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US 2011/0256533 A1Published application
Methods and compositions for the identification of antibiotics that are not susceptible to antibiotic resistance
AssigneeWayne State University
InventorPHILLIP R. CUNNINGHAM
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Abstract
Compositions and methods are provided to identify functional mutant ribosomes that may be used as drug targets. The compositions and methods allow isolation and analysis of mutations that would normally be lethal and allow direct selection of rRNA mutants with predetermined levels of ribosome function. The compositions and methods of the present invention may be used to identify antibiotics to treat a large number of human pathogens through the use of genetically engineered rRNA genes from a variety of species. The invention further provides novel plasmid constructs to be used in the methods of the invention.
Claims22
1-22. (canceled)
A method for identifying functional mutant ribosomes comprising:
- (a) transforming a set of host cells with a set of plasmids, each plasmid comprising a mutant rRNA gene and a selectable marker gene;
- wherein said mutant rRNA gene comprises at least one mutation and a mutant Anti-Shine-Dalgarno sequence;
- and said selectable marker gene comprises a mutant Shine-Dalgarno sequence;
- and
- wherein said mutant Anti-Shine-Dalgarno and said mutant Shine-Dalgarno sequence are a mutually compatible pair;
- thereby forming a set of transformed host cells;
- (b) isolating from the set of transformed host cells those host cells which express the selectable marker gene product;
- and
- (c) sequencing the mutant rRNA gene from each host cell isolated in step (b), thereby identifying functional mutant ribosomes.
6 more elements
A method for identifying functional mutant ribosomes comprising:
- (a) transforming a set of host cells with a set of plasmids, each plasmid comprising a mutant E. coli 16S rRNA gene and a green fluorescent protein gene;
- wherein said mutant E. coli 16S rRNA gene comprises at least one mutation and a Anti-Shine-Dalgarno sequence;
- and
- said green fluorescent protein gene comprises a mutant Shine-Dalgarno sequence;
- and
- wherein said mutant Anti-Shine-Dalgarno and said mutant Shine-Dalgarno sequence are a mutually compatible pair;
- thereby forming a set of transformed host cells;
- (b) isolating from the set of transformed host cells those host cells which express the green fluorescent protein gene product;
- and
- (c) sequencing the mutant E. coli 16S rRNA gene from each host cell isolated in step (b), thereby identifying functional mutant ribosomes.
7 more elements
A method for identifying functional mutant ribosomes that may be suitable as drug targets comprising:
- (a) transforming a first set of host cells with a first set of plasmids, each plasmid comprising a first mutant rRNA gene and a first selectable marker gene;
- wherein said first mutant rRNA gene comprises at least one mutation and a first mutant Anti-Shine-Dalgarno sequence;
- and said first selectable marker gene comprises a first mutant Shine-Dalgarno sequence;
- and
- wherein said first mutant Anti-Shine-Dalgarno and said first mutant Shine-Dalgarno sequence are a mutually compatible pair;
- thereby forming a first set of transformed host cells;
- (b) isolating from the first set of transformed host cells those host cells which express the selectable marker gene product;
- and
- (c) sequencing the first mutant rRNA gene from each host cell isolated in step (b), thereby identifying regions of interest, wherein said regions of interest comprise sequences of one or more nucleic acids which are conserved in each first mutant rRNA gene sequenced;
- (d) generating a second plurality of mutant rRNA genes wherein the regions of interest of step (c) are mutated;
- and each rRNA gene further comprises a second mutant Anti-Shrine-Dalgarno sequence;
- (e) inserting the second plurality of mutant rRNA genes comprising the mutated regions of interest from step (d) into a second plurality of plasmids;
- wherein said plasmids further comprise a second genetically engineered gene which encodes a second selectable marker having a second mutant Shine-Dalgarno sequence, wherein the second mutant Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno sequence are a mutually compatible pair;
- (f) transforming a second set of host cells with the plasmids from step (e), thereby forming a second set of transformed host cells;
- (g) isolating from the second set of transformed host cells of step (f) those host cells which express the selectable marker gene product;
- and
- (h) sequencing the rRNA gene from each host cell isolated in step (g), thereby identifying functional mutant ribosomes that may be suitable as drug targets.
14 more elements
A method for identifying functional mutant ribosomes that may be suitable as drug targets comprising:
- (a) transforming a first set of host cells with a first set of plasmids, each plasmid comprising a first mutant E. coli 16S rRNA gene and a first green fluorescent protein gene;
- wherein said first mutant E. coli 16S rRNA gene comprises at least one mutation and a first mutant Anti-Shine-Dalgarno sequence;
- and said first green fluorescent protein gene comprises a mutant Shine-Dalgarno sequence;
- and
- wherein said first mutant Anti-Shine-Dalgarno and said first mutant Shine-Dalgarno sequence are a mutually compatible pair;
- thereby forming a first set of transformed host cells;
- (b) isolating from the first set of transformed host cells those host cells which express the first green fluorescent protein gene product;
- and
- (c) sequencing the first mutant E. coli 16S rRNA gene from each host cell isolated in step (b), thereby identifying regions of interest, wherein said regions of interest comprise sequences of one or more nucleic acids which are conserved in each first mutant E. coli 16S rRNA gene sequenced;
- (d) generating a second plurality of mutant E. coli 16S rRNA genes wherein the regions of interest of step (c) are mutated;
- and each E. coli 16S rRNA gene further comprises a second mutant Anti-Shrine-Dalgarno sequence;
- (e) inserting the second plurality of mutant E. coli 16S rRNA comprising the mutated regions of interest from step (d) into a second plurality of plasmids;
- wherein said plasmids further comprise a second genetically engineered gene which encodes green fluorescent protein having a second mutant Shine-Dalgarno sequence, wherein the second mutant Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno sequence are a mutually compatible pair;
- (f) transforming a second set of host cells with the plasmids from step (e), thereby forming a second set of transformed host cells;
- (g) isolating from the second set of transformed host cells of step (f) those host cells which express the second genetically engineered gene product;
- and
- (h) sequencing the E. coli 16S rRNA gene from each host cell isolated in step (g), thereby identifying functional mutant ribosomes that may be suitable as drug targets.
14 more elements
27-36. (canceled)
Dependencies are inferred from the claim text.