Plasmids

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Host strain: TOP10
Selection in E. coli: spectinomycin (50 μg/ ml)
Position L0 (Patron et al): B2
Brick name: CTP CDJ1  (N-ter, CDS)
Brick type: Signal and Targeting peptides (CDS)
Brick function: Allows the targetting to the Chloroplast
Source of Brick: Cre12.g507650 
Acceptor plasmid: pAGM1276
Designer / Builder: J. Niemeyer

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: http://www.chlamycollection.org/product/moclo-toolkit/

Niemeyer J, Scheuring D, Oestreicher J, Morgan B, Schroda M. Real-time Monitoring of Subcellular H2O2 Distribution in Chlamydomonas reinhardtii. Plant Cell. 2021 Jul 1:koab176. doi: 10.1093/plcell/koab176. Epub ahead of print. PMID: 34196712.

From Pierre Crozet, Institute of Physico-Chemical Biology-Paris, December 2018

Plasmid used in the Modular Cloning Toolkit (MoClo) from Crozet et al. (2018).

Host strain: TOP10
Selection in E. coli: spectinomycin (50 μg/ ml)
Position L0 (Patron et al): A1-B2
Brick name: P_A*R (Pro + 5’UTR)
Brick type: promoter + 5’UTR
Brick function: engineered pH/R, with RBCS2 5’UTR
Source of Brick: Schroda et al, 2002
Acceptor plasmid: pICH41295
Designer / Builder: L. Westrich and B. Spaniol

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: http://www.chlamycollection.org/product/moclo-toolkit/

Crozet et al. (2018) Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology 7:2074–2086

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: ARpro::CDJ1::roGFP2-Tsa2ΔCR::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: ampicillin (100 μg/ ml)
Type: 2F
Original pl1: pICH47742
Function: H2O2 reporter-no tag targeted to the chloroplast (Stroma)
Brick 1 – pL0: pCM0-011
Brick 2 – pL0: pMBS640
Brick 3 – pL0: pMBS419
Brick 4 – pL0: pCM0-101
Brick 5 – pL0: pCM0-119
Fusion 5′ lvl2: GCAA
Fusion 3′ lvl2: ACTA

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: http://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: PSADpro::aadA::PSAD-T:::: ARpro::HSP70B::roGFP2-Tsa2ΔCR::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-no tag targeted to the chloroplast (Stroma)
TU1: pCM1-01
TU2: pMBS643
End Linker: pICH41744-pL2-EL2

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: http://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Host strain: TOP10
Selection in E. coli: spectinomycin (50 μg/ ml)
Position L0 (Patron et al): B2
Brick name: CTP PSAN  (N-ter, CDS)
Brick type: Signal and Targeting peptides (CDS)
Brick function: Allows the targetting to the Chloroplast
Source of Brick: Cre02.g082500 
Acceptor plasmid: pAGM1276
Designer / Builder: J. Niemeyer

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: http://www.chlamycollection.org/product/moclo-toolkit/

Niemeyer J, Scheuring D, Oestreicher J, Morgan B, Schroda M. Real-time Monitoring of Subcellular H2O2 Distribution in Chlamydomonas reinhardtii. Plant Cell. 2021 Jul 1:koab176. doi: 10.1093/plcell/koab176. Epub ahead of print. PMID: 34196712.

From Jean-David Rochaix to Boynton-Gillham laboratory, 1981

Chloroplast DNA BamH1 14/15 (3.0 kb) from Rochaix’s cw15 strain in pBR313; contains 3′ end of 23S rRNA gene through 5S; see P-124 for same insert in pBR322

host strain: C600
amp resistant

More information

Strain: Rochaix cw15

Insert: chloroplast DNA BamHI 14/15 (3.0 kb)

Genes present: 23S rRNA (3′ end), 5S rRNA

Vector: pBR313

Bacterial host strain: C600

Selectable marker: amp-r tet-s

Origin: Single cell clone of P-3 (Ba1 from Rochaix received 6/81)

