Bacterial luciferase assay

Hastings J.W., Baldwin T.O., Nicoli M.Z., Bacterial luciferase assay, purification and properties, Methods Enzymol. 1978 57 135-152. 

Legocki, R. P., Legocki, M., Baldwin, T. O., and Szalay, A. A. (1986). Bioluminescence in soybean root nodules demonstration of a general approach to assay gene expression in vivo by using bacterial luciferase. Proc. Natl. Acad. Sci. USA 83 9080-9084. 

Bacterial bioluminescence, 30-46 factors required, 31 general scheme, 32 in vivo luminescence, 41 luminescence reaction, 37, 38 Bacterial luciferase, 33-35, 343 assay, 39 cloning, 34 crystal structure, 34 extraction and purification, 34 inactivation, 34, 35 molecular weight, 34 properties, 34 storage, 35 subunits, 34... 

Luciferase assay. In this technique, firefly luciferase is used to measure small amounts of adenosine triphosphate (ATP) in a bacterial culture, ATP levels being reduced by the inhibitory action of aminoglycoside antibiotics. This method may find more application in the future as more active and reliable luciferase preparations become available. 

W. Huang, A. Feltus, A. Witkowski, and S. Daunert, Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase-bacterial luciferase enzyme system. Anal. Chem. 68, 1646-1650 (1996). 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

A typical example of these analytical systems is a manifold using bacterial luciferase for L-phenylalanine assay [228] developed with two separate nylon coils, as shown in Figure 3. The first one contained the specific L-phenylalanine dehydrogenase (L-PheDH) enzyme. 

Andre et al. [8] discuss the determination of adenosine-5 -triphosphate by luciferin-luciferase assay. This method was applied to the determination of adenosine-5 -triphosphate in bacterial colonies filtered from samples of polluted water after incubation for different periods. The adenosine-5 -triphosphate was extracted from the residue in the filter and the amount compared with the biochemical oxygen demand of the filtered water. The oxygen uptake rate and the rate of formation of adenosine-5 -triphosphate were then plotted against time, the two curves being similar in up to three to four days incubation, after which adenosine-5 -triphosphate production declined markedly, although oxygen uptake continued to increase. 

Procedure 8.7 Bioluminescence assay of FMN using bacterial luciferase (EC... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

Bioluminescence-based analytical assays were used to measure various analytes in nanoliter sample volumes. Nanoliter volumes of multiple bioluminescent analytical assays were deposited in an array format and lyophilized. ATP-firefly luciferase (FFL) and NADH-bacterial luciferase (BL) platform reactions were compared. We achieved parallel sample delivery via sample-hydrated membranes. A CCD camera measured the luminescent kinetics for each assay. These miniaturized assays and instruments can he prepared as micro-analytical systems to operate in point-of-care (POC) diagnostic devices. 

Bacterial luciferase coimmobilized with NAD(P)H FMN oxidoreductase on starch gel has been used for bioluminescent assay of aldehydesCo-immobilization of bacterial luciferase, NAD(P)H FMN oxidoreductase and their substrates is referred to as multifunctional immobilized biosensor and is a new trend for use of bioluminescent analysis, e.g. toxicity biotest and bioassay. The main principle of this luciferase biotest is the correlation between toxicity of the sample being studied and changes in bioluminescence parameters in vitro. Toxicity of the sample is measured by the changes in bioliuninescence intensity compared with that of a control. Multifunctional immobilized biosensors based on luciferase have been used for the following bioassays. 

Lei BF, Becvar JE. A new reducing agent of flavins and its application to the assay of bacterial luciferase. Photochem Photobiol 1991 54 473-6. 

A. Nabi and P. J. Worsold, Bioluminescence Assays with Immobilized Bacterial Luciferase Using Flow Injection Analysis. Analyst, 111 (1986) 1321. 

Immunoassay and nucleic acid probe assays Labels. recApoaequorin, firefly luciferase, marine bacterial luciferase, Vargula luciferase Detection reactions, alkaline phosphatase label (luciferin-O-phosphate/firefly luciferase), glucose 6-phosphate dehydrogenase label (marine bacterial luciferase/NADH FMN oxidoreductase reaction)... 

A photon of maximal intensity at 495 nm is produced in the reaction in which the bacterial luciferase catalyzes the oxidation of an aldehyde by oxygen in the presence of FMNHj. The method allows about 100 fmol of NADH to be assayed. The system cam be utilized to measure numerous oxidoreductases which utilize NADH or NADPH. The substrates of such enzymes cam also be assayed by this system. 

Bioluminescent Enzyme Immune Assay (BLEIA), where the enzyme is the marker for the antigen of the immune reaction, which results in light emission. Markers are enzymes that are conjugated to bacterial luciferase, such as peroxidases, acid phosphatases and dehydrogenases. 

Both firefly luciferase (see below) and bacterial luciferase are sensitive reporters of gene expression suitable for the study of regulatory sequences (promoter, enhancer, and terminator) of cellular differentiation and morphogenesis, and of responses to environmental and developmental changes (53,54). The two reporter systems have been used in bacteria, yeast, fungi, insects, plants, and mammalian cells. As far as in vitro assays are concerned, both reporter systems may be considered equally useful. For in vivo applications, however, one system may turn out to be more suitable than the other due to the strengths and weaknesses inherent to each reporter system. 

Bacterial luciferase can be used in the assay of NAD(P)H by a coupling technique. In the first step flavin mononucleotide (FMN) is reduced by NAD (P)H and NADH/FMN-oxidoreductase. FMNH2 then reacts with oxygen, a long chain aldehyde, and bacterial luciferase to give FMN, carboxylic acid, water and light (as described on p. 156) [111]. 

Since ideally, a biosensor should be reagentless, that is, should be able to specifically measure the concentration of an analyte without a supply of reactants, attempts to develop such bioluminescence-based optical fibre biosensors were made for the measurements of NADH28 30. For this purpose, the coreactants, FMN and decanal, were entrapped either separately or together in a polymeric matrix placed between the optical fibre surface and the bacterial oxidoreductase-luciferase membrane. In the best configuration, the period of autonomy was 1.5 h during which about twenty reliable assays could be performed. 

Permeable Cell Assay for high-throughput measurement of cellular ATP synthetic activity (4). In this assay, osmotic shock and Triton X-100 treatment made bacterial cells permeable for ATP Discharged ATP reacted with external luciferase and is detected as bioluminescence. An increased bioluminescence is observed with permeable cells, whereas it is not observed with standard ATP solution and heat-inactivated permeable cells. The cellular ATP synthetic activity is calculated from the slope of increasing bioluminescence. Permeable Cell Assay is simple and rapid with a small amount of cell culmre for quantification of ATP synthesis. 

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