US20150093830A1 - Transformation of algal cells - Google Patents
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- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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- C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
Definitions
- This invention relates to molecular biology, and more specifically to the transformation of algal cells.
- Transformed algae cells may be useful in aquaculture production.
- the transformation of small algal cells with strong cell walls, however, is difficult to achieve. Accordingly, there is a need for improved methods of algal cell transformation.
- 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.
- FIG. 1 shows a sequence of an exemplary transformation construct.
- FIG. 2 illustrates an exemplary method for transforming an algal cell with a particle gun.
- FIG. 3 shows an exemplary polymerase chain reaction result obtained using ble primers as described in connection with Example One.
- FIG. 4 shows the exemplary polymerase chain reaction result obtained as described in connection with Example Two.
- FIG. 5 shows the number of algal cell transformants obtained based on the field strength of electroporation applied to perform the transformation as described in connection with Example Three.
- FIG. 6 shows the number of transformants obtained under exemplary optimal electroporation conditions, while varying the amount of transformation construct DNA used as described in connection with Example Four.
- FIG. 7 illustrates an exemplary method for transforming an algal cell with electroporation.
- Transformed algae cells may be useful in aquaculture production.
- the transformation of small algal cells with strong cell walls, however, is difficult to achieve.
- Various exemplary embodiments of the present invention are useful in the efficient transformation of Nannochloropsis , a microalga of about 3-5 micrometers in size.
- gold particles having a size of about 0.6 micrometers are used in biolistic transformation of algae smaller than about 5 micrometers.
- a transformation vector or transformation construct is precipitated onto the gold particles, and the gold particles carrying the transformation construct are propelled through the cell walls of the algae.
- the transformation efficiency of Nannochloropsis with this method is very low.
- aggregates of gold particles are broken into smaller fragments to increase transformation efficiency.
- 0.6 micrometer gold particles are sonicated for one hour just prior to precipitating a transformation construct onto the gold particles.
- fourteen micrograms of transformation construct may produce approximately twenty zeocine resistant transformants.
- this exemplary embodiment provides about a twenty-fold increase in transformation efficiency over currently available methods.
- An alternative exemplary embodiment uses an improved electroporation method to transform algal cells.
- electroporation algal cells are subjected to an electric current that opens transient pores in the membranes of the algal cells.
- a transformation vector may pass through an open pore, eventually leading to the successful transformation of the algal cell.
- the following electroporation conditions were used: 2200V, 500 ⁇ shunt resistor, 50 ⁇ F capacity, 2 mm gap cuvette.
- iso-osmotic non electrolyte buffer resistance higher than 1100 ⁇
- typical time constants ⁇ of 20-24 ms have been reported by the device. This approach produced hundreds of zeocine resistant transformants per microgram of transformation construct used.
- FIG. 1 shows the sequence of an exemplary transformation construct.
- the exemplary transformation construct 100 includes a promoter sequence 102 , DNA encoding a transcribed but untranslated 5 ′ region (“UTR”) 104 , an intron 106 , a ble gene 108 and DNA encoding a 3′ UTR 110 .
- the transformation construct may comprise any number of promoters, genes, and/or other nucleic acid polymers (naturally occurring or synthetic) and/or their analogs, or other compounds that do not interfere with the ability of the transformation construct to enter the algal cell or the algal genome, or to function.
- additional nucleotides may appear in the transformation construct to facilitate or direct the insertion of the construct (or any part thereof) into a desired location in the genome.
- the various exemplary methods described herein may be employed using the exemplary transformation vectors or transformation constructs PL90, H8 and B9 as described in U.S. Non-Provisional patent application Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-Based Vectors for Algal Cell Transformation,” which is hereby incorporated by reference.
- the difference between the three exemplary vector constructs is the type of selection marker gene (SG) used: the sh ble gene (PL90), the hygromycin B phosphotransferase gene (H8), or the blastocidin S deaminase (B9) gene.
- SG selection marker gene
- FIG. 2 illustrates an exemplary method for transforming an algal cell with a particle gun.
- a desired transformation construct is prepared.
- the transformation construct may be either the PL90 transformation vector, the H8 transformation vector, or the B9 transformation vector.
- small DNA-binding particles are prepared.
- 0.6 ⁇ m gold particles are sonicated in buffer for one hour.
- the transformation construct is adhered to the small DNA-binding particles.
- transformation constructs are precipitated onto the gold particles.
- algal cells are bombarded with the gold particles having the transformation constructs precipitated onto them.
- Nannochloropsis cells according to one exemplary method, are bombarded with fourteen micrograms of transformation construct DNA that has been precipitated onto 0.42 mg of the 0.6 ⁇ m gold particles.
- the algal cells are grown into colonies on selective medium.
- the cells may be further processed to collect the desired product.
- buffers, media, and/or methods of product collection may be used.
- Nannochloropsis oceanica cells were grown in low light (85 ⁇ E/(m2*s)) to mid log phase in F2 media, 50% seawater (See e.g., Guillard, R. R. L. and Ryther, J. H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: 229-239). Nannochloropsis cells were then pelleted at 2500 g for 15 min. The pellet was then resuspended in fresh media.
- Colonies were then analyzed for the presence of the selection marker gene. Colonies on selective media were never obtained in experiments utilizing the control DNA. Only utilization of 1350 PSI rupture disc, stage 1 (uppermost stage, just underneath heptameric adapter) and 1550 PSI rupture disc stage 2 yielded a few transformants under these conditions.
- the PCR program was:
- step 2-4 are amplification stage, 38 cycles
- FIG. 3 shows the exemplary polymerase chain reaction result obtained using ble primers as described in connection with Example One.
- Example One We followed the procedures of Example One, except this time utilizing a 1510 Branson Bath Sonicator, 0.6 ⁇ m gold particles were sonicated for 1-hour just prior to DNA precipitation onto beads. Using the sonicated beads, 14 ⁇ g DNA produced approximately 20 zeocine-resistant transformants.
- the cells were grown in 2 L flasks in F2 medium (containing seawater of half salinity) to early log phase. The cells were then harvested by centrifugation for 10 min. at 2500 and gently resuspended in fresh F2 medium. 5*10 ⁇ 9 cells (in ⁇ 400 ⁇ l) were then plated on an agar plate (containing 1% BactoagarTM from DIFCO) and allowed to dry for ⁇ 20 minutes in a sterile hood.
- F2 medium containing seawater of half salinity
- Tungsten particles 0.7 and 1 ⁇ m average diameter and gold particles 1 ⁇ m or 0.6 ⁇ m average diameter.
