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US 7,008,767 B2Granted
Microorganism genomics, compositions and methods related thereto
AssigneeWisconsin Alumni Research Foundation
InventorsJo Handelsman, Robert M. Goodman, Michelle R. Rondon
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Abstract
The present invention provides methods and composition for accessing, in a generally unbaised manner, a diverse genetic pool for genes involved in biosynthetic pathways. The invention also provides compounds which can be identified by cloning biosynthetic pathways.
Claims32
A method for detecting a compound produced by a biosynthetic pathway, comprising:
- i) isolating genomic DNA from a source containing uncultivated microorganisms, without an intervening step of culturing cells from the source, by mixing the source with an extraction buffer and lysing the microorganisms to obtain isolated genomic DNA;
- and then
- ii) inserting the isolated genomic DNA of step (i) into a replicable vector to obtain clones of the replicable vector comprising the isolated genomic DNA;
- and then
- iii) introducing the clones of step (ii) into host cells to obtain a library of host cells, wherein the isolated genomic DNA in at least some of the clones is at least 50 kb;
- and then
- iv) culturing the host cells of step (iii) under conditions wherein an open reading frame sequence of the isolated genomic DNA is expressed by the host cells;
- and then
- v) detecting a compound produced by the host cells as a result of expression of the open reading frame sequence of step (iv).
6 more elements
A method for detecting a product of a biosynthetic pathway, comprising:
- i) directly cloning into a replicable vector-genomic DNA isolated from a complex microbial sample without an intervening step of culturing cells from the sample, and size-fractionated prior to any enzymatic manipulation of the DNA to facilitate its insertion into the replicable vector, wherein at least some of the genomic DNA cloned into the vector is at least 50 kb long;
- and then
- ii) transforming the replicable vector of step (i) into host cells to obtain a library of host cells;
- and then
- iii) expressing open reading frame sequence(s) of the genomic DNA contained in the library of host cells of step (ii);
- and then
- iv) detecting a compound which is the product of a biosynthetic pathway that is dependent on expression of at least one of the opening reading frames of step (iii).
4 more elements
Dependencies are inferred from the claim text.