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

From Jean-David Rochaix to Boynton-Gillham laboratory, 1981

Chloroplast DNA BamHI 10 (7.6 kb) from Rochaix’s cw15 strain in pBR313; contains entire atpB gene and 3′ portion of ORF1995

host strain: C600
amp resistant

More information

Strain: Rochaix cw15

Insert: chloroplast DNA BamHI 10 (7.6 kb)

Genes present: atpB

Vector: pBR313

Bacterial host strain: C600

Selectable marker: amp-r tet-s

Origin: Single cell clone of P-6 (Ba 5 from Rochaix, received 6/81)

Reference: Boynton et al. (1988). See for use of this plasmid in transformation of atpB gene, and analysis of 12 bp tandem repeats./p>

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

From Jean-David Rochaix to Boynton-Gillham laboratory, 1981

Chloroplast DNA BamHI 8 (9.6 kb) from Rochaix’s cw15 strain in pBR313; contains most of rpoC2 (3′) and 5′ end of psaB; see P-122 for same insert in pBR322

host strain: C600
amp resistant

Additional Information

Strain: Rochaix cw15

Insert: chloroplast DNA BamHI 8 (9.6 kb)

Genes present: rpoC2 (3′ end), psaB (extreme 5′ end), ARS04,02

Vector: pBR313

Bacterial host strain: C600

Selectable marker: amp-r tet-s

Origin: Single cell clone of P-8 (Ba7 from Rochaix, received 6/81)

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

From Jean-David Rochaix to Boynton-Gillham laboratory, 1981

Chloroplast DNA BamHI 6 (13.4 kb) from Rochaix’s cw15 strain in pBR313; contains 3′ end of rps2 rpoA, psbB, psbN, and psbH; P-202 is the same insert in pBR322

host strain: C600
amp resistant

More information

Strain: Rochaix cw15

Insert: chloroplast DNA BamHI 6 (13.4 kb)

Genes present: psbH, psbN, ORF31, psbB, probably rps2

Vector: pBR313

Bacterial host strain: C600

Selectable marker: amp-r tet-s

Origin: Single cell clone of P-10 (Ba10 from Jean-David Rochaix, received 6/81

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA BamHI 9 (8.4 kb) in pUC8, from CC-125 wild type; contains rpl14, rps8, psaA exon 1, and rps4

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA BamHI 9 (8.4 kb)

Genes present: rpl14, rpl5, rps8, psaA (exon 1), rps4

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA BamHI 11/12 (7.0 kb) in pUC8, from CC-125 wild type; contains 16S rRNA through 23S 5′ end

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA BamHI 11/12 (7.0 kb)

Genes present: 16S rRNA, 23s rRNA (5′ end)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

See P-271 for the same insert in pBR322.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 5 (10.5 kb) in pUC8, from CC-125 wild type; contains psbB, rpoA, rps2 and most of rps18

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 5 (10.5 kb)

Genes present: psbB (5′), rps2, atpF

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Jeff Palmer in Boynton/Gillham lab, 1986

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 8 (8.5 kb) in pUC8, from CC-125 wild type; contains rps8, psaA exon 1, rps4 and 5′ end of 16S rRNA

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 8 (8.5 kb)

Genes present: rps8, psaA (exon 1), rps4, 16S rRNA (5′ end)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 9 (7.6 kb) in pUC8, from CC-125 wild type; contains clpP, rpl35, rpl23, and 5′ half of rpl2

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 9 (7.6 kb)

Genes present: clpP, trnL, petB, chlL, rpl23, rpl2 (5′)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 12 (6.5 kb) in pUC8, from CC-125 wild type; contains psaA exon 3

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 12 (6.5 kb)
Genes present: psaA (exon 3)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 15 (6.0 kb) in pUC8, from CC-125 wild type; contains psbA exons 1-4

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 15 (5.8 kb)psbA

Genes present: psbA exons 1-3

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 14 (5.8 kb) in pUC8, from CC-125 wild type; contains psaB, rbcL, and 5′ end of atpA

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 14 (5.5 kb)

Genes present: psaB, rbcL, atpA (5′)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 15 (5.8 kb) in pUC8, from CC-125 wild type; contains psbA exons 1-4

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 15 (5.8 kb) Gene psbA

Genes present: psbA (exons 1, 2, 3)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

Comment Same insert as P-66, but opposite orientation.

