Bioluminescent reporters for catabolic gene expression and pollutant bioavailability

The application of visualized catabolic nah-gene expression using a luxCDABE gene fusion provides a valuable method to measure quantitatively and specifically naphthalene and salicylate bioavailability. It has been demonstrated that the physiological state of the test culture together with the intrinsic regulation mechanisms of the naphthalene degradation pathway as well as the physiological aspects of the lux gene fusion have to be taken into account. The method presented provides a high potential for in situ bioprocess monitoring. In addition, the results obtained with immobilized cells provide a basis for the development of biosensors for environmental applications in specific pollutant monitoring in waste streams and soil slurry systems but, as a general method, also for more conventional biotechnological process control. 8 refs., 2 figs., 1 tab

A destabilized bacterial luciferase for dynamic gene expression studies

Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression

A guide to cost estimating for overseas construction projects

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