Targeted mutagenesis in
This disclosure describes techniques for creating stable, targeted mutations in Spirulina ( Athrospiria ) and Spirulina having stable, targeted mutations.
1. A non-naturally occurring, stable transformant Spirulina comprising:
a. at least one introduced targeted nucleotide mutation incorporated into the Spirulina genome,
wherein the introduced targeted nucleotide mutation is adjacent to a region of nucleotides that is homologous to a nucleotide homology arm contained in a vector utilized to transform said cell via homologous recombination.
2. The Spirulina of claim 1 , wherein the Spirulina is Arthrospira platensis or Arthrospira maxima.
3. The Spirulina of claim 2 , wherein the Spirulina is Arthrospira platensis NIES-39 or Arthrospira sp. PCC 8005.
4. The Spirulina of claim 1 , wherein the introduced targeted nucleotide mutation is inherited for at least 10 generations.
5. The Spirulina of claim 1 , wherein the introduced targeted nucleotide mutation comprises a deletion or disruption of at least a portion of a gene.
6. The Spirulina of claim 1 , wherein the introduced targeted nucleotide mutation comprises addition of an additional copy of an endogenous gene or addition of an exogenous gene.
7. The Spirulina of claim 1 , wherein the introduced targeted nucleotide mutation comprises addition of a gene regulatory element.
8. The Spirulina of claim 7 , wherein the gene regulatory element is a promoter, a regulatory protein binding site, a RNA binding site, a ribosome binding site, or an RNA stability element.
9. The Spirulina of claim 1 , wherein the introduced targeted nucleotide mutation comprises addition of a polynucleotide sequence encoding an exogenous protein domain.
10. The Spirulina of claim 9 , wherein the exogenous protein domain is a phosphorylation site, a stability domain, a protein targeting domain, or protein-protein interaction domain.
11. A method of creating a introduced targeted mutation in Spirulina , the method comprising:
contacting the Spirulina with an osmotic stabilizer;
contacting the Spirulina with a vector having a nucleotide homology arm; and
inducing artificial competence in the Spirulina.
12. The method of claim 11 , wherein the Spirulina is Spirulina platensis.
13. The method of claim 11 , wherein the Spirulina is Arthrospria platensis NIES-39 or Arthrospira sp. PCC 8005.
14. The method of claim 11 , wherein the osmotic stabilizer is at least one of polyethylene glycol, ethylene glycol, glycerol, glucose, or sucrose.
15. The method of claim 11 , wherein the vector is a DNA vector, a linear vector, a circular vector, a single stranded polynucleotide, or a double stranded polynucleotide.
16. The method of claim 11 , wherein the vector comprises a vector having SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4.
17. The method of claim 11 , wherein the nucleotide homology arm is at least 500 bp.
18. The method of claim 11 wherein the nucleotide homology arm is at least 1000 bp.
19. The method of claim 11 , wherein the nucleotide homology arm is at least 2000 bp.
20. The method of claim 11 , wherein the artificial competence is induced by electroporation.
21. The method of claim 11 , further comprising contacting the Spirulina with a pH balancer.
22. The method according to claim 21 , wherein contacting the Spirulina with the pH balancer is prior to contacting the Spirulina with the vector.
23. The method according to claim 21 , wherein a pH of a media containing the Spirulina and the pH balancer is about 7 to about 8.
24. A Spirulina lacking at least one protein as a result of introducing a modification at the loci of the protein by transformation of the Spirulina with at least one DNA construct comprising a sequence homologous with at least a portion of the loci and the modification and integration of the DNA construct at the loci, the Spirulina being otherwise capable of functioning in its native manner.
25. A Spirulina comprising an exogenous gene encoding phycocyanin as a result of introducing the exogenous gene at a targeted loci by transformation of the Spirulina with at least one DNA construct comprising a sequence homologous with at least a portion of the targeted loci and the exogenous gene and integration of the DNA construct at the locus, the Spirulina producing an increased amount of phycocyanin relative to wild-type Spirulina and maintaining the transformation throughout multiple generations.
26. The non-naturally occurring, stable transformant Spirulina of claim 1 , wherein the introduced targeted nucleotide mutation is flanked by two regions of nucleotides that are homologous to nucleotide homology arms contained in a vector utilized to transform said cell via homologous recombination.