Patent
Provided herein are methods for transforming a Pyrococcus furiosus with a polynucleotide. In one embodiment, the method includes contacting a P. furiosus with a polynucleotide under conditions suitable for uptake of the polynucleotide by the P. furiosus , and identifying transformants at a frequency of, for instance, at least 10 3 transformants per microgram DNA. Also provided are isolated Pyrococcus furiosus having the characteristics of Pyrococcus furiosus COM1, and plasmids that include an origin of replication that functions in a Pyrococcus furiosus . The plasmid is stable in a recipient P. furiosus without selection for more than 100 generations and is structurally unchanged after replication in P. furiosus for more than 100 generations.
1. An isolated Pyrococcus furiosus COM1 deposited with the American Type Culture Collection in accordance with the provisions of the Budapest Treaty with the accession number PTA-11303.
2. The isolated Pyrococcus furiosus of claim 1 comprising a heterologous polynucleotide.
3. The isolated Pyrococcus furiosus of claim 2 wherein the heterologous polynucleotide is integrated into the P. furiosus genomic DNA.
4. The isolated Pyrococcus furiosus of claim 2 wherein the heterologous polynucleotide is not integrated into the P. furiosus genomic DNA.
5. A method for transforming a Pyrococcus furiosus with a polynucleotide comprising:
contacting the P. furiosus of claim 1 with a polynucleotide under conditions suitable for uptake of the polynucleotide by the P. furiosus ; and
identifying transformants, wherein at least 10 4 transformants are obtained per microgram DNA.
6. The method of claim 5 wherein at least 10 5 transformants are obtained per microgram DNA.
7. The method of claim 5 wherein the polynucleotide is a linear polynucleotide.
8. The method of claim 7 wherein the linear polynucleotide comprises an intervening region flanked by a first homology region and a second homology region, wherein the first homology region and the second homology region each comprise nucleotide sequences that are homologous to nucleotide sequences present in the P. furiosus genome.
9. The method of claim 8 wherein the first homology region and the second homology region are at least 20 nucleotides in length.
10. The method of claim 5 wherein the polynucleotide is a circular polynucleotide.
11. The method of claim 10 wherein the circular polynucleotide comprises a homology region, wherein the homology region comprises a nucleotide sequence that is homologous to a nucleotide sequence present in the P. furiosus genome.
12. The method of claim 11 wherein the homology region is at least 20 nucleotides in length.
13. The method of claim 12 wherein the circular polynucleotide comprises a first homology region and a second homology region, wherein the first and second homology regions each comprise a different nucleotide sequence, wherein each nucleotide sequence is homologous to a nucleotide sequence present in the P. furiosus genome.
14. The method of claim 13 wherein the first homology region and the second homology region are at least 20 nucleotides in length.
15. The method of claim 5 wherein the conditions suitable for uptake of the polynucleotide do not include exposing the P. furiosus to a heat shock.
16. The method of claim 5 wherein the conditions suitable for uptake of the polynucleotide do not include exposing the P. furiosus to CaCl 2 , an applied electrical field, a liposome, a phage, or conditions resulting in spheroplast formation.
17. The method of claim 5 wherein a portion of the polynucleotide taken up by a transformant is integrated into the P. furiosus genome.
18. The method of claim 17 wherein the integration is by homologous recombination between the polynucleotide and the P. furiosus genome.
19. A method for transforming a Pyrococcus furiosus with a polynucleotide comprising:
contacting the P. furiosus of claim 1 with a polynucleotide under conditions suitable for uptake of the polynucleotide by the P. furiosus , wherein the polynucleotide comprises a homology region, wherein the homology region comprises a nucleotide sequence that is homologous to a nucleotide sequence present in the P. furiosus genome, and wherein the homology region is at least 20 nucleotides in length.