High throughput screening of genetically modified photosynthetic organisms
View Patent ↗The present invention provides a method and compositions for high throughput screening of genetically modified photosynthetic organisms for plasmic state. The present invention provides methods of producing one or more proteins, including biomass degrading enzymes in a plant. Also provided are the methods of producing biomass degradation pathways in alga cells, particularly in the chloroplast. Single enzymes or multiple enzymes may be produced by the methods disclosed. The methods disclosed herein allow for the production of biofuel, including ethanol.
1. A method of determining the plasmic state in a non-vascular photosynthetic organism that has been transformed with an exogenous sequence comprising:
providing in a mixture for a polymerase chain reaction (PCR)
a first primer pair, wherein a first primer of said first primer pair anneals to a selected site in the chloroplast genome of said organism, a second primer of said first primer pair anneals to a flanking region of said selected site, and said first primer pair is capable of amplifying a first endogenous chloroplast sequence,
a second primer pair wherein a first primer of said second primer pair and a second primer of said second primer pair anneal to a second endogenous chloroplast nucleic acid sequence that is not said first endogenous chloroplast sequence,
a polymerase, and
a chloroplast nucleic acid molecule from said organism;
performing a polymerase chain reaction (PCR); and
analyzing the product of said reaction,
wherein said selected site is deleted or disrupted when an exogenous nucleic acid is incorporated into said non-vascular photosynthetic organism.
2. The method of claim 1 , wherein said exogenous nucleic acid sequence comprises at least one gene of interest and a selectable marker.
3. The method of claim 2 , wherein said gene of interest encodes a biomass degrading enzyme.
4. The organism of claim 1 , wherein said non-vascular photosynthetic organism is a microalga and said plasmic state is homoplasmy.
5. The method of claim 1 , wherein said plasmic state is the degree of heteroplasmy.
6. The method of claim 1 , wherein said polymerase chain reaction (PCR) comprises more than 30 cycles.
7. The method of claim 1 , wherein an amplification product of said first primer pair comprises an untranslated region (UTR) and a coding region.
8. The method of claim 7 , wherein said untranslated region is a 5′ untranslated region.
9. The method of claim 1 , further comprising:
providing a second reaction comprising:
a third primer pair wherein a first primer of said third primer pair anneals to said flanking region of said selected site and a second primer of said third primer pair anneals to a said exogenous nucleic acid sequence,
a polymerase, and
a nucleic acid molecule from said organism; and
performing a polymerase chain reaction (PCR); and
analyzing the product of said second reaction.
10. The method of claim 9 , wherein an amplification product of said third primer pair comprises a portion of said flanking region of said selected site and at least a portion of said exogenous nucleic acid sequence.
11. The method of claim 9 , wherein said second polymerase chain reaction (PCR) comprises more than 30 cycles.
12. The method of claim 1 , wherein said reaction further comprises a third primer pair, wherein a first primer of said third primer pair anneals to said flanking region of said selected site and a second primer of said third primer pair anneals to said exogenous nucleic acid sequence.
13. The method of claim 12 , wherein an amplification product of said third primer pair comprises a portion of said flanking region of said selected site and a portion of said exogenous nucleic acid sequence.
14. A method of producing a genetically-modified homoplasmic non-vascular photosynthetic organism comprising:
transforming at least one chloroplast of said non-vascular photosynthetic organism with an exogenous nucleic acid sequence;
providing in a polymerase chain reaction (PCR):
a first primer pair, wherein at least one primer of said first primer pair anneals to a selected site that is deleted or disrupted by the incorporation of said exogenous nucleic acid sequence, and said first primer pair is capable of amplifying a first endogenous chloroplast sequence,
a second primer pair, wherein a first primer of said second primer pair and a second primer of said second primer pair anneal to a second chloroplast nucleic acid sequence that is not deleted or disrupted by said incorporation of said exogenous nucleic acid sequence,
a polymerase; and
a chloroplast DNA from said organism; and
performing a PCR reaction;
analyzing the product of said PCR reaction; and
selecting a homoplasmic non-vascular photosynthetic organism based on the absence of a product from said first primer pair.
15. The method of claim 14 , wherein said exogenous nucleic acid sequence comprises at least one gene of interest and a selectable marker.
16. The method of claim 15 , wherein said gene of interest encodes a biomass degrading enzyme.
17. The method of claim 14 , wherein said non-vascular photosynthetic organism is a microalga.
18. The method of claim 14 , further providing a second reaction comprising a third primer pair wherein at least one of a first primer and a second primer of said third primer pair anneals to said exogenous nucleic acid sequence.
19. The method of claim 18 , wherein an amplification product of said third primer pair comprises a portion of said exogenous nucleic acid sequence and a nucleic acid sequence flanking said exogenous nucleic acid sequence.
20. The method of claim 14 , wherein an amplification product of said first endogenous chloroplast sequence comprises an untranslated region (UTR) and a coding region.
21. The method of claim 20 , wherein said untranslated region is a 5′ untranslated region.
22. The method of claim 14 , wherein said PCR reaction further comprises a third primer pair, wherein at least one of a first primer and a second primer of said third primer pair anneals to said exogenous nucleic acid sequence.
23. The method of claim 22 , wherein an amplification product of said third primer pair comprises at least a portion of said exogenous nucleic acid sequence.
24. The method of claim 14 , wherein said PCR reaction comprises more than 30 cycles.
25. The method of claim 1 , wherein said selected site is deleted when said exogenous nucleic acid is incorporated into said non-vascular photosynthetic organism.
26. The method of claim 14 , wherein said segment of said selected site is deleted when said exogenous nucleic acid is incorporated into said non-vascular photosynthetic organism.