systems via genetic engineering strategies and hence, molecular
tool box for rewiring these organisms has been well established
[4, 5]. Given the fact that physiological characteristics such as cell
size and cell wall thickness and composition vary among the algal
species, it is of importance to develop the electroporation strategy
for oleaginous Nannochloropsis oceanica owing to their promising
characteristics [6].
In this chapter, we have designed the methodology for electroporating the recombinant DNA of about 5000 bp which harbors
the Zeocin resistance gene into N. oceanica. The transformed cells
were preliminary screened by antibiotic resistance and molecular
analyses. The report exemplifies a rapid and effective strategy for
introducing transgenes into N. oceanica, thereby expanding the
microalgal genetic toolkit.
2 Materials
1. Early log phase Nannochloropsis oceanica cells (see Note 1).
2. 0.22 μm filters.
3. 375 mM sorbitol: 0.68 g of sorbitol is dissolved in 10 mL of
water, Filter sterilized and stored at 4
C until use.
4. Daigo IMK medium: 0.253 g of Daigo IMK medium is dissolved in 1 L of water and filter sterilized.
5. Solid IMK medium supplemented with the appropriate antibiotic (see Note 2).
6. Salmon sperm DNA (10 mg/mL).
7. Salmon sperm DNA (30–50 μg) is heat-denatured at 95
C for
1 min and kept on ice.
8. Electroporation cuvette (2 mm).
9. Electroporator (Gene Pulser Xcell from Bio-Rad).
3 Methods
3.1 Preparation
of Electrocompetent
Nannochloropsis
oceanica
1. Nannochloropsis oceanica is inoculated into fresh IMK medium
so that final cell concentration in the culture media at the initial
time would be 1 Â 10
6 cells and cultivated at 25 Æ 1
C in an
artificial climate incubator provided with cool-white fluorescence light of 200 μmol photons/m/s under a photoperiod of
15/9 h light/dark.
2. Thereafter, 200–300 mL of Nannochloropsis cells (11 Â 10
6
cells/mL) at early log phase are harvested by centrifugation at
4000 Â g for 10 min at 4
C.
176
Da-Wei Li et al.
tool box for rewiring these organisms has been well established
[4, 5]. Given the fact that physiological characteristics such as cell
size and cell wall thickness and composition vary among the algal
species, it is of importance to develop the electroporation strategy
for oleaginous Nannochloropsis oceanica owing to their promising
characteristics [6].
In this chapter, we have designed the methodology for electroporating the recombinant DNA of about 5000 bp which harbors
the Zeocin resistance gene into N. oceanica. The transformed cells
were preliminary screened by antibiotic resistance and molecular
analyses. The report exemplifies a rapid and effective strategy for
introducing transgenes into N. oceanica, thereby expanding the
microalgal genetic toolkit.
2 Materials
1. Early log phase Nannochloropsis oceanica cells (see Note 1).
2. 0.22 μm filters.
3. 375 mM sorbitol: 0.68 g of sorbitol is dissolved in 10 mL of
water, Filter sterilized and stored at 4
C until use.
4. Daigo IMK medium: 0.253 g of Daigo IMK medium is dissolved in 1 L of water and filter sterilized.
5. Solid IMK medium supplemented with the appropriate antibiotic (see Note 2).
6. Salmon sperm DNA (10 mg/mL).
7. Salmon sperm DNA (30–50 μg) is heat-denatured at 95
C for
1 min and kept on ice.
8. Electroporation cuvette (2 mm).
9. Electroporator (Gene Pulser Xcell from Bio-Rad).
3 Methods
3.1 Preparation
of Electrocompetent
Nannochloropsis
oceanica
1. Nannochloropsis oceanica is inoculated into fresh IMK medium
so that final cell concentration in the culture media at the initial
time would be 1 Â 10
6 cells and cultivated at 25 Æ 1
C in an
artificial climate incubator provided with cool-white fluorescence light of 200 μmol photons/m/s under a photoperiod of
15/9 h light/dark.
2. Thereafter, 200–300 mL of Nannochloropsis cells (11 Â 10
6
cells/mL) at early log phase are harvested by centrifugation at
4000 Â g for 10 min at 4
C.
176
Da-Wei Li et al.