Luminometry and PCR-based monitoring of gene-tagged cyanobacteria in Baltic Sea microcosms

Abstract The cyanobacterium Synechocystis 6803 was tagged by chromosomal integration of the firefly luciferase gene, lue , resulting in the modified strain Synechocystis 6803- luc . The tagged cells were monitored in Baltic Sea microcosms both by detection of the luc gene by PCR amplification and by measurement of luc gene expression (bioluminescence) in total protein extracted from sediment and water. A new method was developed for isolation and concentration of total protein from sediment for optimization of luciferase quantitation. The detection limit for Synechocystis with a chromosomal luc insertion by bioluminescence was in the order of 4 × 10³ cells per g sediment, a considerable improvement in sensitivity over previous methods. Another improvement was to use an internal luciferase standard to correct for quenching of light output by impurities in the samples. Baltic sea microcosms were inoculated with Synechocystis 6803- luc , and the luc DNA and luciferase protein specific to the tagged cells were monitored over time. A decrease in luminescence in the microcosm water was observed, simultaneously with an increase in luminescence in the sediment, suggesting settling of the luc -tagged cells in the sediment layer.     

Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples

Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 10², by PCR amplification; 10³, by whole cell luminescence; and 10³−10⁴, by measurement of luminescence in cell extracts.     

Stress-activated bioluminescent Escherichia coli sensors for antimicrobial agents detection

A DNA cartridge encoding Photinus pyralis luciferase (luc), lacZ homology extensions and an excisable marker was constructed to facilitate the conversion of Escherichia coli lacZ fusions to luc fusions by lambda Red-mediated recombination. This tool was used to transform a cspA::lacZ strain into a luminescent biosensor for C-group translational inhibitors. Comparison of cspA::lacZ and cspA::luc cells showed native firefly luciferase to be a more rapid and sensitive reporter than beta-galactosidase for chloramphenicol detection. To evaluate the usefulness of a red-shifted variant of P. pyralis luciferase (LucR1) for biosensor development, a single copy translational fusion between the SOS-inducible sulA promoter and the lucR1 gene was inserted at the malP site of the E. coli chromosome. The sulA::lucR1 fusion allowed high signal detection of the quinolone ofloxacin to levels as low as 15% of the minimum inhibitory concentration and could be combined with a cspA::lacZ fusion to yield a biosensor suitable for the independent and dual detection of chloramphenicol and ofloxacin.     

Novel cyanobacterial biosensor for detection of herbicides

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.     

Genetically fused protein A-luciferase for immunological blotting analyses

The gene expression plasmid pMALU5 for the fusion protein of protein A (SpA) with a complete sequence of firefly luciferase (Luc) was constructed. The fused gene was expressed in Escherichia coli, and the resulting SpA-Luc fusion protein was purified by one-step affinity chromatography on IgG-Sepharose. The protein retained both activities: IgG binding capability of protein A and enzymatic activity of luciferase. Blotting analyses were performed with the fusion protein to determine a tumor marker of alpha-fetoprotein (AFP). AFP was detected at the lowest detection limit of 5 pg by dot blotting and Western blotting. The SpA-Luc fusion protein provides a highly selective, sensitive, and versatile marker for blotting analyses.     

The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro

Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L(luc)). We demonstrate that the bioluminescence signal decreases at pO(2) 相似文献   

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

Alternative luciferase for monitoring bacterial cells under adverse conditions

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.     

The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation

Here a screening method is described for transformed tissues and transgenic plants of Dendrobium (Orchidaceae) using the firefly luciferase gene ( luc ) as a combined marker/reporter gene. Protocorm-likebodies (PLB) were bombarded with tungsten particles (1.3 µm) coated with plasmids carrying a 35S-luc chimeric gene. Three weeks after bombardment 1 mM luciferin was added to the tissues and transformed cells were identified by virtue of their bioluminescence as monitored by low-light video microscopy in combination with a real-time photon imaging technique. Transformed tissues were excised, allowed to proliferate, and then subjected to a second round of screening. After three rounds of growth and screening, transformed Dendrobium tissues expressing luciferase were used to generate transgenic plants. Southern blot analysis of several transgenic lines confirmed the integration of the luciferase gene into the orchid genome. It is thought that this procedure can be used for transformation of not only orchids but other species as well.     

Imaging gene expression in live transgenic mice after providing luciferin in drinking water.

