MoClo Toolkit

From Félix de Carpentier, Institute of Physico-Chemical Biology-Paris, February 2020

Plasmid used in the Modular Cloning Toolkit (MoClo) from Crozet et al. (2018) and de Carpentier et al. (2020).

Host strain: NEB^® 10-beta
Selection in E. coli: ampicillin (50 μg/ ml)
Position L1 (Weber et al): 2F
Brick name: PAR-BSR-TRBCS2 (TU)
Brick type: Antibiotic Resistance TU
Brick function: Resistance to Blasticidin in Chlamy
Source of Brick: Assembly of pCM0-020, pCM0-120 and pCM0-115
Acceptor plasmid: pICH47742
Designer / Builder: F. de Carpentier and J. Le Peillet

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

de Carpentier et al. (2020) Blasticidin S deaminase: a new efficient selectable marker for Chlamydomonas reinhardtii. Frontiers in Plant Science 11:242

pCM1-029 sequence

de Carpentier F, Le Peillet J, Boisset ND, Crozet P, Lemaire SD, Danon A. Blasticidin S Deaminase: A New Efficient Selectable Marker for Chlamydomonas reinhardtii. Front Plant Sci. 2020 Mar 5;11:242. doi: 10.3389/fpls.2020.00242. PMID: 32211000; PMCID: PMC7066984.

From Alexander Einhaus and Dr. Thomas Baier, Bielefeld University-Germany, April 2022

Vector: pAGM4673
Host strain: DH5a
Selection: Kanamycin (50 μg/ml)
Origin: synthetic construct
Insert: modified L2 vector, with flanking SspI sites, SspI-containing KanR and LacZa element
Function: modified L2 acceptor vector for L2-assembly, provides SspI sites flanking GOI expression positions and within KanR to linearize and inactivate bacterial resistance gene in a single step, Cred cassette was replaced by LacZa for blue/white screening after assembly, see Einhaus et al. 2022 for detailed description.

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/

Sequence

Crozet P, Navarro FJ, Willmund F, Mehrshahi P, Bakowski K, Lauersen KJ, Pérez-Pérez ME, Auroy P, Gorchs Rovira A, Sauret-Gueto S, Niemeyer J, Spaniol B, Theis J, Trösch R, Westrich LD, Vavitsas K, Baier T, Hübner W, de Carpentier F, Cassarini M, Danon A, Henri J, Marchand CH, de Mia M, Sarkissian K, Baulcombe DC, Peltier G, Crespo JL, Kruse O, Jensen PE, Schroda M, Smith AG, Lemaire SD. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth Biol. 2018 Sep 21;7(9):2074-2086. doi: 10.1021/acssynbio.8b00251. Epub 2018 Sep 5. PMID: 30165733.

Einhaus A, Steube J, Freudenberg RA, Barczyk J, Baier T, Kruse O. Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production. Metab Eng. 2022 Jun 16;73:82-90. doi: 10.1016/j.ymben.2022.06.002. Epub ahead of print. PMID: 35717002.

From Elena Kreis, Michael Schroda lab, University of Kaiserslautern,  January 2023

Host strain: TOP10
Selection in E. coli: Spectinomycin (50 μg / ml)
Position L0 (Patron et al): B2
Brick name: cp70B-BioID (N-Term, CDS)
Brick type: Reporter gene (CDS)
Brick function: BioID chloroplast targeted by CTP of HSP70B for N-terminal fusion
Source of Brick: Sythesized
Acceptor plasmid: pAGM1276
Designer / Builder: E. Kreis

Sequence

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 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): B3-B4
Brick name: roGFP2-Orp1 (CDS)
Brick type: Reporter gene (CDS)
Brick function: roGFP2-Orp1 in CDS without STOP and that can be fused in C-Ter
Source of Brick: Synthetized
Acceptor plasmid: pAGM1287
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 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): B3-B4
Brick name: roGFP2-Tsa2ΔCR (CDS)
Brick type: Reporter gene (CDS)
Brick function: roGFP2-Tsa2ΔCR in CDS without STOP and that can be fused in C-Ter
Source of Brick: Synthetized
Acceptor plasmid: pAGM1287
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 Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: PSADpro::roGFP2-Tsa2ΔCR::3xHA::RPL23-T
Host strain: TOP10
Selection in E. coli: ampicillin (100 μg/ ml)
Type: 2F
Original pl1: pICH47742
Function: H2O2 reporter-3xHA tag
Brick 1 – pL0: pCM0-016
Brick 2 – pL0: pMBS419
Brick 3 – pL0: pCM0-093
Brick 4 – 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:::: PSADpro::roGFP2-Tsa2ΔCR::3xHA::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-3xHA tag
TU1: pCM1-01
TU2: pMBS425
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 Elena Kreis, Michael Schroda lab, University of Kaiserslautern,  January 2023

Host strain: TOP10
Selection in E. coli: Spectinomycin (50 μg / ml)
Position L0 (Patron et al): B2
Brick name: cp70B-APEX2 (N-Term, CDS)
Brick type: Reporter gene (CDS)
Brick function: APEX2 chloroplast targeted by CTP of HSP70B for N-terminal fusion
Source of Brick: Sythesized
Acceptor plasmid: pAGM1276
Designer / Builder: E. Kreis

Sequence

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): B3-B4
Brick name: roGFP2
Brick type: Reporter gene (CDS)
Brick function: roGFP2 in CDS without STOP and that can be fused in C-Ter
Source of Brick: PCR on pMBS419
Acceptor plasmid: pAGM1287
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 Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: ARpro::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
Brick 1 – pL0: pCM0-017
Brick 2 – pL0: pMBS419
Brick 3 – pL0: pCM0-101
Brick 4 – 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: ARpro::roGFP2::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: ampicillin (100 μg/ ml)
Type: 2F
Original pl1: pICH47742
Function: roGFP2 unfused-no tag
Brick 1 – pL0: pCM0-017
Brick 2 – pL0: pMBS467
Brick 3 – pL0: pCM0-101
Brick 4 – 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::roGFP2-Tsa2ΔCR::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-no tag
TU1: pCM1-01
TU2: pMBS469
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

Name: PSADpro::aadA::PSAD-T:::: ARpro::roGFP2::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: roGFP2 unfused-no tag
TU1: pCM1-01
TU2: pMBS470
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 Elena Kreis, Michael Schroda lab, University of Kaiserslautern,  January 2023

Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg / ml)
Position L0 (Patron et al): B5
Brick name: TurboID/TID (C-Term, CDS)
Brick type: Reporter gene (CDS)
Brick function: TurboID/TID without STOP for C-terminal fusion
Source of Brick: Sythesized
Acceptor plasmid: pUC57-Kan-BsaI/BpiI-free
Designer / Builder: E. Kreis

Sequence

From Elena Kreis, Michael Schroda lab, University of Kaiserslautern,  January 2023

Host strain: TOP10
Selection in E. coli: Spectinomycin (50 μg / ml)
Position L0 (Patron et al): B2
Brick name: cp70B-TurboID/cp70B-TID (N-Term, CDS)
Brick type: Reporter gene (CDS)
Brick function: TurboID/TID chloroplast targeted by CTP of HSP70B for N-terminal fusion
Source of Brick: Sythesized
Acceptor plasmid: pAGM1276
Designer / Builder: E. Kreis

Sequence