Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae |
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Authors: | Gupta Rakesh K Patterson Stacey S Ripp Steven Simpson Michael L Sayler Gary S |
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Institution: | The Center for Environmental Biotechnology and Department of Microbiology, University of Tennessee, 676 Dabney Hall, Knoxville, TN 37996-1605, USA. |
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Abstract: | The luxA, B, C, D, and E genes from Photorhabdus luminescens were cloned and functionally expressed in Saccharomyces cerevisiae to construct a bacterial lux-based yeast bioreporter capable of autonomous bioluminescence emission. The bioreporter was engineered using a series of pBEVY yeast expression vectors that allowed for bi-directional constitutive or inducible expression of the individual luxA, B, C, and E genes. The luxD gene, encoding the acyl-ACP transferase that ultimately supplies the requisite aldehyde substrate for the bioluminescent reaction, was fused to a yeast internal ribosomal entry site (IRES) sequence to ensure high bi-cistronic expression. Although self-generation of bioluminescence was achieved by the bioreporter, the signal was relatively weak and decayed rapidly. To overcome this instability, a flavin oxidoreductase gene (frp) from Vibrio harveyi was co-expressed to provide sufficient concentrations of the FMNH(2) co-factor required for the bioluminescent reaction. Expression of frp with the lux genes not only stabilized but also enhanced bioluminescence to levels approaching 9.0x10(5) times above background. |
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Keywords: | Bioluminescence Bioreporter Biosensor lux Saccharomyces |
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