Mice expressing the firefly luciferase gene luc under the control of various gene promoters are used to image long-term changes in tumor growth, infection, development, and circadian rhythms. This novel approach enables ongoing regulation of gene expression to be visualized through repeated imaging of luciferase bioluminescence. Typically, luciferin, the luciferase substrate, is injected into mice before they are anaesthetized for imaging. To avoid the effects of handling and stress from injection on expression of the transgene, oral luciferin delivery methods were tested as an alternative to current methods. For unobscured imaging, a transgenic mouse line containing luc controlled by the enhancer and promoter for the major immediate-early gene of human cytomegalovirus (CMV) was crossed with a hairless albino mouse stock (HRS/J), resulting in the Hr-CMV line. Mice given food and water ad libitum readily drank 1-5 mM luciferin in water or apple juice and could be imaged repeatedly on subsequent days without any apparent adverse effects. Oral and injected luciferin produced similar patterns of luminescence in the body areas examined: abdomen, tail vertebrae, gonads, hind leg, foreleg and others, although the tail showed a slightly brighter relative luminescence after oral luciferin. These results show that luciferin is not appreciably degraded in the digestive tract and can be easily administered orally to avoid injection and any concomitant effects on behavior that could alter gene expression.     

Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein

We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)–fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET–FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.     

Second-generation triple reporter for bioluminescence, micro-positron emission tomography, and fluorescence imaging

Bioluminescence, positron emission tomography (PET), and fluorescence modalities are currently available for noninvasive imaging in vivo, each with its own merits. To exploit the combined strengths of each and facilitate multimodality imaging, we engineered a dual-reporter construct in which firefly luciferase (FLuc) and a 12-amino acid nonstructural linker were fused in frame to the N-terminus of a mutant herpes simplex virus thymidine kinase (mNLS-SR39TK) kinetically enhanced for positron emission tomography (PET). Furthermore, a triple-reporter construct was developed in which monster green fluorescent protein (MGFP), a recently available enhanced fluorescent protein, was introduced into the fusion vector downstream of an internal ribosome entry site (IRES) to allow analysis by fluorescence microscopy or flow cytometry without compromising the specific activities of the upstream fusion components. FLuc bioluminescence was measured with a cooled charge-coupled device camera and mNLS-SR39TK activity by 9-[4-[(18)F]fluoro-3-(hydroxymethyl) butyl guanine ((18)F-FHBG) microPET or (3)H-penciclovir net accumulation. Importantly, HeLa cells transiently transfected with the FLuc-mNLS-SR39TK-IRES-MGFP triple reporter retained the same specific activities of the FLuc-mNLS-SR39TK heteroenzyme and the individual unfused enzymes with no change in protein half-lives. The presence of the IRES-MGFP modestly decreased upstream heteroprotein expression. In living mice, somatic gene transfer of a ubiquitin promoter-driven FLuc-mNLS-SR39TK-IRES-MGFP plasmid showed a > 1,000-fold increase in liver photon flux and a > 2-fold increase in liver retention of (18)F-FHBG by microPET compared with mice treated with control plasmid. Multifocal hepatocellular fluorescence was readily observed by standard confocal microscopy. This second-generation triple reporter incorporating enhanced components enables bioluminescence, PET, and fluorescence imaging of cells and living animals.     

Construction of streptavidin-luciferase fusion protein for ATP sensing with fixed form

A fusion protein consisting of streptavidin and firefly luciferase was constructed to establish an accurate measuring technique of local ATP concentration. The fusion protein retained the binding ability of streptavidin and enzymatic activity of luciferase. Also, it could detect the concentration of antigens and could determine nanomolar concentrations of ATP in its fixed form via interactions with biotin-conjugated antibodies.     

A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.     

ATP生物发光测定试剂研究进展

萤火虫荧光素酶是ATP生物发光试剂的关键组成部分,可通过萤火虫尾提取纯化或基因工程技术制备,酶的活力和纯度决定了ATP生物发光试剂的性能。迄今许多先进技术在ATP生物发光试剂的制备中均有应用,包括酶基因工程改造技术、ATP循环的酶法放大技术、荧光素酶蛋白的活力及发光稳定技术,特异的细胞ATP提取技术等。ATP生物发光试剂的研究焦点主要集中在提高发光试剂的检测灵敏度和性能、增加产品的适应性等方面。