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 16 (5.0 kb) in pUC8, from CC-125 wild type; contains 3′ end of rpl2, rps19, rpl16, rpl14, and rpl5

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 16 (5.0 kb)

Genes present: rpl2 (part), rps19, rpl16, oriA, rpl14, rpl5

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 17 (4.9 kb) in pUC8, from CC-125 wild type; contains rps12, psaJ, atpI, and psbJ

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 17 (4.9 kb)

Genes present: psaJ, rps12

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 18 (4.9 kb) in pUC8, from CC-125 wild type; contains rps11, tscA, and most of chlN

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert:

Genes present:

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 19 (4.4 kb) in pUC8; contains psbH, psbN, and psbT

host strain: JM83
amp resistant

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 24 (3.3 kb) in pUC8, from CC-125 wild type; contains ycf12 and atpE

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 24 (3.3 kb)

Genes present: atpE

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab

Comment The 350 bp EcoRI-Bgl II subclone of this plasmid is a good probe for atpE.

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 26 (3.0 kb) in pUC8, from CC-125 wild type; internal to ORF2971

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 26 (3.0 kb)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 31 (1.4 kb) in pUC8, from CC-125 wild type; internal to rpoC2

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 31 (1.4 kb)

Genes present: rpoC2 (part)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA EcoRI 32 (1.3 kb) in pUC8, from CC-125 wild type; contains 3′ half of tufA

host strain: JM83
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA EcoRI 32 (1.3 kb)

Genes present: tufA (3′ end)

Vector: pUC8

Bacterial host strain: JM83

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

Origin: Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA PstI 4 (15.1 kb) in pBR322, from CC-125 wild type; contains rps2, rps18, ycf3, ycf4, rps9, psbE, rpoB and psbF

host strain: SK2267
tetracycline resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA PstI 4 (15.1 kb)

Genes present: rps2 (probably), atpF, psbE, rpoB, psbF

Vector: pBR322

Bacterial host strain: SK2267

Selectable marker: tet-r, amp-s

Origin: David Lamb in Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Orientation of P-78

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA PstI 14 (3.8 kb) in pBR322, from CC-125 wild type; contains psbT and psbB

host strain: SK2267
tetracycline resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: chloroplast DNA PstI 14 (3.8 kb)

Genes present: psbB

Vector: pBR322

Bacterial host strain: SK2267

Selectable marker: tet-r, amp-s

Origin: David Lamb in Boynton/Gillham lab, 1983

This map is from the original file for the plasmid and was drawn before the complete sequence was known.

Boynton-Gillham laboratory, Duke University, 1983

Chloroplast DNA PstI 19 (2.9 kb) in pBR322, from CC-125 wild type; contains rpoC1b

host strain: SK2267
tetracycline resistant

Boynton-Gillham laboratory, Duke University, 1983

Mitochondrial Bam-Sal fragment (5.6 kb) in pBR322, from CC-125 wild type; contains cox1, nd2, nd6, W, L6, L7, Q, and S1

host strain: SK2267
amp resistant

More information

Strain: CC-125 wild type mt+ 137c

Insert: mitochondrial DNA BamHI-SalI (5.6 kb)

Genes present: cox1, nd2, nd6, W, L6, L5, L7, Q, S1

Vector: pBR322

Bacterial host strain: SK2267

Selectable marker: amp-r

Origin: Boynton/Gillham lab, 1983