths, the protein aggregates in a rapidly reversible manner as the solubility limit is approached (69). 

Several vectors for expressing CAT in transformed flies and tissue culture cells have been constructed (see Ashburner 1989a, chapter 33). For primary references, see Mismer and Rubin (1987) and Thummel et al. (1988). A number of vectors for expressing firefly luciferase are available, including vectors that express luc from the per, CRE, hsp70, and tint promoters, and a UAS-responsive promoter (Lockett et al. 1992 Brandes et al. 1996 Emery et al. 1997 Plautz et al. 1997a,b Stanewsky et al. 1997,1998). 

HEK-293 hGH High-Five hsp70 HSV IPTG IRES kb Lac LCR LoxP LUC MCS human embryonic kidney cells human growth hormone TM BTI-TN-5B1-4 (cell line derived from the insect Trichoplusia ni) heat shock protein 70 herpes simplex virus isopropyl 1 -thio-fi-D-galactopyranoside internal ribosomal entry site kilobases lactose operon/repressor locus control region locus of crossover of PI luciferase isolated from firefly multiple cloning site... 

The reporter gene is the luc operon from the firefly Photinus pyralis. This operon codes for an enzyme, luciferase, that catalyses the reaction between D-luciferin (the substrate), and oxygen, and requires ATP as a cofactor. This reaction produces the emission light that provides the measure of enzyme activity. The oxidation of one molecule of luciferin involves one ATP molecule and releases one photon (a stoichiometric reaction) (DeLuca and McElroy, 1974 Wood el al. 1989). The reaction can be summarized as follows ... 

Some new luminescent and fluorescent reporters (some of them even non-substrate proteins ) are very attractive because of their easy and fast detection, explaining their current frequent use. The bacterial luciferase isolated from the Vibrio fischeri lux operon contains luxAB encoding the functional subunits and luxCDE for the synthesis and recycling of the aldehyde substrate (Prosser, 1996). Firefly (Photinus pyralis) luciferase, encoded by the luc gene catalyses the oxidative carboxylation of beetle luciferin, in which photons are emitted (LaRossa, 1998). Its short half-life and lack of any post-translational modification makes it ideal to look after effects in gene expression (Naylor, 1999). Detection of... 

Two types of ludferase genes, cloned from bacteria and firefly, are used as sensitive reporter systems in a wide variety of cells such as barterial, yeast, insect, animal, and plant cells. Bacterial luciferases are flavoenzymes composed of two subunits each encoded by the luxA and luxB genes, while the firefly ludferase is a single polypeptide spedfied by the luc gene. The two types of luciferase catalyze... 

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