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Bioluminescent Reporter plasmid to express Firefly luciferase (FFLuc) in Mycobacteria 

The signal produced by recombinant mycobacteria expressing Firefly luciferase can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.

Has been previously licensed to commercial parties for research into tuberculosis treatments or the pathogenesis of M. tuberculosis.

Background

Bioluminescence, the production of light by luciferase-catalysed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localisation of luciferase-expressing cells within an animal. Applied to the study of infectious diseases, BLI permits the detection of microorganisms from within living animals thus allowing the spatiotemporal study of infection in real-time in the same host. Moreover, using luciferase as a reporter of gene expression, it is possible to establish when and where a gene function is needed, shedding light on bacterial pathogenesis. Finally, BLI constitutes an easy and rapid method to test novel antimicrobial compounds in vivo.

Three main luciferin-luciferase systems have been utilised for BLI. The first system is represented by the firefly luciferase (FFluc) from Photinus pyralis which uses D-luciferin (a benzothiazole) as substrate, is dependent on ATP and results in the production of yellow-green light (557 nm). The second system includes the luciferases from the marine organisms Renilla reniformis (a cnidarian) and Gaussia princeps (a copepod) and the substrate coelenterazine. The signal produced by the G. princeps luciferase (Gluc) has been reported to be stronger than that of FFluc, even though the light emitted is in the blue range (480 nm) and is therefore more susceptible to tissue absorption and scattering. The fact that Gluc is strongly resistant to heat and extreme pH, and that it is secreted by eukaryotic cells also make this system very attractive. Bacterial luciferases, found in the terrestrial bacterium Photorhabdus luminescens and marine bacteria from the genera Vibrio and Photobacterium, constitute the third luciferin-luciferase system. These luciferases are heterodimeric enzymes that use FMNH2 and a long-chain aldehyde as substrates. Bacterial luciferases are encoded by the genes luxAB that form an operon (luxCDABE) together with three additional genes (luxCDE) whose products synthesise the longchain aldehyde. The main advantage of this system is that it does not need exogenously added substrate, but again the light produced is in the blue range (490 nm).

 

 

Figure 1. In vivo bioluminescence imaging of ffluc-expressing M. tuberculosis. Mice were inoculated endotracheally with 56106 CFU of either wild-type M. tuberculosis (control) or FFluc-producing M. tuberculosis. 20 ml of 30 mg ml21 luciferin was administered intranasally and mice were imaged 5–10 min after. Mice were contained in a large air-tight box for safety considerations. The image was obtained using an IVIS Spectrum and is displayed as a pseudocolour image of peak bioluminescence (given as photons s21 cm22 sr21 ). Red represents the most intense light emission while blue correspond to the weakest signal. The colour bar indicates relative signal intensity. Mice were imaged with an integration time of 1 min. doi:10.1371/journal.pone.0010777.g014

Figure from Nuria Andreu,Andrea Zelmer, Taryn Fletcher, Paul T. Elkington, Theresa H. Ward, Jorge Ripoll, Tanya Parish, Gregory J. Bancroft, Ulrich Schaible, Brian D. Robertson, Siouxsie WilesOptimisation of bioluminescent reporters for use with mycobacteria. PLoS One. 2010 May 24;5(5):e10777.

 

These plasmids and associated plasmids are available through Addgene to academics and non-profits.

For commercial enquiries, please make your request through the Quicktech portal or contact the Quicktech team directly (quicktech@imperialinnovations.co.uk) to discuss the release of these supplies.

The following plasmids are available:

-       pMV306hspFFLuc reporter plasmid. Please find more information through the following link: https://www.addgene.org/26156/.

-       pMV306hspFFLux reporter plasmid. Please find more information through the following link: https://www.addgene.org/26159/

-       pMV306hspGluc reporter plasmid. Please find more information through the following link: https://www.addgene.org/26161/

 

Internal case number: 5737

 

pMV306hspFFLuc reporter plasmid

Reporter plasmids for research into tuberculosis treatments or the pathogenesis of M. tuberculosis

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