Transformation of Algal Cells

• US 20090317857A1
• Filed: 06/08/2009
• Published: 12/24/2009
• Est. Priority Date: 06/06/2008
• Status: Active Grant

• Current Assignee: Aurora Algae, Incorporated (Reliance Industries Limited)
• Original Assignee: Aurora Algae, Incorporated (Reliance Industries Limited)
• Inventors: Kilian, Oliver, Vick, Bertrand
• Granted Patent: US 8,119,859 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/8206 by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8237 Externally regulated expression systems
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome

Abstract

Exemplary methods include a method for transforming an algal cell by preparing a transformation construct, preparing a particle for bombarding the algal cell, adhering the transformation construct to the particle, bombarding the algal cell with the particle, and growing the algal cell into a colony. The transformation construct is replicated within a nuclear genome of the algal cell and the growing of the algal cell is in a nutrient medium. Another exemplary method may include a method for genetically modifying an algal cell, by adding nucleic acid to the algal cell while the algal cell is suspended in a solution of low conductivity, introducing the nucleic acid into the algal cell by application of an electrical pulse resulting in a transformed algal cell, and selecting a colony that includes the transformed algal cell.

Claims

1. A method for transforming an algal cell, the method comprising:

preparing a transformation construct;

preparing a particle for bombarding the algal cell;

adhering the transformation construct to the particle;

bombarding the algal cell with the particle; and

growing the algal cell into a colony.

2. The method of claim 1, wherein the transformation construct is replicated within a nuclear genome of the algal cell.

3. The method of claim 1, wherein the growing of the algal cell is in a nutrient medium.

4. The method of claim 1, wherein the transformation construct is a nucleic acid in an expression cassette.

5. The method of claim 4, wherein the expression cassette further comprises:

a promoter functional in the algal cell;

a selectable transformation marker gene; and

a termination region functional in the algal cell.

6. The method of claim 5, wherein the selectable transformation marker gene is a gene capable of conferring antibiotic resistance to the algal cell.

7. The method of claim 6, the method further comprising:

selecting the algal cell growing in a selective medium having an antibiotic the gene has conferred resistance upon to the algal cell.

8. The method of claim 1, wherein the algal cell is an auxotrophic mutant.

9. The method of claim 1, wherein the transformation construct includes a sh ble gene.

10. The method of claim 1, wherein the transformation construct includes a hygromycin B phosphotransferase gene.

11. The method of claim 1, wherein the transformation construct includes a nitrate reductase gene.

12. The method of claim 1, wherein the transformation construct includes a blastocidin S deaminase gene.

13. The method of claim 1, wherein the particle is a gold particle smaller than or equal to approximately 1 micrometer average diameter.

14. The method of claim 13, wherein the preparing of the gold particle includes sonicating the gold particle with ultrasound prior to the adhering of the transformation construct to the gold particle.

15. The method of claim 1, wherein the adhering of the transformation construct to the particle includes precipitating DNA of the transformation construct to the particle.

22. The method of claim 1, wherein the algal cell is of genus Nannochloropsis.

16. A method for genetically modifying an algal cell, the method comprising:

adding nucleic acid to the algal cell while the algal cell is suspended in a solution of low conductivity; and

introducing the nucleic acid into the algal cell by application of an electrical pulse resulting in a transformed algal cell; and

selecting a colony that includes the transformed algal cell.

17. The method of claim 16, wherein the nucleic acid includes a transformation construct.

18. The method of claim 16, wherein the electrical pulse is applied with a field strength of higher than approximately 7500 V/cm.

19. The method of claim 16, wherein the electrical pulse is applied with a field strength of higher than approximately 9000 V/cm.

20. The method of claim 16, wherein the electrical pulse is applied with a field strength of higher than approximately 10,000 V/cm.

21. The method of claim 16, wherein the electrical pulse is applied with a shunt resistor of greater than approximately 100 OHM.

23. The method of claim 16, wherein the algal cell is of genus Nannochloropsis.