- the particles Prior to the precipitation of the DNA onto the particles (the micro carrier), the particles were resuspended in ddH2O and sonicated for 1 hour in a 1510 Branson Bath Sonicator. 3 mg particles were coated with 100 ⁇ g linearized transformation construct DNA as described elsewhere (see user manual Biolistic PDS-1000/He particle gun from Bio-Rad) and resuspended in 500 ⁇ l Ethanol after 2 washing steps in the same volume ethanol.
- the petri dish containing the cells was placed on the corresponding stage within the particle delivery system (stage 1 being the nearest to the heptamer adapter, stage 3 being the one most far away).
- stage 1 being the nearest to the heptamer adapter, stage 3 being the one most far away.
- One of three different rupture discs Biorad was used: 1100 psi, 1350 psi, 1550 psi. Vacuum was generally applied to 29 mm Hg (obtained within ⁇ 20 sec.) and the He pressure was increased at max speed in order to provide the He shock wave after rupture of the rupture disc. The pressure in the delivery chamber was instantly increased at maximal rate (less than 10 sec until atmospheric pressure was reached) and the petri dish with the bombarded cells was recovered.
- the cells were gently scraped off the plate resuspended in 10 ml of F2 medium (half salinity compared to seawater) and allowed to recover in low light ( ⁇ 14 ⁇ E/(m2*s)) over night.
- F2 medium half salinity compared to seawater
- 0.5*10 ⁇ 7 cells were plated on F2 agar plates (petri dish, F2 medium with half salinitiy compared to seawater and 0.8% BactoagarTM from DIFCO) and the respective selection agent. Plates were incubated at 25° C. at 50 ⁇ E/(m2*sec). Colonies were visible after ⁇ 2-3 weeks.
- stage position within the particle delivery system
- rupture disc
- the following table reflects the quantity of transformants observed when using different experimental parameters.
- the vector used in these experiments was PL90 conferring resistance against the drug zeocine.
- Gold particles pretreatment 1 no no no yes yes yes yes Rupture 1100 1350 1550 1100 1350 1550 disc psi psi psi psi psi psi Stage 1 0 1 0 11 4 77 2 0 0 2 25 4 22 3 0 0 0 8 4 5 1 gold particles were washed and then disrupted by 1 h ultra sound treatment
- the vector PL90 may be used for the nuclear transformation of Nannochloropsis by conferring resistance against the drug zeocine.
- FIG. 4 shows the exemplary polymerase chain reaction result obtained as described in connection with Example Two.
- Nannochloropsis oceanica was grown in 2 L flasks in F2 medium (containing seawater of half salinity) to mid log phase. The cells were then harvested by centrifugation for 10 min. at 2500 and gently resuspended in 375 mM sorbitol. The cells were washed several times in 375 mM sorbitol solution in order to minimize conductivity of remaining medium. The cells were finally resuspended to a final concentration of 10 ⁇ 10 cells/ml and used for transformation within an hour.
- Electroporation was performed in a Biorad GenPulser I Electroporator utilizing 2 mm cuvettes. 100 ⁇ l cells at 10 ⁇ 10 cells/ml were pipetted into a 2 mm cuvette and varying amounts of transformation construct in ⁇ 5 ⁇ l volume ddH2O added. The cuvette containing the DNA-cell mixture was gently snipped for mixing and then placed into the electroporation chamber.
- the cells were allowed to stay in the cuvette for 3 minutes before they were recovered and resuspended in 10 ml F2 medium (half salinity). After an overnight incubation in low light ( ⁇ 14 ⁇ E/(m2*s), 0.5*10 ⁇ 7 cells were plated on F2 agar plates and the respective selection agent. Plates were incubated at 25° C. at 50 ⁇ E/(m2*sec). Colonies were visible after ⁇ 2-3 weeks.
- FIG. 5 shows the number of algal cell transformants obtained based on the field strength of electroporation applied to perform the transformation as described in connection with Example Three.
- the result of this experiment as shown in FIG. 5 was very surprising: transformation was successful at field strengths equal or higher than 8 kV/cm and efficiency peaked at 10.5 kV/cm. Note that applied field strengths for the electroporation of algae are typically factor 10 lower. Higher Field strengths than 12000V/cm were not possible to apply (cuvettes arced).
- Nannochloropsis with the vector PL90 if we treat the cells as indicated above.
- FIG. 6 shows the number of transformants obtained under exemplary optimal electroporation conditions, while varying the amount of transformation construct DNA used as described in connection with Example Four.
- FIG. 7 illustrates an exemplary method for transforming an algal cell with electroporation.
- a desired transformation construct is prepared.
- the transformation construct may be either the PL90 transformation vector, the H8 transformation vector, or the B9 transformation vector.
- the transformation construct is added to algal cells, while the algal cells are suspended in a solution of low conductivity.
- the solution is a sorbitol solution.
- the transformation construct is introduced into the algal cells by application of an electrical pulse, resulting in transformation of the algal cells.
- the electrical pulse is applied with a field strength of higher than approximately 10,000 V/cm, and with a shunt resistor of greater than approximately 100 OHM.
- the algal cells are grown into colonies on selective medium.
- the cells may be further processed to collect the desired product.
- buffers, media, and/or methods of product collection may be used.
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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.
Description
- The present application is a continuation of U.S. Non-Provisional patent application Ser. No. 12/480,611 filed on Jun. 8, 2009, titled “Transformation of Algal Cells,” which claims the benefit and priority of U.S. Provisional Patent Application Ser. No. 61/059,672 filed on Jun. 6, 2008, titled “VCP-Based Vector for Nannochloropsis Transformation,” all of which are hereby incorporated by reference.
- The present application is related to U.S. Non-Provisional patent application Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-Based Vectors for Algal Cell Transformation,” which is hereby incorporated by reference.
- The present application is filed with sequence listing(s) attached hereto and incorporated herein by reference.
- 1. Field of the Invention
- This invention relates to molecular biology, and more specifically to the transformation of algal cells.
- 2. Description of Related Art
- Transformed algae cells may be useful in aquaculture production. The transformation of small algal cells with strong cell walls, however, is difficult to achieve. Accordingly, there is a need for improved methods of algal cell transformation.
- 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.
