10. Centrifuge the cells at 1500 Â g for 10 min, resuspend in
0.6 mL f/2 medium, and plate 0.2 mL of this suspension
onto solid medium containing 75 μg/mL Zeocin (see Note 6).
11. Parafilm the plate and incubate the plate for 10~15 days at
22
C with continuously light. Then colonies appear and can
be further processed after 1–2 weeks.
4 Notes
1. The restriction site used for plasmid linearization should locate
away from the expression cassettes of the plasmid.
2. Check for complete linearization using agarose gel electrophoresis (0.8% gel).
3. Sorbitol could dissolve easily at room temperature when stirred
well. If necessary, warm the solution to about 37
C to accelerate and obtain complete dissolution.
4. Resuspend the pellet on ice when the room temperature is
above 22
C.
5. The concentration of linearized plasmid DNA determined by
spectrophotometry should not be less than 0.1 μg/μL.
6. Plate gently once and avoid repeated plating.
References
1. Apt KE, Kroth-Pancic PG, Grossman A (1996)
Stable nuclear transformation of the diatom
Phaeodactylum tricornutum. Mol Gen Genet
252:572–579
2. Falciatore A, Casotti R, Leblanc C, Abrescia C,
Bowler C (1999) Transformation of nonselectable reporter genes in marine diatoms. Mar
Biotechnol 1:239–251
3. Miyagawa A, Okami T, Kira N, Yamaguchi H,
Ohnishi K, Adachi M (2009) High efficiency
transformation of the diatom Phaeodactylum
tricornutum with a promoter from the diatom
Cylindrotheca
fusiformis.
Phycol
Res
57:142–146
4. Zaslavskaia LA, Lippmeier JC, Kroth PG,
Grossman AR, Apt KE (2000) Transformation
of the diatom Phaeodactylum tricornutum
(Bacillariophyceae) with a variety of selectable
marker and reporter genes. J Phycol
36:379–386
5. Fischer H, Robl I, Sumper M, Kro ¨ger N
(1999) Targeting and covalent modification
of cell wall and membrane proteins heterologously expressed in the diatom Cylindrotheca
fusiformis (Bacillariophyceae). J Phycol
35:113–120
6. Poulsen N, Kro ¨ger N (2005) A new molecular
tool for transgenic diatoms—control of mRNA
and protein biosynthesis by an inducible
promoter-terminator
cassette.
FEBS
J
272:3413–3423
7. Poulsen N, Chesley PM, Kro ¨ger N (2006)
Molecular genetic manipulation of the diatom
Thalassiosira pseudonana (Bacillariophyceae). J
Phycol 42:1059–1065
8. Miyagawa-Yamaguchi A, Okami T, Kira N,
Yamaguchi H, Ohnishi K, Adachi M (2011)
Stable nuclear transformation of the diatom
Chaetoceros sp. Phycol Res 59:113–119
9. Muto M, Fukuda Y, Nemoto M, Yoshino T,
Matsunaga T, Tanaka T (2013) Establishment
of a genetic transformation system for the
marine pennate diatom Fistulifera sp. strain
JPCC DA0580—a high triglyceride producer.
Mar Biotechnol 15:48–55
10. Du ¨nahay TG, Jarvis EE, Roessler PG (1995)
Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J Phycol
31:1004–1012
166
Hanhua Hu and Yufang Pan
0.6 mL f/2 medium, and plate 0.2 mL of this suspension
onto solid medium containing 75 μg/mL Zeocin (see Note 6).
11. Parafilm the plate and incubate the plate for 10~15 days at
22
C with continuously light. Then colonies appear and can
be further processed after 1–2 weeks.
4 Notes
1. The restriction site used for plasmid linearization should locate
away from the expression cassettes of the plasmid.
2. Check for complete linearization using agarose gel electrophoresis (0.8% gel).
3. Sorbitol could dissolve easily at room temperature when stirred
well. If necessary, warm the solution to about 37
C to accelerate and obtain complete dissolution.
4. Resuspend the pellet on ice when the room temperature is
above 22
C.
5. The concentration of linearized plasmid DNA determined by
spectrophotometry should not be less than 0.1 μg/μL.
6. Plate gently once and avoid repeated plating.
References
1. Apt KE, Kroth-Pancic PG, Grossman A (1996)
Stable nuclear transformation of the diatom
Phaeodactylum tricornutum. Mol Gen Genet
252:572–579
2. Falciatore A, Casotti R, Leblanc C, Abrescia C,
Bowler C (1999) Transformation of nonselectable reporter genes in marine diatoms. Mar
Biotechnol 1:239–251
3. Miyagawa A, Okami T, Kira N, Yamaguchi H,
Ohnishi K, Adachi M (2009) High efficiency
transformation of the diatom Phaeodactylum
tricornutum with a promoter from the diatom
Cylindrotheca
fusiformis.
Phycol
Res
57:142–146
4. Zaslavskaia LA, Lippmeier JC, Kroth PG,
Grossman AR, Apt KE (2000) Transformation
of the diatom Phaeodactylum tricornutum
(Bacillariophyceae) with a variety of selectable
marker and reporter genes. J Phycol
36:379–386
5. Fischer H, Robl I, Sumper M, Kro ¨ger N
(1999) Targeting and covalent modification
of cell wall and membrane proteins heterologously expressed in the diatom Cylindrotheca
fusiformis (Bacillariophyceae). J Phycol
35:113–120
6. Poulsen N, Kro ¨ger N (2005) A new molecular
tool for transgenic diatoms—control of mRNA
and protein biosynthesis by an inducible
promoter-terminator
cassette.
FEBS
J
272:3413–3423
7. Poulsen N, Chesley PM, Kro ¨ger N (2006)
Molecular genetic manipulation of the diatom
Thalassiosira pseudonana (Bacillariophyceae). J
Phycol 42:1059–1065
8. Miyagawa-Yamaguchi A, Okami T, Kira N,
Yamaguchi H, Ohnishi K, Adachi M (2011)
Stable nuclear transformation of the diatom
Chaetoceros sp. Phycol Res 59:113–119
9. Muto M, Fukuda Y, Nemoto M, Yoshino T,
Matsunaga T, Tanaka T (2013) Establishment
of a genetic transformation system for the
marine pennate diatom Fistulifera sp. strain
JPCC DA0580—a high triglyceride producer.
Mar Biotechnol 15:48–55
10. Du ¨nahay TG, Jarvis EE, Roessler PG (1995)
Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J Phycol
31:1004–1012
166
Hanhua Hu and Yufang Pan