Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon

Abstract

Bioluminescent bacterial bioreporters harbor a fusion of bacterial bioluminescence genes (luxCDABE), acting as the reporting element, to a stress-response promoter, serving as the sensing element. Upon exposure to conditions that activate the promoter, such as an environmental stress or the presence of an inducing chemical, the promoter::reporter fusion generates a dose-dependent bioluminescent signal. In order to improve bioluminescent bioreporter performance we have split the luxCDABE genes of Photorhabdus luminescens into two smaller functional units: luxAB, that encode for the luciferase enzyme, which catalyzes the luminescence reaction, and luxCDE that encode for the enzymatic complex responsible for synthesis of the reaction's substrate, a long-chain aldehyde. The expression of each subunit was put under the control of either an inducible stress-responsive promoter or a synthetic constitutive promoter, and different combinations of the two units were tested for their response to selected chemicals in Escherichia coli. In all cases tested, the split combinations proved to be superior to the native luxCDABE configuration, suggesting an improved efficiency in the transcription and/or translation of two small gene units instead of a larger one with the same genes. The best combination was that of an inducible luxAB and a constitutive luxCDE, indicating that aldehyde availability is limited when the five genes are expressed together in E. coli, and demonstrating that improved biosensor performance may be achieved by rearrangement of the lux operon genes.

Application of Bioluminescence Imaging forIn VivoMonitoring of Fungal Infections

“…Here, although the reporter system is functional when all genes are located in a single operon, some substrate limitations can occur that limit the linearity of the reporter system. Recent studies have shown that this limitation is circumvented by independent and constitutive expression of the substrate-producing genes luxC , D , E and by only controlling expression of the luciferase-encoding genes by the promoter of interest [20]. …”

Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

“…Compared to ?uorescent signals, bioluminescent signals show lower illumination intensity (Troy et al 2004). A number of studies have attempted to increase bioluminescence signals (Chang et al 2004;Yagur-Kroll and Belkin 2011), including those by MEB (Eltzov et al 2009). The technology developed in this study aims at improving this on two fronts; the ?rst, includes optimizing the known silver deposition methodology (Eltzov et al 2009) and the second, enhancing signals of the different bioreporters.…”

Molecular Manipulations for Enhancing Luminescent Bioreporters Performance in the Detection of Toxic Chemicals

“…The P. luminescens luxCDABE cassette was also instrumental in a demonstration of an improvement of bioreporter performance by rearranging the lux-CDABE genes and splitting them into two independent functional units: luxAB, coding for the luciferase enzyme, and luxCDE, coding for the reductase complex [27]. The hypothesis that splitting the five-genes operon will be beneficial was driven by three main assumptions:…”