4 Notes
1. The diatom cells are obtained from the University of Texas
Culture Collection. They are maintained by weekly or biweekly
subculture at a 12-fold dilution with fresh growth medium.
2. The f/2 + Si medium [11] can also be used instead of IMK.
3. DNA vectors for transformation of C. gracilis: The vector
pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein ( fcp) gene and the
vector pCgNRp contains the inducible promoter of a nitrate
reductase (NR) gene to drive the expression of introduced
genes. In both vectors, an acetyl-CoA acetyltransferase
(ACAT) promoter drives nat gene expression for antibiotic
selection [9]. The vectors having a bleomycin-resistant gene
cassette are also available [12].
4. The transformed C. gracilis cells are selected on IMK agar
plates containing 1% agar and 400 μg/mL of nourseothricin.
5. Two days before transformation, 10 mL of pre-cultured cells
are transferred to 110 mL IMK medium in a 300 mL-flask.
6. Use 3.0 Â 10
7 cells per sample for P. tricornutum; 4.5 Â 10
7
cells per sample for C. gracilis.
7. A centrifuge with a swing-bucket rotor is preferable.
8. A linearized DNA can be prepared either by restriction-enzyme
digestion or by PCR. The pPha-T1can be linearized with NdeI,
when there is no NdeI site in the inserted gene. The plasmids
for C. gracilis can be linearized by HindIII.
9. The electrical impedance of 250–400 Ω is affordable.
10. For transformation of C. gracilis: Square electric poring pulses:
300 V, 5 ms pulse duration, 9 pulses, 25 ms interval, 10% decay
rate. Transfer pulses: 8 V, pulse duration, 50 ms pulse duration,
40 pulses for each direction, interval 50 ms, 40% decay rate.
11. Do not dry the plate thoroughly to prevent the damage to the
cells.
12. Colonies appear within 14 days usually.
13. In general, half of antibiotics-resistant colonies have the genes
inserted in the vector. The copy number of insertion would be
less than 3. The expression of inserted gene should be analyzed
by RT-PCR or other methods.
Transformation of Diatoms with Multi-Pulse Electroporation
173
1. The diatom cells are obtained from the University of Texas
Culture Collection. They are maintained by weekly or biweekly
subculture at a 12-fold dilution with fresh growth medium.
2. The f/2 + Si medium [11] can also be used instead of IMK.
3. DNA vectors for transformation of C. gracilis: The vector
pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein ( fcp) gene and the
vector pCgNRp contains the inducible promoter of a nitrate
reductase (NR) gene to drive the expression of introduced
genes. In both vectors, an acetyl-CoA acetyltransferase
(ACAT) promoter drives nat gene expression for antibiotic
selection [9]. The vectors having a bleomycin-resistant gene
cassette are also available [12].
4. The transformed C. gracilis cells are selected on IMK agar
plates containing 1% agar and 400 μg/mL of nourseothricin.
5. Two days before transformation, 10 mL of pre-cultured cells
are transferred to 110 mL IMK medium in a 300 mL-flask.
6. Use 3.0 Â 10
7 cells per sample for P. tricornutum; 4.5 Â 10
7
cells per sample for C. gracilis.
7. A centrifuge with a swing-bucket rotor is preferable.
8. A linearized DNA can be prepared either by restriction-enzyme
digestion or by PCR. The pPha-T1can be linearized with NdeI,
when there is no NdeI site in the inserted gene. The plasmids
for C. gracilis can be linearized by HindIII.
9. The electrical impedance of 250–400 Ω is affordable.
10. For transformation of C. gracilis: Square electric poring pulses:
300 V, 5 ms pulse duration, 9 pulses, 25 ms interval, 10% decay
rate. Transfer pulses: 8 V, pulse duration, 50 ms pulse duration,
40 pulses for each direction, interval 50 ms, 40% decay rate.
11. Do not dry the plate thoroughly to prevent the damage to the
cells.
12. Colonies appear within 14 days usually.
13. In general, half of antibiotics-resistant colonies have the genes
inserted in the vector. The copy number of insertion would be
less than 3. The expression of inserted gene should be analyzed
by RT-PCR or other methods.
Transformation of Diatoms with Multi-Pulse Electroporation
173