Plasmids

From Kyle Lauersen, Olaf Kruse lab, Bielefeld University-Germany, November 2017

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: Zeocin
Reporter: GFP

Sequence (.gb file)

Wichmann J, Baier T, Wentnagel E, Lauersen KJ, Kruse O. Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab Eng. 2017 Dec 16. pii: S1096-7176(17)30395-6. doi: 10.1016/j.ymben.2017.12.010. [Epub ahead of print]

From Kyle Lauersen, Olaf Kruse lab, Bielefeld University-Germany, November 2017

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: Zeocin
Reporter: RFP

Sequence (.gb file)

Wichmann J, Baier T, Wentnagel E, Lauersen KJ, Kruse O. Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab Eng. 2017 Dec 16. pii: S1096-7176(17)30395-6. doi: 10.1016/j.ymben.2017.12.010. [Epub ahead of print]

From Kyle Lauersen, Olaf Kruse lab, Bielefeld University-Germany, November 2017

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: Zeocin
Reporter: Gaussia princeps Luciferase

Sequence (.gb file)

Wichmann J, Baier T, Wentnagel E, Lauersen KJ, Kruse O. Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab Eng. 2017 Dec 16. pii: S1096-7176(17)30395-6. doi: 10.1016/j.ymben.2017.12.010. [Epub ahead of print]

From Kyle Lauersen, Olaf Kruse lab, Bielefeld University-Germany, November 2017

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: spectinomycin
Reporter: Gaussia princeps Luciferase

Sequence (.gb file)

Wichmann J, Baier T, Wentnagel E, Lauersen KJ, Kruse O. Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab Eng. 2017 Dec 16. pii: S1096-7176(17)30395-6. doi: 10.1016/j.ymben.2017.12.010. [Epub ahead of print]

From Elizabeth Specht, Stephen Mayfield Lab, University of California-San Diego, May 2018

Vector: pBluescript

Origin: Created by seamless cloning of several different fragments; fully sequence-verified.

Insert: This construct contains two intact cassettes and one truncated cassette. The first intact cassette is the ARS2 coding sequence, driven by the chimeric HSP70-RBCS2 promoter and followed by its endogenous 3’ UTR; the CDS and 3’ UTR were amplified from cDNA harvested from a strain under sulfur starvation conditions. The second intact cassette is the ARG7 CDS, surrounded by its endogenous promoter/5’ UTR and 3’ UTR, which were amplified from genomic DNA of wild type CC1010 strain. The final, truncated cassette contains the 3’ half of the CDS of hygromycin resistance gene followed by the RBCS2 3’ UTR; upstream of this lies a 1.6kb intron that was amplified from CC1010 genomic DNA.

Total insert size is 9,093 bp.

Comment: This plasmid represents one half of the pair of plasmids designed to detect homologous recombination in C. reinhardtii. Upon recombination with its partner, pHR23, it will produce an intact, functional hygromycin resistance cassette. pHR18 is the plasmid that has been transformed into CC-1820 to create the strain B12.

Host strain: DH5α

Selection: Ampicillin resistant in E. coli; restores arginine prototrophy in arg7 Chlamydomonas mutants.

Sequence

From Elizabeth Specht, Stephen Mayfield Lab, University of California-San Diego, May 2018

Vector: pBluescript

Host strain: DH5a E. coli

Origin: Created by seamless cloning of several different fragments; fully sequence-verified.

Insert: This plasmid contains 4,360bp of homology to its complementing partner, pHR18, followed by one intact cassette for paromomycin resistance and one truncated cassette. The truncated cassette contains the 5’ half of the hygromycin resistance CDS, preceded by the beta-tubulin promoter and 5’ UTR, followed by a 1.6kb intron amplified from wild type strain CC-1010 genomic DNA.

Total insert size is 8,847 bp.

Comment: This plasmid represents one half of the pair of plasmids designed to detect homologous recombination in C. reinhardtii. Upon recombination with its partner, pHR18, it will produce an intact, functional hygromycin resistance cassette. pHR23 can be transformed directly into strain B12, which already harbors pHR18 in its genome, to screen for homologous recombination rate. Paromomycin resistance can be used to assess the ratio of recombinants to total transformation efficiency.

Host strain: DH5α

Selection: Ampicillin resistant in E. coli; paromomycin resistant in Chlamydomonas.

Sequence

From Kyle Lauersen, Olaf Kruse lab, Bielefeld University-Germany, May 2018

This is a minimized version of the pOpt2 vector which contains only a gene of interest cassette for reporter fusion to the aadA spectinomycin resistance gene. The vector has full complementarity to the pOpt2 vectors and contains the PsaD chloroplast targeting peptide between NdeI-BamHI. Fusion proteins can be generated for desired targets in fusion to the aadA and spectinomycin resistance allows direct selection for expressing transformants. This selection works whether fusion proteins are targeted to the algal chloroplast or the cytoplasm.

