Genetically modified bacterial strains and novel vectors for use in expressing and assaying natural products
OwnerAsuncion Martinez, Steve Kolvekinventors
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Abstract
Provided herein are heretofore-unknown strains of Streptomyces that are unable to express undecylprodrodigiosin, actinorhodin, or both, as well as novel vectors that permit the creation of chromosomal mutations in Streptomyces and do not insert selection markers in the genome of Streptomyces, along with vectors and methods for transferring large segments of DNA into the Streptomyces and Psuedomonas chromosome by conjugation.
Claims30
A vector for introducing genetically unmarked mutations into the chromosome of a unicellular host, comprising:
- (a) an origin of replication;
- (b) a counterselectable marker for the unicellular host;
- and
- (c) a selectable marker for the unicellular host,
- wherein said selectable marker is exciseable by crossover in the unicellular host, so that the selectable marker does not remain in the genome of said unicellular host after crossover.
A genetically modified S. lividans strain that is unable to produce actinorhodin, wherein the modification comprises the unmarked deletion of the act gene cluster in the genome of said strain.
A genetically modified S. lividans strain that is unable to produce undecylprodigiosin, wherein the modification comprises the deletion of the red gene cluster in the genome of said strain.
A genetically modified S. lividans strain that is unable to produce actinorhodin and undecylprodigiosin, wherein the modification comprises the deletion of the act and red gene clusters in the genome of said strain.
A genetically modified Pseudomonas strain having a genome containing an attB site.
A DNA cassette for conjugative transfer of a vector from E. coli into a Streptomyces or Pseudomonas spp., and subsequent integration of said vector into the chromosome of said Streptomyces or Pseudomonas spp., wherein said cassette is inserted into said vector, said cassette comprising:
- (a) a first loxP site;
- (b) a DNA sequence that encodes a phage integrase operably associated with a promoter;
- (c) an attP site for integration of a DNA sequence contained in the vector into the chromosome of the Pseudomonas or Streptomyces spp.;
- (d) an origin of transfer for conjugation;
- and
- (e) a second loxP site,
- such that elements (b), (c), and (d) lay between elements (a) and (e) in the DNA cassette.
A vector comprising a DNA cassette for the conjugal transfer of said vector from E. coli into Streptomyces where said cassette comprises:
- (a) a first loxP site;
- (b) a gene that encodes for integrase operably associated with a promoter;
- (c) an attP site for integration of said vector into the chromosome of Streptomyces spp.;
- (d) an origin of transfer for conjugation;
- and
- (e) a second loxP site,
- wherein elments (b), (c), and (d) lay between elments (a) and (e) in the DNA cassette;
- and
- (f) a counterselectable marker sacB,
- such that said vector can be used as a donor for in vitro transfer of the said cassette to a new vector by site-specific recombination.
A bacterial artificial chromosome that can be used to clone large DNA fragments and transfer them from E. coli to Pseudomonas or Streptomyces by conjugation, wherein said bacterial artificial chromosome comprises:
- (a) a first loxP site;
- (b) a gene that encodes for integrase operably associated with a promoter
- (c) an attP site for integration of said vector into the chromosome of Pseudomonas or Streptomyces spp.;
- (d) an origin of transfer for conjugation;
- and
- (e) a second loxP site,
- wherein elements (b), (c) and (d) lay between elements (a) and (e) in the bacterial artificial chromosome.
A method for the high-throughput transfer of large DNA fragments from E. coli to Streptomyces or Pseudomonas, comprising the steps of:
- (a) providing an E. coli cell having a recombinant bacterial artificial chromosome (BAC) containing a large DNA fragment;
- (b) providing a Streptomyces or Pseudomonas cell into which the large DNA fragment is to be transferred;
- (c) contacting the E. coli cell and the Streptomyces or Pseudomonas cell so that they undergo conjugation, in which the recombinant BAC is transferred to the Streptomyces or Pseudomonas cell;
- and
- (d) selecting the Streptomyces or Pseudomonas cell that contains the recombinant BAC.
Dependencies are inferred from the claim text.