-
FIG. 1 shows a sequence of an exemplary transformation construct. -
FIG. 2 illustrates an exemplary method for transforming an algal cell with a particle gun. -
FIG. 3 shows an exemplary polymerase chain reaction result obtained using ble primers as described in connection with Example One. -
FIG. 4 shows the exemplary polymerase chain reaction result obtained as described in connection with Example Two. -
FIG. 5 shows the number of algal cell transformants obtained based on the field strength of electroporation applied to perform the transformation as described in connection with Example Three. -
FIG. 6 shows the number of transformants obtained under exemplary optimal electroporation conditions, while varying the amount of transformation construct DNA used as described in connection with Example Four. -
FIG. 7 illustrates an exemplary method for transforming an algal cell with electroporation. - Provided herein are exemplary methods for transforming algal cells. Transformed algae cells may be useful in aquaculture production. The transformation of small algal cells with strong cell walls, however, is difficult to achieve. Various exemplary embodiments of the present invention are useful in the efficient transformation of Nannochloropsis, a microalga of about 3-5 micrometers in size.
- Typically, gold particles having a size of about 0.6 micrometers are used in biolistic transformation of algae smaller than about 5 micrometers. A transformation vector or transformation construct is precipitated onto the gold particles, and the gold particles carrying the transformation construct are propelled through the cell walls of the algae. The transformation efficiency of Nannochloropsis with this method is very low.
- According to the various exemplary methods provided herein, aggregates of gold particles are broken into smaller fragments to increase transformation efficiency. Using a 1510 Branson Bath Sonicator, 0.6 micrometer gold particles are sonicated for one hour just prior to precipitating a transformation construct onto the gold particles. Using the sonicated gold particles or beads, fourteen micrograms of transformation construct may produce approximately twenty zeocine resistant transformants. Thus, this exemplary embodiment provides about a twenty-fold increase in transformation efficiency over currently available methods.
- An alternative exemplary embodiment uses an improved electroporation method to transform algal cells. In electroporation, algal cells are subjected to an electric current that opens transient pores in the membranes of the algal cells. A transformation vector may pass through an open pore, eventually leading to the successful transformation of the algal cell. Using a Biorad Genpulser I electroporator set to the exponential protocol, the following electroporation conditions were used: 2200V, 500Ω shunt resistor, 50 μF capacity, 2 mm gap cuvette. When 10̂9 cells have been used in iso-osmotic non electrolyte buffer (resistance higher than 1100Ω) in a volume of 100 microliters, typical time constants τ of 20-24 ms have been reported by the device. This approach produced hundreds of zeocine resistant transformants per microgram of transformation construct used.
-
FIG. 1 shows the sequence of an exemplary transformation construct. Theexemplary transformation construct 100 includes apromoter sequence 102, DNA encoding a transcribed but untranslated 5′ region (“UTR”) 104, anintron 106, able gene 108 and DNA encoding a 3′ UTR 110. The transformation construct may comprise any number of promoters, genes, and/or other nucleic acid polymers (naturally occurring or synthetic) and/or their analogs, or other compounds that do not interfere with the ability of the transformation construct to enter the algal cell or the algal genome, or to function. In some embodiments, additional nucleotides may appear in the transformation construct to facilitate or direct the insertion of the construct (or any part thereof) into a desired location in the genome. - The various exemplary methods described herein may be employed using the exemplary transformation vectors or transformation constructs PL90, H8 and B9 as described in U.S. Non-Provisional patent application Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-Based Vectors for Algal Cell Transformation,” which is hereby incorporated by reference. The difference between the three exemplary vector constructs is the type of selection marker gene (SG) used: the sh ble gene (PL90), the hygromycin B phosphotransferase gene (H8), or the blastocidin S deaminase (B9) gene.
-
FIG. 2 illustrates an exemplary method for transforming an algal cell with a particle gun. - At
step 205, a desired transformation construct is prepared. According to one exemplary embodiment, the transformation construct may be either the PL90 transformation vector, the H8 transformation vector, or the B9 transformation vector. - At
step 210, small DNA-binding particles are prepared. According to one exemplary embodiment, 0.6 μm gold particles are sonicated in buffer for one hour. - At
step 215, the transformation construct is adhered to the small DNA-binding particles. In one exemplary method, transformation constructs are precipitated onto the gold particles. - At
step 220, algal cells are bombarded with the gold particles having the transformation constructs precipitated onto them. Nannochloropsis cells, according to one exemplary method, are bombarded with fourteen micrograms of transformation construct DNA that has been precipitated onto 0.42 mg of the 0.6 μm gold particles. - At
step 225, the algal cells are grown into colonies on selective medium. According to one exemplary embodiment, if the transformed cells produce a large quantity of a desired gene product, the cells may be further processed to collect the desired product. One having ordinary skill in the art will recognize that many appropriate buffers, media, and/or methods of product collection may be used. - Nannochloropsis oceanica cells were grown in low light (85 μE/(m2*s)) to mid log phase in F2 media, 50% seawater (See e.g., Guillard, R. R. L. and Ryther, J. H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: 229-239). Nannochloropsis cells were then pelleted at 2500 g for 15 min. The pellet was then resuspended in fresh media. 5*10̂9 resuspended cells were spread on petri dishes (F2 media, 50% seawater, 1% Bactoagar™ from DIFCO) and allowed to dry. 0.6 μm gold particles (Biorad) were coated essentially as recommended by Biorad with transformation vector PL90. The plated cells were then bombarded with microparticles (coated with transformation construct DNA as recommended by Biorad) by a particle gun (Biolistic PDS-1000/He particle gun Bio-Rad) at vacuum pressure of 29 mm Hg utilizing the heptameric adapter from Biorad. All different stages were used in combination with 3 different rupture discs (1100 PSI, 1350 PSI, 1550 PSI) provided by Biorad. Cells were subsequently resuspended in 10 ml fresh F2 Medium (half salinity) and allowed to recover overnight under low light (˜10-25 μE/(m2*s)). 0.5*10̂7 cells were then plated on agar plates (0.8% Bactoagar™ from DIFCO) containing the selection agent (2 μg/ml zeocine, μg/
ml 300 hygromycin or 50 μg/ml blasticidin, depending on the transformation construct used) and incubated at 25° C. at ˜50 μE/(m2*sec). Negative control DNA was linearized pJet1 vector DNA (Fermentas). Growth of colonies could be observed after ˜2-3 weeks. Colonies were then analyzed for the presence of the selection marker gene. Colonies on selective media were never obtained in experiments utilizing the control DNA. Only utilization of 1350 PSI rupture disc, stage 1 (uppermost stage, just underneath heptameric adapter) and 1550 PSIrupture disc stage 2 yielded a few transformants under these conditions. - To proof for the presence of transformation construct within the cells (and thus that the transformation experiment was a success), a single colony was picked and restreaked on unselective medium and allowed to grow for approximately one week. Cells were then scraped (˜10 μg cells), washed 3 times in F2-50% seawater, incubated with 30 units DNAse I in DNAse I buffer (Fementas) 1 hour at 37° C. Cells were then washed once in F2-50% seawater and resuspended in 50 μl F2-50% seawater. Cells were pelleted and the supernatant collected; cells were resuspended in 50 μl A ddH20. Cell and supernatant fractions were incubated 7 minutes at 95° C.; 5 μl each fraction were subjected to PCR (50 μl reaction volume) with specific primers against the ble gene (rev: 5′TTA GTC CTG CTC CTC GGC CAC GAA3′, for: 5′ATG GCC AAG TTG ACC AGT GCC GT3′).
