metalloprotease gametolysin for cell wall degradation [6]. However,
both options are associated with technical difficulties because cw
mutants are fragile and cell-wall removal is a time-consuming process. To overcome these difficulties, we have developed a rapid
transformation method without cell-wall removal using a square
electric pulses-generating electroporator [7], providing a more
convenient method for Chlamydomonas transformation and
enhancing its usability as a model organism.
2 Materials
2.1 Preparation
of aph7
00 DNA Cassette
1. PrimeSTAR GXL DNA Polymerase (Takara). 5Â PCR buffer
and 2.5 mM dNTP mixture are packaged together.
2. pHyg3 plasmid (1 ng/μL): This plasmid harbors the aph7
00
expression cassette [8], which confers hygromycin B resistance.
The pHyg3 plasmid is available from the Chlamydomonas
Resource Center (http://www.chlamycollection.org/).
3. Primers for amplification of the aph7
00 expression cassette:
pHyg3-F1 primer (5
0 -GCACCCCAGGCTTTACACTTTATGCTTCC-3
0 ) and pHyg3-R1 primer (5
0 -CCATTCAGG
CTGCGCAACTGTTGG-3
0 ).
4. Thermal Cycler.
5. QIAquick PCR Purification Kit (QIAGEN).
6. 1% agarose/TAE gel.
2.2 Culture Media
1. Nutrient solution: For a 100Â stock solution, weigh 242 g of
Tris, 40 g of NH 4 Cl, 10 g of MgSO 4 ·7H 2 O, 5.0 g of
CaCl 2 ·2H 2 O and dissolve in 900 mL deionized water. Add
100 mL of glacial acetic acid. Sterilize the solution by autoclaving for 20 min and store at 4
C.
2. Hutner’s trace elements: For a 100Â stock solution, weigh
5.0 g of Na 2 ·EDTA, 2.2 g of ZnSO 4 ·7H 2 O, 1.14 g of
H 3 BO 3 , 0.51 g of MnCl 2 ·4H 2 O, 0.16 g of CoCl 2 ·6H 2 O,
0.16 g of CuSO 4 ·5H 2 O, 0.11 g of (NH 4 )6MO 7 O 24 ·4H 2 O,
and 0.50 g of FeSO 4 ·7H 2 O and dissolve in this order in 1 L
deionized water. Sterilize the solution by autoclaving for
20 min and store at 4
C. The color of the mixture will turn
purple from green.
3. Phosphate solution: For a 1000Â stock solution, weigh 108 g
of K 2 HPO 4 and 56 g of KH 2 PO 4 and dissolve in 1 L deionized
water. Sterilize the solution by autoclaving for 20 min and store
at 4
C.
4. TAP (Tris–acetate–phosphate) medium: Mix 10 mL of nutrient
solution, 10 mL of Hutner’s trace elements, and 1 mL of
156
Takashi Yamano and Hideya Fukuzawa
both options are associated with technical difficulties because cw
mutants are fragile and cell-wall removal is a time-consuming process. To overcome these difficulties, we have developed a rapid
transformation method without cell-wall removal using a square
electric pulses-generating electroporator [7], providing a more
convenient method for Chlamydomonas transformation and
enhancing its usability as a model organism.
2 Materials
2.1 Preparation
of aph7
00 DNA Cassette
1. PrimeSTAR GXL DNA Polymerase (Takara). 5Â PCR buffer
and 2.5 mM dNTP mixture are packaged together.
2. pHyg3 plasmid (1 ng/μL): This plasmid harbors the aph7
00
expression cassette [8], which confers hygromycin B resistance.
The pHyg3 plasmid is available from the Chlamydomonas
Resource Center (http://www.chlamycollection.org/).
3. Primers for amplification of the aph7
00 expression cassette:
pHyg3-F1 primer (5
0 -GCACCCCAGGCTTTACACTTTATGCTTCC-3
0 ) and pHyg3-R1 primer (5
0 -CCATTCAGG
CTGCGCAACTGTTGG-3
0 ).
4. Thermal Cycler.
5. QIAquick PCR Purification Kit (QIAGEN).
6. 1% agarose/TAE gel.
2.2 Culture Media
1. Nutrient solution: For a 100Â stock solution, weigh 242 g of
Tris, 40 g of NH 4 Cl, 10 g of MgSO 4 ·7H 2 O, 5.0 g of
CaCl 2 ·2H 2 O and dissolve in 900 mL deionized water. Add
100 mL of glacial acetic acid. Sterilize the solution by autoclaving for 20 min and store at 4
C.
2. Hutner’s trace elements: For a 100Â stock solution, weigh
5.0 g of Na 2 ·EDTA, 2.2 g of ZnSO 4 ·7H 2 O, 1.14 g of
H 3 BO 3 , 0.51 g of MnCl 2 ·4H 2 O, 0.16 g of CoCl 2 ·6H 2 O,
0.16 g of CuSO 4 ·5H 2 O, 0.11 g of (NH 4 )6MO 7 O 24 ·4H 2 O,
and 0.50 g of FeSO 4 ·7H 2 O and dissolve in this order in 1 L
deionized water. Sterilize the solution by autoclaving for
20 min and store at 4
C. The color of the mixture will turn
purple from green.
3. Phosphate solution: For a 1000Â stock solution, weigh 108 g
of K 2 HPO 4 and 56 g of KH 2 PO 4 and dissolve in 1 L deionized
water. Sterilize the solution by autoclaving for 20 min and store
at 4
C.
4. TAP (Tris–acetate–phosphate) medium: Mix 10 mL of nutrient
solution, 10 mL of Hutner’s trace elements, and 1 mL of
156
Takashi Yamano and Hideya Fukuzawa