Abstracts of the 14th International Symposium on Bioluminescence and Chemiluminescence.

S 269 Copyright © 2006 John Wiley & Sons, Ltd. Luminescence 2006; 21: 269–298 DOI: 10.1002/bio Luminescence 2006; 21: 269–298 ABSTRACTS Abstracts of the 14th International Symposiums of the 14th International Symposium on Bioluminescence and Chemiluminescence School of Biosciences, University of Nottingham, Nottingham, UK The nucleoside analogue cytosine arabinoside (Ara-C) is the cornerstone of acute myeloid leukaemia (AML) therapy, but approximately 70% of patients fail to achieve complete remission with Ara-C. Current methods of testing drug sensitivity of AML are time consuming and unsuitable for routine screening. This project has developed a bioluminescent bacterial biosensor for use in a simple and rapid assay to determine the Ara-C sensitivity of patient cells, enabling clinicians to consider drug efficacy prior to treatment. An Ara-C-sensitive Escherichia coli was constructed and made self-bioluminescent by introducing the pBBR1MCS-2 plasmid carrying the lux operon (luxCDABE). The biosensor was evaluated with AML cell lines exposed to therapeutic levels of Ara-C. A significant increase in light level was observed in cell-lines known to be Ara-C sensitive. An Ara-C-resistant cell line showed no significant increase in light production in the presence of Ara-C compared to untreated controls. The assay was completed within 8–10 h and correlated well with a standard cytotoxicity assay taking 3–4 days. Samples of bone marrow and peripheral blood from AML patients were tested by both methods and the rapid bacterial biosensor assay correlated well with standard tests and also patient outcomes, indicating potential for clinical use. Quantitative luminescence yield spectra of firefly bioluminescence Y. Ando, K. Niwa, N. Yamada, T. Enomoto, T. Irie, H. Kubota, Y. Ohmiya and H. Akiyama Institute for Solid State Physics, University of Tokyo, Chiba, Japan CREST-JST, Saitama, Japan Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan ATTO Corporation, Tokyo, Japan We have developed a quantitative bio/chemiluminescence measurement system to determine the luminescence yield spectrum, which is the spectrum of total luminescence intensity emitted by the reaction in absolute light units of photons/ nm or photons/eV. To quantify the intriguing colour change in firefly bioluminescence from yellow-green to red with decreasing pH, we measured the luminescence yield spectra for Photinus pyralis at various pH values. The spectral shapes were well reproduced by one pH-sensitive and two pH-insensitive components with Gaussian energy broadening, peaking around 560, 620 and 670 nm. We found that the pH-sensitive 560 nm peak component contributes mostly to the change in the spectral shape and quantum yield with pH. Abstracts are in alphabetical sequence of first author Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications D. Ablamsky, M. M. Murtiashaw, L. Uzasci, T. L. Southworth, H. Fraga and B. R. Branchini Department of Chemistry, Connecticut College, 270 Mohegan Avenue, New London, CT, 06320 USA Departamento de Química, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, 687, 4169-007, Porto, Portugal Luciferase from the North American firefly Photinus pyralis (λmax = 557 nm) is widely used for monitoring gene expression. Previously, we developed a set of red (S284T)and green (V241I/G246A/F250S)-emitting luciferase mutants with spectral characteristics suitable for bioluminescence reporter applications. We undertook a site-directed mutagenesis approach to improve the thermostability of these enzymes at 37°C. Inspired by the reports of Tisi and co-workers that several mutations combine to significantly improve the thermostability of wild-type P. pyralis luciferase, we made variants of the redand green-emitting enzymes, each containing the additional changes T214A, A215L, I232A, F295L and E354K. The new proteins retained the favourable spectral properties and had enhanced thermostability. The green-emitting mutant (λmax = 546 nm) had a half-life at 37°C of 10.5 h, a 46-fold increase over V241I/G246A/F250S, and became resistant to red-shifting at low pH. The red-emitting mutant (λmax = 610 nm) had a half-life at 37°C of 8.8 h, an 11-fold increase over S284T. Moreover, the additional mutations improved the 37°C expression yields of both thermostable variants ~10-fold. Additionally, model single-colour reporter assays demonstrated that the thermostable mutants could be detected at the attomol level. These results demonstrate the feasibility of using the thermostable enzymes in mammalian cell reporter