- The PCR program was:
- 1. 94 C, 3 min, 1 cycle;
- 2. 94 C, 15 sec;
- 3. 58 C, 30 sec;
- 4. 72 C, 30 sec (steps 2-4 are amplification stage, 38 cycles);
- 5. 72 C, 5 min; and
- 6. 4 C, cool down and keep temperature.
-
FIG. 3 shows the exemplary polymerase chain reaction result obtained using ble primers as described in connection with Example One. - We followed the procedures of Example One, except this time utilizing a 1510 Branson Bath Sonicator, 0.6 μm gold particles were sonicated for 1-hour just prior to DNA precipitation onto beads. Using the sonicated beads, 14 μg DNA produced approximately 20 zeocine-resistant transformants.
- Detailed Procedure.
- Growth and preparation of cells: the cells were grown in 2 L flasks in F2 medium (containing seawater of half salinity) to early log phase. The cells were then harvested by centrifugation for 10 min. at 2500 and gently resuspended in fresh F2 medium. 5*10̂9 cells (in ˜400 μl) were then plated on an agar plate (containing 1% Bactoagar™ from DIFCO) and allowed to dry for ˜20 minutes in a sterile hood.
- Preparation of Particles.
- Different sizes and materials of particles were used (all obtained from Biorad), i.e. Tungsten particles 0.7 and 1 μm average diameter and
gold particles 1 μm or 0.6 μm average diameter. Prior to the precipitation of the DNA onto the particles (the micro carrier), the particles were resuspended in ddH2O and sonicated for 1 hour in a 1510 Branson Bath Sonicator. 3 mg particles were coated with 100 μg linearized transformation construct DNA as described elsewhere (see user manual Biolistic PDS-1000/He particle gun from Bio-Rad) and resuspended in 500 μl Ethanol after 2 washing steps in the same volume ethanol. 10 μl of this DNA coated particle solution (‘the microcarrier’) was pipetted onto each macrocarrier which was fitted subsequently into the Hepta Adapter (optional component of the Biolistic® PDS-1000/He Particle Delivery system). Thus, each shot with the particle gun provided 7×2 μg=14 μg DNA precipitated on micro carrier. Coated particles were used for biolistic transformation within an hour. - Delivery of Particles to Cells.
- The petri dish containing the cells was placed on the corresponding stage within the particle delivery system (
stage 1 being the nearest to the heptamer adapter,stage 3 being the one most far away). One of three different rupture discs (Biorad) was used: 1100 psi, 1350 psi, 1550 psi. Vacuum was generally applied to 29 mm Hg (obtained within ˜20 sec.) and the He pressure was increased at max speed in order to provide the He shock wave after rupture of the rupture disc. The pressure in the delivery chamber was instantly increased at maximal rate (less than 10 sec until atmospheric pressure was reached) and the petri dish with the bombarded cells was recovered. - Post Delivery Treatment of Cells.
- The cells were gently scraped off the plate resuspended in 10 ml of F2 medium (half salinity compared to seawater) and allowed to recover in low light (˜14 μE/(m2*s)) over night. Next day, 0.5*10̂7 cells were plated on F2 agar plates (petri dish, F2 medium with half salinitiy compared to seawater and 0.8% Bactoagar™ from DIFCO) and the respective selection agent. Plates were incubated at 25° C. at 50 μE/(m2*sec). Colonies were visible after ˜2-3 weeks.
- Results.
- 1. From all the particles tested, only the 0.6 μm gold particles produced transformants.
- 2. We tested all different combinations of stage position (within the particle delivery system) and rupture disc.
- 3. A major difference was observed when we pretreated the gold particles with ultrasound.
- The following table reflects the quantity of transformants observed when using different experimental parameters. The vector used in these experiments was PL90 conferring resistance against the drug zeocine.
-
Gold particles pretreatment1 no no no yes yes yes Rupture 1100 1350 1550 1100 1350 1550 disc psi psi psi psi psi psi Stage 1 0 1 0 11 4 77 2 0 0 2 25 4 22 3 0 0 0 8 4 5 1gold particles were washed and then disrupted by 1 h ultra sound treatment - These results indicate that:
- 1. Pretreatment of the 0.6 μm gold particles by ultra sound dramatically increases the transformation efficiency.
- 2. That the transformation efficiency is highest at 1100 Psi pressure (rupture disc) and if the petri dish containing the cells is localized on
stage 2. - 3. The vector PL90 may be used for the nuclear transformation of Nannochloropsis by conferring resistance against the drug zeocine.
-
FIG. 4 shows the exemplary polymerase chain reaction result obtained as described in connection with Example Two. - Detailed Procedure.
- Growth and Preparation of Cells.
- Nannochloropsis oceanica was grown in 2 L flasks in F2 medium (containing seawater of half salinity) to mid log phase. The cells were then harvested by centrifugation for 10 min. at 2500 and gently resuspended in 375 mM sorbitol. The cells were washed several times in 375 mM sorbitol solution in order to minimize conductivity of remaining medium. The cells were finally resuspended to a final concentration of 10̂10 cells/ml and used for transformation within an hour.
- Electroporation.
- Electroporation was performed in a Biorad GenPulser I Electroporator utilizing 2 mm cuvettes. 100 μl cells at 10̂10 cells/ml were pipetted into a 2 mm cuvette and varying amounts of transformation construct in <5 μl volume ddH2O added. The cuvette containing the DNA-cell mixture was gently snipped for mixing and then placed into the electroporation chamber.
- Device settings were exponential decay protocol with 500 Ohm Shunt resistance and 50 μF capacity, 2 mm Gap
- Post Delivery Treatment of Cells.
