Engineering microorganisms
The present disclosure provides, inter alia, Thraustochytrium and relevant methods and reagents, including engineered regulatory sequences from and/or operative in Thraustochytrid or Thraustochytrium , selectable markers useful for engineering microorganisms such as Thraustochytrids, methods for mutagenizing microorganisms, novel strains produced by mutagenesis and methods and compositions related to production of particular compounds in microorganisms.
1. A method for transforming a Thraustochytrium cell comprising the steps of:
(a) providing a competent Thraustochytrium cell;
(b) delivering a recombinant nucleic acid into the competent Thraustochytrium cell, wherein the recombinant nucleic acid comprises a selectable marker and a tubulin promoter that is at least 80% identical to SEQ ID NO:10; and
(c) culturing the competent Thraustochytrium cell in a culturing medium containing a selection agent that reduces growth of cells without the selectable marker.
2. The method of claim 1 , wherein selectable marker is an antibiotic resistance gene.
3. The method of claim 2 , wherein selection agent is an antibiotic.
4. The method of claim 3 , wherein antibiotic is zeocin.
5. The method of claim 4 , wherein the zeocin is present at a concentration greater than 50 μg/mL.
6. The method of claim 4 , wherein the zeocin is present at a concentration of about 100 μg/mL.
7. The method of claim 2 , wherein the antibiotic resistance gene is a zeocin resistance gene.
8. The method of claim 1 , wherein the recombinant nucleic acid further comprises a gene expression cassette distinct from the selectable marker.
9. The method of claim 1 , further comprising: (d) isolating a competent Thraustochytrium cell containing the selectable marker.
10. The method of claim 1 , wherein the step of delivering comprises biolistic delivery of particles coated with the recombinant nucleic acid.
11. The method of claim 10 , wherein the particles comprise gold particles.
12. The method of claim 1 , wherein the culturing medium contains between about 10 g/L and about 40 g/L of salt.
13. The method of claim 1 , wherein the recombinant nucleic acid comprises a tubulin terminator.
14. The method of claim 13 , wherein the nucleotide sequence of the terminator is at least 80% identical to SEQ ID NO:14.
15. The method of claim 1 , wherein the recombinant nucleic acid comprises a tubulin promoter operably linked to the selectable marker.
16. The method of claim 15 , wherein the selectable marker is a zeocin resistance gene.
17. The method of claim 15 , wherein the selectable marker comprises able gene.
18. The method of claim 17 , wherein the ble gene is a Sh ble gene, a Tn5 ble gene or a Sa ble gene.
19. The method of claim 1 , wherein the cell is ONC-T18.
20. The method of claim 1 , wherein the culturing medium contains between about 15 g/L and about 35 g/L salt.
21. The method of claim 1 , wherein the culturing medium contains between about 18 g/L and about 35 g/L salt.
22. A method for transforming a Thraustochytrium cell comprising the steps of:
(a) providing a competent Thraustochytrium cell;
(b) delivering a recombinant nucleic acid into the competent Thraustochytrium cell, wherein the recombinant nucleic acid comprises a selectable marker and a tubulin terminator that is at least 80% identical to SEQ ID NO:14; and
(c) culturing the competent Thraustochytrium cell in a culturing medium containing a selection agent that reduces growth of cells without the selectable marker.
23. The method of claim 22 , wherein selectable marker is an antibiotic resistance gene.
24. The method of claim 23 , wherein the selection agent is an antibiotic.
25. The method of claim 24 , wherein the antibiotic is zeocin.
26. The method of claim 25 , wherein the zeocin is contained in the culture medium at a concentration greater than 50 μg/mL.
27. The method of claim 25 , wherein the zeocin is contained in the culture medium at a concentration of about 100 μg/mL.
28. The method of claim 22 , wherein the recombinant nucleic acid further comprises a gene expression cassette distinct from the selectable marker.
29. The method of claim 22 , further comprising: (d) isolating a competent Thraustochytrium cell containing the selectable marker.
30. The method of claim 22 , wherein the step of delivering comprises biolistic delivery of particles coated with the recombinant nucleic acid.
31. The method of claim 30 , wherein the particles comprise gold particles.
32. The method of claim 22 , wherein the culturing medium contains between about 10 g/L and about 40 g/L of salt.
33. The method of claim 22 , wherein the culturing medium contains between about 15 g/L and about 35 g/L salt.
34. The method of claim 22 , wherein the culturing medium contains between about 18 g/L and about 35 g/L salt.
35. The method of claim 22 , wherein the recombinant nucleic acid comprises a tubulin promoter.
36. The method of claim 35 , wherein the nucleic acid sequence of the tubulin promoter is at least 80% identical to SEQ ID NO:10.
37. The method of claim 22 , wherein the selectable marker is a zeocin resistance gene.
38. The method of claim 22 , wherein the selectable marker comprises a ble gene.
39. The method of claim 38 , wherein the ble gene is a Sh ble gene, a Tn5 ble gene or a Sa ble gene.
40. The method of claim 22 , wherein the cell is ONC-T18.