We have developed a multipulse electroporation method to
achieve nuclear transformation of the pennate diatom Phaeodactylum tricornutum [8] and the centric diatom Chaetoceros gracilis
[9]. P. tricornutum is a model species widely used in the diatom
research, while C. gracilis is a more practical species commercially
used in fisheries and also attracting interest as a feedstock for
biofuels production and biorefinery [10]. Our transformation process is very simple and its efficiency is higher than previously
reported diatom transformation system. In this chapter, we
describe our detailed procedure to transform P. tricornutum as an
example.
2 Materials
2.1 Diatom Strain
and Culture
1. Phaeodactylum tricornutum Bohlin (UTEX 642) (see Note 1).
2. The IMK culture medium containing 0.025% (w/v) Daigo’s
IMK, 4% (w/v) sea salts, and 0.2 mM Na 2 SiO 3 (see Note 2).
2.2 Multipulse
Electroporation
1. Resuspension buffer: 0.77 M mannitol mixed with 1.5% IMK
(v/v).
2. NEPA21 multipulse electroporation apparatus (NEPAGENE,
Chiba, Japan) (Fig. 1).
3. A DNA vector for transformation of P. tricornutum: The plasmid pPha-T1 having a bleomycin-resistant gene cassette with
the fcpB promoter [5]. The reporter genes encoding GFP or
luciferase can be inserted downstream of the fcpA promoter in
the vector (see Note 3).
4. A linearized DNA is prepared.
5. IMK agar plates containing 1% agar and 100 μg/mL of Zeocin™ for selection (see Note 4).
3 Methods
A simple scheme of the transformation process is shown in Fig. 2.
Carry out all procedures at room temperature, in a clean bench
when necessary. The following procedure is for six independent
electroporation samples. Diligently follow all regulations when
disposing of waste and genetically modified organisms.
3.1 Preparation
of Diatom Cells
1. Culture diatoms at 20
C in an artificial climate incubator with
gentle shaking under continuous light (30 μmol photons/m
2
/s)
(see Note 5).
2. Use Diatom cells at exponential growth phase
(OD 730 ¼ 0.25–0.38).
170
Kentaro Ifuku and Dongyi Yan
achieve nuclear transformation of the pennate diatom Phaeodactylum tricornutum [8] and the centric diatom Chaetoceros gracilis
[9]. P. tricornutum is a model species widely used in the diatom
research, while C. gracilis is a more practical species commercially
used in fisheries and also attracting interest as a feedstock for
biofuels production and biorefinery [10]. Our transformation process is very simple and its efficiency is higher than previously
reported diatom transformation system. In this chapter, we
describe our detailed procedure to transform P. tricornutum as an
example.
2 Materials
2.1 Diatom Strain
and Culture
1. Phaeodactylum tricornutum Bohlin (UTEX 642) (see Note 1).
2. The IMK culture medium containing 0.025% (w/v) Daigo’s
IMK, 4% (w/v) sea salts, and 0.2 mM Na 2 SiO 3 (see Note 2).
2.2 Multipulse
Electroporation
1. Resuspension buffer: 0.77 M mannitol mixed with 1.5% IMK
(v/v).
2. NEPA21 multipulse electroporation apparatus (NEPAGENE,
Chiba, Japan) (Fig. 1).
3. A DNA vector for transformation of P. tricornutum: The plasmid pPha-T1 having a bleomycin-resistant gene cassette with
the fcpB promoter [5]. The reporter genes encoding GFP or
luciferase can be inserted downstream of the fcpA promoter in
the vector (see Note 3).
4. A linearized DNA is prepared.
5. IMK agar plates containing 1% agar and 100 μg/mL of Zeocin™ for selection (see Note 4).
3 Methods
A simple scheme of the transformation process is shown in Fig. 2.
Carry out all procedures at room temperature, in a clean bench
when necessary. The following procedure is for six independent
electroporation samples. Diligently follow all regulations when
disposing of waste and genetically modified organisms.
3.1 Preparation
of Diatom Cells
1. Culture diatoms at 20
C in an artificial climate incubator with
gentle shaking under continuous light (30 μmol photons/m
2
/s)
(see Note 5).
2. Use Diatom cells at exponential growth phase
(OD 730 ¼ 0.25–0.38).
170
Kentaro Ifuku and Dongyi Yan