- After electroporation the cells were allowed to stay in the cuvette for 3 minutes before they were recovered and resuspended in 10 ml F2 medium (half salinity). After an overnight incubation in low light (˜14 μE/(m2*s), 0.5*10̂7 cells were plated on F2 agar plates and the respective selection agent. Plates were incubated at 25° C. at 50 μE/(m2*sec). Colonies were visible after ˜2-3 weeks.
- Results.
- In initial experiments, we used 0.5 μg DNA/100 μl cell suspension and varied the voltage. We routinely measured electrical resistance of the cell-DNA mixture with the respective option in the Biorad GenPulser I Electroporator to ensure that resistance was >1100 Ohm. The actual electroporation usually returned exponential decay times τ of 20-24 ms. Initially, when performing these experiments with varying field strengths we got a single transformant (again we used the PL90 vector linearized and performed selection on agar plates containing zeocine) at the highest field strength initially tried (which was 1000V, =5 kV/cm). 5 kV/cm is a very high field strength and we did not expect that we might obtain transformants at all at such a high field strength. Since we obtained a single transformant at this high voltage we performed electroporations in additional experiments at very high voltages.
-
FIG. 5 shows the number of algal cell transformants obtained based on the field strength of electroporation applied to perform the transformation as described in connection with Example Three. The result of this experiment as shown inFIG. 5 was very surprising: transformation was successful at field strengths equal or higher than 8 kV/cm and efficiency peaked at 10.5 kV/cm. Note that applied field strengths for the electroporation of algae are typically factor 10 lower. Higher Field strengths than 12000V/cm were not possible to apply (cuvettes arced). - We conclude that we can transform Nannochloropsis with the vector PL90 if we treat the cells as indicated above.
- Parameters are (within Biorad GenPulser I Electroporator): 10̂9 cells in 100 μl 375 mM sorbitol (washed several times), 500 Ohm shunt resistor, 2 mm gap cuvette, 10500 volt/cm (=2100V/2 mm), exponential decay program.
- We then tested the efficiency of transformation by adding varying amounts of DNA to the electroporation cuvette (again containing 10̂9 cells in 100 μl).
-
FIG. 6 shows the number of transformants obtained under exemplary optimal electroporation conditions, while varying the amount of transformation construct DNA used as described in connection with Example Four. We observed a linear increase in the number of transformants obtained based on increasing the amount of transformation construct DNA used. For example, 3 μg of transformation construct DNA added during a single electroporation experiment yielded approximately 9000 transformants, 1.5 μg of transformation construct DNA yielded approximately 4500 transformants. This result indicates that no saturation with DNA occurred and that the transformation method via electroporation which we established is extremely efficient. -
FIG. 7 illustrates an exemplary method for transforming an algal cell with electroporation. - At
step 705, a desired transformation construct is prepared. According to one exemplary embodiment, the transformation construct may be either the PL90 transformation vector, the H8 transformation vector, or the B9 transformation vector. - At
step 710, the transformation construct is added to algal cells, while the algal cells are suspended in a solution of low conductivity. In various exemplary embodiments, the solution is a sorbitol solution. - At
step 715, the transformation construct is introduced into the algal cells by application of an electrical pulse, resulting in transformation of the algal cells. According to some exemplary embodiments, the electrical pulse is applied with a field strength of higher than approximately 10,000 V/cm, and with a shunt resistor of greater than approximately 100 OHM. - At
step 720, the algal cells are grown into colonies on selective medium. According to one exemplary embodiment, if the transformed cells produce a large quantity of a desired gene product, the cells may be further processed to collect the desired product. One having ordinary skill in the art will recognize that many appropriate buffers, media, and/or methods of product collection may be used. - While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of a preferred embodiment should not be limited by any of the above-described exemplary embodiments.
Claims (7)
1. 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, the nucleic acid including a gene for a fatty acid;
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.
2. The method of claim 1 , wherein the nucleic acid includes a transformation construct.
3. The method of claim 1 , wherein the electrical pulse is applied with a field strength of higher than approximately 7500 V/cm.
4. The method of claim 1 , wherein the electrical pulse is applied with a field strength of higher than approximately 9000 V/cm.
5. The method of claim 1 , wherein the electrical pulse is applied with a field strength of higher than approximately 10,000 V/cm.
6. The method of claim 1 , wherein the electrical pulse is applied with a shunt resistor of greater than approximately 100 OHM.
7. The method of claim 1 , wherein the algal cell is of genus Nannochloropsis.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11834650B1 (en) | 2018-06-22 | 2023-12-05 | National Technology & Engineering Solutions Of Sandia, Llc | Methods of transfection using sonoporation |
Families Citing this family (36)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2009255947B2 (en) | 2008-06-06 | 2014-12-18 | Aurora Algae, Inc. | Vcp-based vectors for algal cell transformation |
US20100022393A1 (en) * | 2008-07-24 | 2010-01-28 | Bertrand Vick | Glyphosate applications in aquaculture |
US8940340B2 (en) * | 2009-01-22 | 2015-01-27 | Aurora Algae, Inc. | Systems and methods for maintaining the dominance of Nannochloropsis in an algae cultivation system |
