Plasmids

From Saul Purton, 1993

Chloroplast DNA EcoRI 34 (ca. 1.1 kb) in pUC9; contains psaA exon 2 and 3′ end of psbD

host strain: unknown
amp resistant

Boynton-Gillham laboratory, Duke Univesrsity, 1993

Chloroplast DNA PstI 11 (7.8 kb) in BSKS+; contains atpA (3′ end), atpH, rps11, rpoA

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+

Insert: PstI 11 (7.8 kb)

Genes present: atpA (part), atpH, rpoA (part)

Vector: BSKS+

Bacterial host strain: JM83

Selectable marker: amp-r, white on X-gal

Origin: Barbara Randolph-Anderson in Boynton/Gillham lab, 1993

This plasmid was derived from P-80, which was the same insert in a tetracycline-resistant vector. This map is from the original file for plasmid P-80 and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke Univesrsity, 1993

Chloroplast DNA PstI 13 (4.0 kb) in BSKS+; internal to ORF2971

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+

Insert: PstI 13 (4.0 kb)

Genes present: internal to ORF2971

Vector: BSKS+

Bacterial host strain: JM83

Selectable marker: amp-r, white on X-gal

Origin: Barbara Randolph-Anderson in Boynton/Gillham lab, 1993

This plasmid was derived from P-81, which was the same insert in a tetracycline-resistant vector. This map is from the original file for the plasmid and was drawn before the complete sequence was known.

From Sabeeha Merchant, 1993

ATPC cDNA (CF1 ATP synthase, gamma subunit), EcoRI (1.8 kb) in pTZ18R

host strain: JM101
amp resistant

Yu LM, Merchant S, Theg SM, Selman BR (1988) Isolation of a cDNA clone for the gamma subunit of the chloroplast ATP synthase of Chlamydomonas reinhardtii: import and cleavage of the precursor protein.Proc Natl Acad Sci USA 85:1369-1373

From Sabeeha Merchant, 1993

Cytochrome c6 cDNA, 710 bp EcoRI fragment in pGEM1

host strain: HB101
amp resistant

Merchant S, Bogorad L (1987) The Cu(II)-repressible plastidic cytochrome c. Cloning and sequence of a complementary DNA for the pre-apoprotein. J Biol Chem 262:9062-9067

From Sabeeha Merchant, 1993

Plastocyanin cDNA, 577 bp EcoRI fragment in pTZ18R

host strain: HB101
amp resistant

Merchant S, Hill K, Kim JH, Thompson J, Zaitlin D, Bogorad L (1990) Isolation and characterization of a complementary DNA clone for an algal pre-apoplastocyanin. J Biol Chem 265:12372-12379

From Sabeeha Merchant, 1993

Cytochrome c6 genomic, ca. 5 kb SstI fragment in pTZ19R

host strain: JM101
amp resistant

Hill KL, Li HH, Singer J, Merchant S (1991) Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis of the kinetics and metal specificity of its copper-responsive expression. J Biol Chem 266:15060-15067

From Sabeeha Merchant, 1993

Plastocyanin genomic, ca. 6.5 kb KpnI fragment in pTZ19R

host strain: DH5 alpha
amp resistant

Quinn J, Li HH, Singer J, Morimoto B, Mets L, Kindle K, Merchant S (1993) The plastocyanin-deficient phenotype of Chlamydomonas reinhardtii Ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin. J Biol Chem 268:7832-7841

Boynton-Gillham laboratory, Duke University, 1993

Chloroplast construct containing E. coli recA with an N-end deletion, flanked by Chlamydomonas atpA 5′ and rbcL 3′; 1.97 kb in BSKS+

host strain: XL1-Blue
amp resistant

Boynton-Gillham laboratory, Duke University, 1993

Chloroplast expression cassette, 0.62 kb in BSSK+, with 5′ and 3′ ends of psbA, and NcoI/XbaI sites for insertion of any coding sequence in frame

host strain: XL1-Blue
amp resistant

Boynton-Gillham laboratory, Duke University, 1993

Chloroplast cassette, aadA flanked by psbA 5′ and 3′ regions; ca. 1.5 kb in BSKS+

host strain: XL1-Blue
amp resistant

Boynton-Gillham laboratory, Duke University, 1993

aadA flanked by psbA 5′ and atpB 3′; 3.6 kb in BSKS+

host strain: XL1-Blue
amp resistant

From Jean-David Rochaix, 1993

Chloroplast DNA EcoRI 6 (10 kb) in pBR328; contains psbC 3′, psaC, and rpoC1b; P-583 is the same insert in a different vector

host strain: XL1-Blue
amp resistant

From Jean-David Rochaix, 1993

Chloroplast DNA EcoRI 13 (6.0 kb) in pBR328; contains psbA exons 1-4 and extreme 3′ end of chlN

host strain: unknown
amp resistant

From Jean-David Rochaix, 1993

Thought to be chloroplast DNA Eco RI fragment 23; 3.4 kb in pBR328;
could be Eco 24 instead

host strain: unknown
amp resistant

From Jean-David Rochaix, 1993

Chloroplast DNA EcoRI 28 (2.0 kb) in pCRI; internal to ORF1995

host strain: unknown
kanamycin resistant

From Jean-David Rochaix, 1993

Chloroplast DNA EcoRI 6 (10 kb) in pCRI; contains psbC 3′, psaC, and rpoC1b; P-578 is the same insert in pBR328

host strain: unknown
kanamycin resistant

From Jean-David Rochaix, 1993

Chloroplast DNA EcoRI 7 (8.7 kb) in pCRI; contains ccsA, psbZ, psbM, rps14, rps7, and the 5′ end of atpE

