Homologous recombination in an algal nuclear genome

• US 8,865,468 B2
• Filed: 10/19/2009
• Issued: 10/21/2014
• Est. Priority Date: 10/19/2009
• Status: Active Grant

• Current Assignee: Aurora Algae, Incorporated (Reliance Industries Limited)
• Original Assignee: Aurora Algae, Incorporated (Reliance Industries Limited)
• Inventors: Kilian, Oliver, Vick, Bertrand
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US12/581,812
• Publication Number:
  US 20110091977A1
• Time in Patent Office: 1,828 Days
• Field of Search: None
• US Class Current: 435/471
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts

Abstract

Exemplary transformation methods are provided for introducing deoxyribonucleic acid (DNA) into the nucleus of an algal cell. A transformation construct may be prepared, with the transformation construct having a first sequence of DNA similar to a corresponding first sequence of nuclear DNA, a second sequence of DNA similar to a corresponding second sequence of the nuclear DNA, and a sequence of DNA of interest inserted between the first and second sequences of DNA of the transformation construct. A target sequence of DNA inserted between the first and second corresponding sequences of the nuclear DNA may be transformed, resulting in replacement of the target sequence of DNA with the sequence of DNA of interest. Also provided are exemplary transformation constructs, with some transformation constructs having a first sequence of DNA similar to a corresponding first sequence of nuclear DNA of an algal cell, a second sequence of DNA similar to a corresponding second sequence of nuclear DNA of the algal cell, and a sequence of DNA of interest inserted between the first and second sequences of the transformation construct.

Claims

1. A transformation method for introducing deoxyribonucleic acid (DNA) into the nucleus of an algal cell, the method comprising:

preparing a transformation construct, the transformation construct having a first sequence of DNA similar to a corresponding first sequence of nuclear DNA, the transformation construct having a second sequence of DNA similar to a corresponding second sequence of the nuclear DNA, the transformation construct having a sequence of DNA of interest inserted between the first and second sequences of DNA of the transformation construct,

transforming a target sequence of DNA inserted between the first and second corresponding sequences of the nuclear DNA, resulting in replacement of the target sequence of DNA with the sequence of DNA of interest, wherein the algal cell is of algal genus Nannochloropsis.

2. The method of claim 1, wherein the sequence of DNA of interest separates the first and second sequences of DNA similar to the corresponding respective first and second sequence of the nuclear DNA by approximately 4.5 kb.

3. The method of claim 1, wherein either the first or second sequence of DNA similar to the corresponding respective first or second sequence of the nuclear DNA comprises approximately 1000 base pairs (bps).

4. The method of claim 1, wherein either the first or second sequence of DNA similar to the corresponding respective first or second sequence of the nuclear DNA comprises approximately less than 1000 bps.

5. The method of claim 1, wherein either the first or second sequence of DNA similar to the corresponding respective first or second sequence of the nuclear DNA comprises approximately greater than 1000 bps.

6. The method of claim 1, wherein either the first or second sequence of DNA similar to the corresponding respective first or second sequence of the nuclear DNA comprises approximately greater than 10,000 bps.

7. The method of claim 1, wherein the sequence of DNA of interest further comprises a regulatory or promoter sequence.

8. The method of claim 7, wherein the promoter is uni-directional or bi-directional.

9. The method of claim 1, wherein the sequence of DNA of interest further comprises a promoter sequence and an antibiotic resistance marker.

10. The method of claim 1, wherein the sequence of DNA of interest further comprises a gene for nutrient assimilation or biosynthesis of a metabolite.

11. The method of claim 10, wherein the gene codes for nitrate reductase or nitrite reductase.

12. The method of claim 1, wherein the sequence of DNA of interest is approximately 0 bps, resulting in deletion or near deletion of the target sequence of DNA.

13. The method of claim 1, wherein the sequence of DNA of interest is transcribed but does not encode a polypeptide.

14. The method of claim 1, wherein the sequence of DNA of interest encodes a peptide that is added to a peptide encoded by either the first or the second sequence of nuclear DNA.

15. The method of claim 1, wherein the sequence of DNA of interest encodes a non-coding regulatory DNA sequence.

16. The method of claim 1, wherein the algal cell of the algal genus Nannochloropsis is used for the production of biofuels.

17. The method of claim 1, wherein the algal cell is haploid.

18. The method of claim 1, the method further comprising:

changing a phenotypic characteristic of the algal cell or imparting new characteristics to the algal cell.

19. The method of claim 1, wherein the first sequence of DNA similar to the corresponding first sequence of the nuclear DNA has a first length in base pairs which does not equal a second length in base pairs of the second sequence of DNA similar to the corresponding second sequence of the nuclear DNA.

20. The method of claim 1, wherein the target sequence of DNA is less than 1 kb.