US8314228B2 (en) | 2009-02-13 | 2012-11-20 | Aurora Algae, Inc. | Bidirectional promoters in Nannochloropsis |
CA2756035A1 (en) * | 2009-03-20 | 2010-09-23 | Algal Scientific Corporation | System and method for treating wastewater via phototactic heterotrophic microorganism growth |
US8865468B2 (en) * | 2009-10-19 | 2014-10-21 | Aurora Algae, Inc. | Homologous recombination in an algal nuclear genome |
US8809046B2 (en) | 2011-04-28 | 2014-08-19 | Aurora Algae, Inc. | Algal elongases |
US8865452B2 (en) * | 2009-06-15 | 2014-10-21 | Aurora Algae, Inc. | Systems and methods for extracting lipids from wet algal biomass |
US9101942B2 (en) * | 2009-06-16 | 2015-08-11 | Aurora Algae, Inc. | Clarification of suspensions |
US8747930B2 (en) * | 2009-06-29 | 2014-06-10 | Aurora Algae, Inc. | Siliceous particles |
US8404473B2 (en) | 2009-06-30 | 2013-03-26 | Aurora Algae, Inc. | Cyanobacterial isolates having auto-flocculation and settling properties |
WO2011011463A2 (en) * | 2009-07-20 | 2011-01-27 | Aurora Biofuels, Inc. | Manipulation of an alternative respiratory pathway in photo-autotrophs |
US8765983B2 (en) * | 2009-10-30 | 2014-07-01 | Aurora Algae, Inc. | Systems and methods for extracting lipids from and dehydrating wet algal biomass |
EP2389799A1 (en) | 2010-05-25 | 2011-11-30 | BioMass Booster, S.L. | Method for increasing plant biomass |
US8722359B2 (en) | 2011-01-21 | 2014-05-13 | Aurora Algae, Inc. | Genes for enhanced lipid metabolism for accumulation of lipids |
US8926844B2 (en) | 2011-03-29 | 2015-01-06 | Aurora Algae, Inc. | Systems and methods for processing algae cultivation fluid |
US8569530B2 (en) | 2011-04-01 | 2013-10-29 | Aurora Algae, Inc. | Conversion of saponifiable lipids into fatty esters |
CN103974966A (en) | 2011-04-28 | 2014-08-06 | 奥罗拉藻类股份有限公司 | Algal desaturases |
US9018013B2 (en) | 2011-08-12 | 2015-04-28 | Life Technologies Corporation | Molecular biology tools for algal engineering |
WO2013166065A1 (en) | 2012-04-30 | 2013-11-07 | Aurora Algae, Inc. | ACP Promoter |
US10612034B2 (en) | 2012-06-01 | 2020-04-07 | Exxonmobil Research And Engineering Company | Promoters and terminators for use in eukaryotic cells |
WO2013188462A2 (en) | 2012-06-12 | 2013-12-19 | Synthetic Genomics, Inc. | Regulatory elements and uses thereof |
WO2014027995A1 (en) * | 2012-08-12 | 2014-02-20 | Life Technologies Corporation | Molecular biology tools for algal engineering |
EP2909304A4 (en) | 2012-10-16 | 2016-09-21 | Exxonmobil Res & Eng Co | Dgat genes and methods of use for triglyceride production in recombinant microorganisms |
US9309523B2 (en) | 2012-12-05 | 2016-04-12 | Exxonmobil Research And Engineering Company | Nannochloropsis kozak consensus sequence |
US8835149B2 (en) | 2012-12-06 | 2014-09-16 | Exxonmobil Research And Engineering Company | DGAT genes comprising pleckstrin homology domains and methods of use for triglyceride production in recombinant microorganisms |
US10123986B2 (en) | 2012-12-24 | 2018-11-13 | Qualitas Health, Ltd. | Eicosapentaenoic acid (EPA) formulations |
US9629820B2 (en) | 2012-12-24 | 2017-04-25 | Qualitas Health, Ltd. | Eicosapentaenoic acid (EPA) formulations |
US9266973B2 (en) | 2013-03-15 | 2016-02-23 | Aurora Algae, Inc. | Systems and methods for utilizing and recovering chitosan to process biological material |
WO2015126992A1 (en) | 2014-02-19 | 2015-08-27 | The Regents Of The University Of California | Colostrum/milk protein compositions |
WO2016109840A2 (en) | 2014-12-31 | 2016-07-07 | Synthetic Genomics, Inc. | Compositions and methods for high efficiency in vivo genome editing |
WO2016168756A1 (en) | 2015-04-15 | 2016-10-20 | Synthetic Genomics, Inc. | Algal chloroplastic srp54 mutants |
EP3246401A1 (en) | 2016-05-20 | 2017-11-22 | Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives | New fatty acid decarboxylase and its uses |
MX2020005939A (en) | 2017-12-08 | 2020-08-24 | Synthetic Genomics Inc | Improving algal lipid productivity via genetic modification of a tpr domain containing protein. |
BR112020010329A2 (en) | 2017-12-29 | 2020-11-17 | Synthetic Genomics, Inc. | genetic modulation of photosynthetic organisms for enhanced growth |
US11891640B1 (en) | 2021-01-20 | 2024-02-06 | National Technology & Engineering Solutions Of Sandia, Llc | Crop protection in algae by exogenous terpene expression |
Family Cites Families (94)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US1926780A (en) | 1931-11-11 | 1933-09-12 | John W Lippincott | Endless water course |
FR94705E (en) | 1966-06-01 | 1969-10-24 | Inst Francais Du Petrole | Improved method for cultivating algae and implementing device. |
US3962466A (en) * | 1972-11-10 | 1976-06-08 | Dai-Nippon Sugar Manufacturing Co., Ltd. | Method for treatment of microorganisms |
US4003337A (en) | 1974-10-23 | 1977-01-18 | Kerry Lamar Moore | Fish growing tank and method |
US4267038A (en) | 1979-11-20 | 1981-05-12 | Thompson Worthington J | Controlled natural purification system for advanced wastewater treatment and protein conversion and recovery |
US4365938A (en) | 1980-01-14 | 1982-12-28 | Warinner Archie F | Modular low head high volume water pump and aquaculture system |
US4535060A (en) | 1983-01-05 | 1985-08-13 | Calgene, Inc. | Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase, production and use |
US5668298A (en) * | 1984-12-24 | 1997-09-16 | Eli Lilly And Company | Selectable marker for development of vectors and transformation systems in plants |
US4658757A (en) | 1985-11-14 | 1987-04-21 | Ocean Ventures-1 | Method and apparatus for improved aquaculture/mariculture |
US5105085A (en) * | 1989-11-17 | 1992-04-14 | Mcguire Danny G | Fluid analysis system |
US7037692B1 (en) | 1990-03-16 | 2006-05-02 | Calgene, Inc. | Plant desaturases compositions and uses |
US5527456A (en) | 1992-06-02 | 1996-06-18 | Jensen; Kyle R. | Apparatus for water purification by culturing and harvesting attached algal communities |
US5661017A (en) * | 1993-09-14 | 1997-08-26 | Dunahay; Terri Goodman | Method to transform algae, materials therefor, and products produced thereby |