A gel run in December 1993 suggested that this insert also includes the adjacent fragment EcoRI 25.

host strain: unknown
kanamycin resistant

From Steve Surzycki, 1993

Chloroplast DNA BglII-SalI (4.3 kb) in pUC18; contains 3′ end of rps3 and 5′ end of rpoC2

host strain: JM83
amp resistant

Fong SE, Surzycki SJ (1992) Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement. Curr Genet 21:485-497

From Steve Surzycki, 1993

Chloroplast DNA PstI-BglII (3.1 kb) in pUC8; contains psbL, petG, and 5′ portion of rps3; subclone of chloroplast DNA fragment BglII 8

host strain: JM83
amp resistant

Fong SE, Surzycki SJ (1992) Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement. Curr Genet 21:485-497

From Patrick Ferris, 1994

Gulliver K (left end); 4.1 kb HindIII in pUC13; used as a probe for the Gulliver transposon

host strain: unknown
amp resistant

Ferris PJ (1989) Characterization of a Chlamydomonas transposon, Gulliver, resembling those in higher plants. Genetics 122:363-377

From Patrick Ferris, 1994

Gulliver N (right end); EcoRI/HindIII 0.6 kb in pUC13; contains ca. 130 bp of non-Gulliver DNA,= probe C for MT- locus

host strain: unknown
amp resistant

Ferris PJ (1989) Characterization of a Chlamydomonas transposon, Gulliver, resembling those in higher plants. Genetics 122:363-377

Boynton-Gillham laboratory, Duke University, 1994

Chloroplast DNA PstI 22 (2.3 kb) in BSKS+, intergenic region between rpoC1a and rpoC1b

host strain: JM83
amp resistant

Boynton-Gillham laboratory, Duke University, 1994

Chloroplast DNA PstI 12 (7.5 kb) in BSKS+; contains tscA, chlN, and psbA exons 1 and 2

host strain: JM83
amp resistant

Boynton-Gillham laboratory, Duke University, 1994

Chloroplast DNA BamHI 17 (800 bp) from CC-503 cw92 in pTZ18R; contains 3′ end and UTR of ccsA

host strain: DH5 alpha
amp resistant

From Steve Mayfield, 1994

OEE2 cDNA, 1.4 kb EcoRI fragment in pUC19

host strain: JM83
amp resistant

Mayfield SP, Rahire M, Frank G, Zuber H, Rochaix JD (1987) Expression of the nuclear gene encoding oxygen-evolving enhancer protein 2 is required for high levels of photosynthetic oxygen evolution in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 84:749-753

From Steve Mayfield, 1994

OEE2 genomic, EcoRI/KpnI fragment in pUC19

host strain: JM83
amp resistant

Mayfield SP, Rahire M, Frank G, Zuber H, Rochaix JD (1987) Expression of the nuclear gene encoding oxygen-evolving enhancer protein 2 is required for high levels of photosynthetic oxygen evolution in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 84:749-753

From Pete Lefebvre, 1994

CRY1 marker; RPS14 gene, ca. 4 kb EcoRI fragment in pUC119

Derived by the following steps: pCRY1-1 was constructed by using PCR to amplify the 3.6 kb genomic region containing the entire CRY1-1 transcription unit and 250 bp of upstream sequence. The 3.6 kb product was cloned into pUC119 digested with XbaI and SacI. Plasmid pRS2-1 was constructed by amplifying a 1073 bp region of the RBCS2 promoter and cloning the PCR product into pUC119 deigested with SmaI and dT tailed with Taq polymerase. pJN1 was constructed by inserting the 3.0 kb BfrI-EcoRI fragment of pCRY1-1 into the HincII site of pRS2-1. pJN4 was constructed by ligating the 474 bp Alw26I-ApaI fragment of pCRY1-1 to the 7.2 kb ApaI-BamHI fragment of pJN1.

Lefebvre included the following suggestions on use of this plasmid:

The protocol we use for transformation is included in the Nelson et al. paper. As you will notice in the paper, the RBCS2 promoter was deleted and inverted during the PCR steps of constructing pJN4, but it still works well for transformation.

The key point to be stressed is the need to determine the appropriate killing concentration for emetine for your strain under your conditions. Emetine is variable in potency between batches, and it is light sensitive. You should determine, by replica plating, the killing concentration for your strain, and then attempt your selection on a concentration 2-3x that needed to kill all of your wild-type. We grow our cells on plates in the light, but not very strong light (about 15 inches away from a two-bulb fluorescent shop light). Make your plates only a few days in advance, and keep in the dark until use.

You might want to try selection in minimal media as well as acetate-containing media. Expression from the RBCS2 promoter is maximal in light in media without acetate.

host strain: DH4 alpha
amp resistant

Nelson JA, Savereide PB, Lefebvre PA (1994) The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. Mol Cell Biol 14:4011-4019

From Sabeeha Merchant, 1994

845 bp of cytochrome c6 5′ UTR, in BSKS-; sequence sufficient to drive copper-dependent expression

host strain: DH5 alpha
amp resistant

Hill KL, Li HH, Singer J, Merchant S (1991) Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis of the kinetics and metal specificity of its copper-responsive expression. J Biol Chem 266:15060-15067

From Sabeeha Merchant, 1994

120 bp of cytochrome c6 5′ UTR in BSKS-; sequence sufficient to drive copper-dependent expression

host strain: DH5 alpha
amp resistant

Hill KL, Li HH, Singer J, Merchant S (1991) Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis of the kinetics and metal specificity of its copper-responsive expression. J Biol Chem 266:15060-15067