Luciferase expression cassettes and methods of use

Details Luciferase expression cassettes and methods of use Luciferase expression cassettes and methods of use

2000-09-07

2004-05-18

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving luciferase

C435S189000, C435S252300, C435S252310, C435S253400, C435S253500, C435S320100, C436S002000, C536S023200, C536S024100

Reexamination Certificate

active

06737245

ABSTRACT:

TECHNICAL FIELD
The present invention relates to luciferase expression vectors, methods of making same and methods of use thereof.
BACKGROUND OF THE INVENTION
Bioluminescent bacteria are widely found in both marine and terrestrial environments. Interestingly, all identified species of naturally occurring marine and terrestrial bioluminescent bacteria are Gram-negative. To date, at least eleven species in four Gram-negative genera have been described: Vibrio, Photobacterium, Shewanella (Altermonas) and Photorhabdus (Xenorhabdus). In all these species, the five genes responsible for bioluminescence are clustered in the lux operon (luxCDABE).
The bioluminescence (emitted blue-green light having a wavelength of about 490 nm) is thought to result from a luciferase-catalyzed oxidation of reduced flavin mononucleotide (FMNH
2
) and a long-chain fatty aldehyde. The luciferase enzyme is encoded by two subunits (luxAB), whereas the fatty acid reductase polypeptides responsible for the biosynthesis of the aldehyde substrate for the luminescent reaction are encoded by the three genes luxCDE. The genes encoding luciferase and the fatty acid reductase polypeptides have been cloned from the lux operons of Vibrio, Photobacterium and Photorhabdus and sequenced. In each case, the luxCDE genes flank the luxAB genes, with transcription in the order luxCDABE. Although a number of additional lux genes have been identified in each of these three bacteria, only luxAE are essential for the biosynthesis of light (reviewed by Meighen, E., (1993
, The FASEB Journal
7:1016-1022 and Ulitzur, S., (1997),
J. Biolumin Chemilumin
12:179-192).
Methods described in U.S. Pat. No. 5,650,135, make possible the detection of bioluminescent bacteria in a living animal without dissecting or otherwise opening the animal up (“in vivo monitoring”)—the light is detected through muscle, skin, fur & other traditionally “opaque” tissues using a highly sensitive camera. In this context and others, it would therefore be desirable to confer bioluminescence properties on a bacterium of one's choice, so that the bacterium could be followed with in vivo monitoring in various models of infection. In particular, it would be desirable to confer such bioluminescence properties on Gram-positive bacteria, since many bacteria pathogenic to mammals are in fact Gram-positive. For example, infections caused by Stapholococcus, a Gram-positive cocci, are ubiquitous and include, e.g., abscesses, mastitis, pneumonia, bacteremia, osteomyletis, enterocolitis and toxic shock syndrome (TSS). Another Gram-positive cocci, Streptococcus is the primary cause of pharyngeal infections (“strep” throat). Gram-positive bacilli such as Anthrax and Listeria (which causes meningitis) can cause severe, and even fatal infections in humans and other mammals.
While a non-bioluminescent Gram-negative bacterium can typically be engineered to have bioluminescence properties by cloning into it a luxCDABE operon (under control of a suitable promoter) from a bioluminescent species (see, e.g., Contag, et al., U.S. Pat. No. 5,650,135), previous attempts to make bioluminescent Gram-positive bacteria have met with limited success. For example, one approach employed an expression cassette encoding a functional LuxAB fusion protein (Jacobs, M., et al., (1991)
Mol. Gen. Genet
. 230:251-256). In this cassette, a Gram-positive ribosome binding site (RBS) was inserted upstream of luxA, with the luxB gene cloned in frame downstream of luxA. Although this approach has been successful in generating a number of novel genera of bioluminescent Gram-positive bacteria useful for certain environmental and food safety studies (e.g., the assessment of food products for contamination by such bacteria), these bacteria are not useful for studying pathogenicity. A major reason for this limitation is that the LuxAB fusion proteins described in the prior art not stable at mammalian body temperatures, and are thus capable of catalyzing only minimal light production in bacterial cells at 37° C.
In fact, none of the bioluminescent Gram-positive bacteria which have been published to date produce enough light in vivo to make them useful for the in vivo monitoring applications discussed above. It would therefore be desirable to have a method by which Gram-positive bacteria could be made to bioluminescence at temperatures found in mammalian host cells, and at levels of brightness suitable for monitoring in living animals. The present invention provides, inter alia, such methods, expression cassettes, and other tools useful for generating bioluminescent Gram-positive bacteria suitable for studies relating to infection and/or pathogenesis.
SUMMARY OF THE INVENTION
In one aspect, the invention includes an expression cassette comprising a polynucleotide encoding luxA, luxB, luxC, luxD and luxE gene products, wherein (a) the arrangement of coding sequences for the gene products is in the following relative order 5′-luxA-luxB-luxC-luxD-luxE-3′; (b) transcription of the polynucleotide results in a polycistronic RNA encoding all the gene products; and (c) each of the luxA, luxB, luxC, luxD and luxE gene products is expressed as an individual polypeptide. In one embodiment, the expression cassette includes a multiple-insertion site located adjacent the 5′ end of the luxA coding sequences. In another embodiment, the expression cassette further comprises at least one Gram-positive ribosome binding site sequence (SEQ ID NO:1) upstream of each of the polynucleotide sequences encoding each of the luxA, luxB, luxC, luxD and luxE gene products. The coding sequences of the gene products preferably encode a luciferase that is stable at 37° C., such as the luciferase of
Photorhabdus luminescens
. Accordingly, the nucleotide coding sequences for the luciferase are preferably derived from such organisms. In one series of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sa1-Sa6; such as Sa2 or Sa4. In a related series of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sp1, Sp5, Sp6, Sp9, Sp16 and Sp17 (e.g., Sp16).
In another aspect, the invention includes an expression cassette comprising a polynucleotide encoding luxA, and luxB gene products, wherein (a) transcription of the polynucleotide results in a polycistronic RNA encoding both gene products, and (b) polynucleotide sequences comprising Gram-positive ribosome-binding site sequences are located adjacent the 5′ end of the luxA coding sequences and adjacent the 5′ end of the luxB coding sequences. In one embodiment, the expression cassette further comprises an insertion site 5′ to at least one of either the luxA or luxB coding sequences. The insertion site may, for example, further comprise a multiple-insertion site. In one embodiment, the multiple-insertion site is located 5′ to the luxA coding sequences. In a related embodiment, the multiple-insertion site is located 5′ to the luxB coding sequences. In another embodiment, the polynucleotide further encodes luxC, luxD and luxE gene products. The arrangement of the coding sequences for the lux gene products may be, for example, in the following relative order 5′-luxA-luxB-luxC-luxD-luxE-3′. Preferably, Gram-positive bacterial Shine-Dalgarno sequences are 5′ to all of the lux coding sequences. In one group of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sa1-Sa6, e.g., Sa2 or Sa4. In another group of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sp1, Sp5, Sp6, Sp9, Sp16 and Sp17, such as Sp16. As was described above, the coding sequences for luxA and luxB are preferably obtained from an organism with a lucife

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