US5417550A (en) | 1993-11-02 | 1995-05-23 | Marine Gikens Co., Ltd. | Submersed jet pump for generating a stream of water |
MX9707666A (en) * | 1995-04-06 | 1997-11-29 | Seminis Vegetables | Process for selection of transgenic plant cells. |
WO1997039106A1 (en) * | 1996-04-12 | 1997-10-23 | Martek Biosciences Corporation | Methods and tools for transformation of eukaryotic algae |
JP2000503858A (en) * | 1996-06-14 | 2000-04-04 | ラトガーズ・ザ・ステイト・ユニバーシティ・オブ・ニュージャージー | Plastid marker genes that can be selected based on editing |
US5823781A (en) * | 1996-07-29 | 1998-10-20 | Electronic Data Systems Coporation | Electronic mentor training system and method |
US6117313A (en) | 1996-12-27 | 2000-09-12 | Goldman; Joshua | Method and apparatus for aquaculture and for water treatment related thereto |
ATE435288T1 (en) | 1997-02-27 | 2009-07-15 | Suntory Holdings Ltd | DELTA-9 DESATURASE GENE |
ATE305048T1 (en) | 1997-08-01 | 2005-10-15 | Martek Biosciences Corp | NUTRIENT COMPOSITIONS CONTAINING DHA AND METHOD FOR THE PRODUCTION THEREOF |
US7834855B2 (en) | 2004-08-25 | 2010-11-16 | Apple Inc. | Wide touchpad on a portable computer |
US6166231A (en) | 1998-12-15 | 2000-12-26 | Martek Biosciences Corporation | Two phase extraction of oil from biomass |
CN100460513C (en) | 2000-01-19 | 2009-02-11 | 马泰克生物科学公司 | Solventless extraction process |
US6831040B1 (en) * | 2000-01-27 | 2004-12-14 | The Regents Of The University Of California | Use of prolines for improving growth and other properties of plants and algae |
EP1158051B1 (en) | 2000-05-24 | 2005-12-14 | DSM IP Assets B.V. | Process for producing astaxanthin |
US20030022359A1 (en) | 2000-06-20 | 2003-01-30 | Sayre Richard T. | Transgenic algae for delivering antigens to an animal |
US20070178451A1 (en) * | 2001-08-02 | 2007-08-02 | Molian Deng | Nucleic acid sequences from Chlorella sarokiniana and uses thereof |
DE10040814A1 (en) | 2000-08-21 | 2002-03-07 | Thor Gmbh | Synergistic biocide composition |
US6871195B2 (en) | 2000-09-13 | 2005-03-22 | E-Promentor | Method and system for remote electronic monitoring and mentoring of computer assisted performance support |
EP1415160A2 (en) | 2000-09-30 | 2004-05-06 | Diversa Corporation | Whole cell engineering by mutagenizing a substantial portion of a starting genome, combining mutations, and optionally repeating |
FR2821855B1 (en) * | 2001-03-09 | 2004-04-02 | Cayla | SYNTHETIC GENES AND BACTERIAL PLASMIDS WITHOUT GIC |
US6723243B2 (en) | 2001-04-19 | 2004-04-20 | Aquafiber Technologies Corporation | Periphyton filtration pre- and post-treatment system and method |
US7547551B2 (en) * | 2001-06-21 | 2009-06-16 | University Of Antwerp. | Transfection of eukaryontic cells with linear polynucleotides by electroporation |
US6736572B2 (en) | 2001-07-18 | 2004-05-18 | Brian Geraghty | Method and apparatus for reducing the pollution of boat harbors |
US7238477B2 (en) | 2001-09-24 | 2007-07-03 | Intel Corporation | Methods to increase nucleotide signals by Raman scattering |
US7381326B2 (en) | 2002-02-15 | 2008-06-03 | Israel Haddas | Mega flow system |
US7449568B2 (en) | 2002-02-28 | 2008-11-11 | Takara Bio Inc. | Alga-origin promoter, intron and terminator |
EP1503715A4 (en) | 2002-05-03 | 2005-07-06 | Martek Biosciences Corp | High-quality lipids and methods for producing by enzymatic liberation from biomass |
US20050064577A1 (en) | 2002-05-13 | 2005-03-24 | Isaac Berzin | Hydrogen production with photosynthetic organisms and from biomass derived therefrom |
WO2003094598A1 (en) | 2002-05-13 | 2003-11-20 | Greenfuel Technologies Corporation | Photobioreactor and process for biomass production and mitigation of pollutants in flue gases |
US7821425B2 (en) | 2002-07-12 | 2010-10-26 | Atmel Corporation | Capacitive keyboard with non-locking reduced keying ambiguity |
US20040161364A1 (en) | 2003-02-10 | 2004-08-19 | Carlson Peter S. | Carbon sequestration in aqueous environments |
WO2004106238A2 (en) | 2003-05-27 | 2004-12-09 | Fmc Corporation | Method for control of aquatic vegetation |
AU2003903453A0 (en) | 2003-07-07 | 2003-07-17 | The University Of Queensland | Production of hydrogen |
US20050095569A1 (en) | 2003-10-29 | 2005-05-05 | Patricia Franklin | Integrated multi-tiered simulation, mentoring and collaboration E-learning platform and its software |
KR100754687B1 (en) | 2003-12-12 | 2007-09-03 | 삼성전자주식회사 | Multi input device of wireless terminal and his control method |
US20050181345A1 (en) | 2003-12-19 | 2005-08-18 | Edumedia Development Corporation | Mentor based computer assisted learning |
CA3023314C (en) | 2004-04-22 | 2019-12-10 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
CN103451246B (en) | 2004-04-22 | 2018-02-16 | 联邦科学技术研究组织 | With recombinant cell synthesis of long-chain polyunsaturated fatty acids |
US20080194029A1 (en) | 2004-05-07 | 2008-08-14 | Peter Hegemann | Method for Increasing the Ratio of Homologous to Non-Homologous Recombination |
WO2006017624A2 (en) | 2004-08-03 | 2006-02-16 | Mentornet | Mentor-protege matching system and method |
US7874808B2 (en) | 2004-08-26 | 2011-01-25 | Pentair Water Pool And Spa, Inc. | Variable speed pumping system and method |
US7402428B2 (en) * | 2004-09-22 | 2008-07-22 | Arborgen, Llc | Modification of plant lignin content |
US7678931B2 (en) | 2004-10-22 | 2010-03-16 | Martek Biosciences Corporation | Process for preparing materials for extraction |
US8039230B2 (en) | 2004-11-08 | 2011-10-18 | Chromagenics B.V. | Selection of host cells expressing protein at high levels |
US20060155558A1 (en) | 2005-01-11 | 2006-07-13 | Sbc Knowledge Ventures, L.P. | System and method of managing mentoring relationships |
KR100606803B1 (en) | 2005-05-16 | 2006-08-01 | 엘지전자 주식회사 | Mobile communication terminal with performing function using scroll wheel device and method of performing function using this |
EP2270132A3 (en) | 2005-06-07 | 2012-07-18 | HR Biopetroleum, Inc. | Continuous-batch hybrid process for production of oil and other useful products from photosynthetic microbes |
CN1956335B (en) | 2005-10-27 | 2010-06-23 | 盛群半导体股份有限公司 | Adjacent induction device and its induction method |
