MoClo Level 0 Parts

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: spectinomycin (50 μg/ ml)
Position L0 (Patron et al): B3-B5
Brick name: BSR (CDS)
Brick type: Antibiotic Resistance gene (CDS)
Brick function: Resistance to Blasticidin in Chlamy
Source of Brick: Synthetized
Acceptor plasmid: pICH41308
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: https://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

pCM0-120 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

Host strain: DH5a
Selection in E. coli: Streptomycin (50 μg/ml)
Position L0 (Patron et al): B6-C1
Brick name: FDX (3´UTR + Ter)
Brick type: 3’UTR + Terminator
Brick function: 3’UTR and Terminator for expression (Lopez-Paz et al. 2017)
Source of Brick: Einhaus et al. 2022
Acceptor plasmid: pICH41276
Designer / Builder: A. Einhaus and T. Baier

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: https://www.chlamycollection.org/product/moclo-toolkit/

Sequence

López-Paz C, Liu D, Geng S, Umen JG. Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression. Plant J. 2017 Dec;92(6):1232-1244. doi: 10.1111/tpj.13731. Epub 2017 Nov 18. PMID: 28980350; PMCID: PMC5718938.

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, Baier T, Rosenstengel M, Freudenberg RA, Kruse O. Rational Promoter Engineering Enables Robust Terpene Production in Microalgae. ACS Synth Biol. 2021 Apr 16;10(4):847-856. doi: 10.1021/acssynbio.0c00632. Epub 2021 Mar 25. PMID: 33764741.

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

Host strain: DH5a
Selection in E. coli: Streptomycin (50 μg/ml)
Position L0 (Patron et al): B5
Brick name: StrepII (i2)  (C-ter, CDS)
Brick type: Immuno- and purification tag
Brick function: Add a StrepII tag (with RBCS2 intron 2) + STOP in C-terminal
Source of Brick: Einhaus et al. 2022
Acceptor plasmid: pAGM1301
Designer / Builder: A. Einhaus and T. Baier

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: https://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 Alexander Einhaus and Dr. Thomas Baier, Bielefeld University-Germany, April 2022

Host strain: DH5a
Selection in E. coli: Streptomycin (50 μg/ml)
Position L0 (Patron et al): B4
Brick name: mCerulean3 (2xi1) (CDS)
Brick type: Reporter gene (CDS)
Brick function: mCerulean3 reporter (with 2 copies of the RBCS2 intron 1) + GSG-Linkers in B4
Source of Brick: Einhaus et al. 2022
Acceptor plasmid: pAGM1299
Designer / Builder: A. Einhaus and T. Baier

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: https://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 Nick Jacobebbinghaus and Dr. Thomas Baier, Bielefeld University, Germany, April 2024

Host strain: DH5a
Selection in E. coli: Streptomycin (50 μg/ml)
Position L0 (Patron et al.): B5
Brick name: pCM0-TB288
Brick type: Antibiotic Resistance gene (CDS)
Brick function: A synthetic construct, which allows bicistronic co-expression from a joint mRNA encoding an upstream-located GOI and the downstream-located selection marker aphVIII. A variable ORF 1 (B3 and B4 position) will be terminated by addition of a GS-Linker (8 aa long), the RBCS intron 2 and a C-terminal FLAG-tag including a Stop-codon. A 249 bp long, native intergenic sequence from the C. reinhardtii nuclear genome (gene locus Cre01.g022450/Cre01.g022462) allows transcriptional read through and ribosome assembly for individual translation of downstream aphVIII. A suitable terminator including a 3’UTR is required (in B6_C1 position) and terminates transcription. This construct transfers the aphVIII selection pressure on a joint mRNA for GOI expression, allows bicistronic expression designs and simplifies screening procedure after nuclear transformations due to increased expression level of the upstream-located GOI. Plasmid size: 3681 bp, see Jacobebbinghaus et al. 2024 for detailed description and application guidelines
Source of Brick: Jacobebbinghaus et al., 2024 https://doi.org/10.1111/tpj.16677
Acceptor plasmid: pAGM1301
Designer / Builder: N. Jacobebbinghaus and T. Baier

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-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: https://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: https://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: https://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 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: https://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 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

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): B5
Brick name: APEX2 (C-Term, CDS)
Brick type: Reporter gene (CDS)
Brick function: APEX2 without STOP for C-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 HSP70B  (N-ter, CDS)
Brick type: Signal and Targeting peptides (CDS)
Brick function: Allows the targetting to the Chloroplast
Source of Brick: Cre06.g250100.t1.2
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: https://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): 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: https://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.