WO2007084078A1 (en) | 2006-04-22 | 2007-07-26 | Simlab Inventions & Consultancy Private Limited | A keyboard for a mobile phone or other portable communication devices |
US7745696B2 (en) | 2006-06-12 | 2010-06-29 | The Regents Of The University Of California | Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae |
IL184971A0 (en) | 2006-08-01 | 2008-12-29 | Brightsource Energy Inc | High density bioreactor system, devices and methods |
US8088614B2 (en) | 2006-11-13 | 2012-01-03 | Aurora Algae, Inc. | Methods and compositions for production and purification of biofuel from plants and microalgae |
US20080160488A1 (en) | 2006-12-28 | 2008-07-03 | Medical Simulation Corporation | Trainee-as-mentor education and training system and method |
US9637714B2 (en) | 2006-12-28 | 2017-05-02 | Colorado State University Research Foundation | Diffuse light extended surface area water-supported photobioreactor |
KR20100051041A (en) | 2007-03-07 | 2010-05-14 | 엔벤타 바이오파마슈티칼스 코퍼레이션 | Double-stranded locked nucleic acid compositions |
US20090061493A1 (en) * | 2007-06-01 | 2009-03-05 | Solazyme, Inc. | Lipid Pathway Modification in Oil-Bearing Microorganisms |
US8993314B2 (en) | 2007-07-28 | 2015-03-31 | Ennesys Sas | Algae growth system for oil production |
WO2009018498A2 (en) | 2007-08-01 | 2009-02-05 | Bionavitas, Inc. | Illumination systems, devices, and methods for biomass production |
KR101442542B1 (en) | 2007-08-28 | 2014-09-19 | 엘지전자 주식회사 | Input device and portable terminal having the same |
US8598378B2 (en) | 2008-03-14 | 2013-12-03 | University Of Hawaii | Methods and compositions for extraction and transesterification of biomass components |
WO2009124070A1 (en) | 2008-03-31 | 2009-10-08 | Kuehnle Agrosystems, Inc. | Nuclear based expression of genes for production of biofuels and process co-products in algae |
AU2009255947B2 (en) | 2008-06-06 | 2014-12-18 | Aurora Algae, Inc. | Vcp-based vectors for algal cell transformation |
CN101289659B (en) | 2008-06-19 | 2010-12-22 | 中国海洋大学 | Delta6 fatty acid desaturated enzyme of marine microalgae and applications thereof |
US20090319338A1 (en) | 2008-06-23 | 2009-12-24 | Parks Eric J | Method and system for virtual mentoring |
US20090325270A1 (en) | 2008-06-25 | 2009-12-31 | Bertrand Vick | Use of 2-hydroxy-5-oxoproline in conjunction with algae |
US20100022393A1 (en) | 2008-07-24 | 2010-01-28 | Bertrand Vick | Glyphosate applications in aquaculture |
WO2010027455A1 (en) | 2008-09-04 | 2010-03-11 | Ciris Energy, Inc. | Solubilization of algae and algal materials |
US8170976B2 (en) | 2008-10-17 | 2012-05-01 | The Boeing Company | Assessing student performance and providing instructional mentoring |
US20100198659A1 (en) | 2009-02-04 | 2010-08-05 | Sirota Consulting LLC | Methods for matching and managing mentors and mentees and systems thereof |
US8314228B2 (en) | 2009-02-13 | 2012-11-20 | Aurora Algae, Inc. | Bidirectional promoters in Nannochloropsis |
US8434626B2 (en) | 2009-02-16 | 2013-05-07 | Combined Power, Llc | System and related method for concentrating biological culture and circulating biological culture and process fluid |
US8865468B2 (en) | 2009-10-19 | 2014-10-21 | Aurora Algae, Inc. | Homologous recombination in an algal nuclear genome |
US8809046B2 (en) | 2011-04-28 | 2014-08-19 | Aurora Algae, Inc. | Algal elongases |
US9101942B2 (en) | 2009-06-16 | 2015-08-11 | Aurora Algae, Inc. | Clarification of suspensions |
US20100323387A1 (en) | 2009-06-19 | 2010-12-23 | Shaun Bailey | Optimization of Response to Light |
US8404473B2 (en) | 2009-06-30 | 2013-03-26 | Aurora Algae, Inc. | Cyanobacterial isolates having auto-flocculation and settling properties |
WO2011011463A2 (en) | 2009-07-20 | 2011-01-27 | Aurora Biofuels, Inc. | Manipulation of an alternative respiratory pathway in photo-autotrophs |
US20120107801A1 (en) | 2009-10-19 | 2012-05-03 | Oliver Kilian | High-efficiency homologous recombination in the oil-producing alga, nannochloropsis |
US8722359B2 (en) | 2011-01-21 | 2014-05-13 | Aurora Algae, Inc. | Genes for enhanced lipid metabolism for accumulation of lipids |
CN103974966A (en) | 2011-04-28 | 2014-08-06 | 奥罗拉藻类股份有限公司 | Algal desaturases |
US8709766B2 (en) | 2011-10-17 | 2014-04-29 | Colorado School Of Mines | Use of endogenous promoters in genetic engineering of Nannochloropsis gaditana |
WO2013166065A1 (en) | 2012-04-30 | 2013-11-07 | Aurora Algae, Inc. | ACP Promoter |
-
2009
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- 2009-06-08 US US12/480,635 patent/US8318482B2/en not_active Expired - Fee Related
- 2009-06-08 US US12/480,611 patent/US8119859B2/en not_active Expired - Fee Related
- 2009-06-08 EP EP09759628A patent/EP2297326A4/en not_active Ceased
- 2009-06-08 WO PCT/US2009/046650 patent/WO2009149465A1/en active Application Filing
- 2009-06-08 WO PCT/US2009/046656 patent/WO2009149470A1/en active Application Filing
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2012
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- 2012-11-26 US US13/685,659 patent/US8685723B2/en not_active Expired - Fee Related
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2013
- 2013-06-11 US US13/915,555 patent/US8753879B2/en not_active Expired - Fee Related
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2014
- 2014-05-13 US US14/276,743 patent/US20150093830A1/en not_active Abandoned
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11834650B1 (en) | 2018-06-22 | 2023-12-05 | National Technology & Engineering Solutions Of Sandia, Llc | Methods of transfection using sonoporation |
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US20130078716A1 (en) | 2013-03-28 |
WO2009149465A1 (en) | 2009-12-10 |
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AU2009255947B2 (en) | 2014-12-18 |
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WO2009149465A8 (en) | 2010-07-29 |
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US8318482B2 (en) | 2012-11-27 |
US20090317857A1 (en) | 2009-12-24 |
US20120208279A1 (en) | 2012-08-16 |
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