Host strain: DH5α
Selection in E. coli: ampicillin

Sequence

Wichmann J, Baier T, Wentnagel E, Lauersen KJ, Kruse O (2018) Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab. Eng. 45:211–222

From Luke Mackinder, University of York-UK, December 2018

pLM017: pLM005-EPYC1-Venus-3xFLAG

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin

Sequence

ackinder LC, Meyer MT, Mettler-Altmann T, Chen VK, Mitchell MC, Caspari O, Freeman Rosenzweig ES, Pallesen L, Reeves G, Itakura A, Roth R, Sommer F, Geimer S, Mühlhaus T, Schroda M, Goodenough U, Stitt M, Griffiths H, Jonikas MC (2016) A Repeat Protein Links Rubisco to Form the Eukaryotic Carbon-Concentrating Organelle. Proc Natl Acad Sci U S A. 113:5958–63

From Luke Mackinder, University of York-UK, December 2018

pLM035: pLM006-RBCS1-mCherry-6xHis

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: Hygromycin

Sequence

Mackinder LC, Meyer MT, Mettler-Altmann T, Chen VK, Mitchell MC, Caspari O, Freeman Rosenzweig ES, Pallesen L, Reeves G, Itakura A, Roth R, Sommer F, Geimer S, Mühlhaus T, Schroda M, Goodenough U, Stitt M, Griffiths H, Jonikas MC (2016) A Repeat Protein Links Rubisco to Form the Eukaryotic Carbon-Concentrating Organelle. Proc Natl Acad Sci U S A. 113:5958–63

From Paula Barjona, University of Erlangen-Nuremberg, December 2018

The constitutive promoter HSP70A/Rbcs2 in plasmid pBR9 was replaced by the PCR-amplified FEA1 inducible promoter from plasmid p5’Fea1-ARS2 yielding Fea1_Ble2A-mCherry.

Host strain: DH5α
Selection in E. coli: ampicillin

Sequence

Barjona do Nascimento Coutinho P, Friedl C, Buchholz R, Stute SC (2017) Chemical regulation of Fea1 driven transgene expression in Chlamydomonas reinhardtii. Algal Research. 26:323–329

Rasala BA, Barrera DJ, Ng J, Plucinak TM, Rosenberg JN, Weeks DP, Oyler GA, Peterson TC, Haerizadeh F, Mayfield SP (2013) Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. Plant J. 74:545-56

From Jessica Hennacy, Martin Jonikas lab, Princeton University, August 2020

pRAM118 is derived from pLM005, but contains a hygromycin selectable marker in lieu of the paromomycin cassette. All other features remain the same.

Selection: ampicillin resistant in E. coli; hygromycin in Chlamydomonas

takura AK, Chan KX, Atkinson N, Pallesen L, Wang L, Reeves G, Patena W, Caspari O, Roth R, Goodenough U, McCormick AJ, Griffiths H, Jonikas MC. A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 2019 Sep 10;116(37):18445-18454. doi: 10.1073/pnas.1904587116. Epub 2019 Aug 27. PMID: 31455733; PMCID: PMC6744930.

From Erin Dymek, Elizabeth Smith lab, Dartmouth College, November 2019

C terminally BCCP tagged PF16 genomic clone in pBluescript

Fu G, Zhao L, Dymek E, Hou Y, Song K, Phan N, Shang Z, Smith EF, Witman GB, Nicastro D. Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus. J Cell Biol. 2019 Dec 2;218(12):4236-4251. doi: 10.1083/jcb.201906006. Epub 2019 Oct 31. PMID: 31672705; PMCID: PMC6891083.

From Erin Dymek, Elizabeth Smith lab, Dartmouth College, November 2019

C terminal HA tagged PF15 genomic clone in pBluescript

Dymek EE, Lefebvre PA, Smith EF. PF15p is the chlamydomonas homologue of the Katanin p80 subunit and is required for assembly of flagellar central microtubules. Eukaryot Cell. 2004 Aug;3(4):870-9. doi: 10.1128/EC.3.4.870-879.2004. PMID: 15302820; PMCID: PMC500881.

From Erin Dymek, Elizabeth Smith lab, Dartmouth College, November 2019

Katanin p60 genomic clone in pBluescript

Dymek EE, Smith EF. PF19 encodes the p60 catalytic subunit of katanin and is required for assembly of the flagellar central apparatus in Chlamydomonas. J Cell Sci. 2012 Jul 15;125(Pt 14):3357-66. doi: 10.1242/jcs.096941. Epub 2012 Mar 30. PMID: 22467860; PMCID: PMC3516377.

From Erin Dymek, Elizabeth Smith lab, Dartmouth College, November 2019

GFP tagged katanin p60 genomic clone in pBluescript

Dymek EE, Smith EF. PF19 encodes the p60 catalytic subunit of katanin and is required for assembly of the flagellar central apparatus in Chlamydomonas. J Cell Sci. 2012 Jul 15;125(Pt 14):3357-66. doi: 10.1242/jcs.096941. Epub 2012 Mar 30. PMID: 22467860; PMCID: PMC3516377.

From Erin Dymek, Elizabeth Smith lab, Dartmouth College, November 2019

PACRG genomic clone in pUC57

Dymek EE, Lin J, Fu G, Porter ME, Nicastro D, Smith EF. PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility. Mol Biol Cell. 2019 Jul 15;30(15):1805-1816. doi: 10.1091/mbc.E19-01-0063. Epub 2019 May 22. PMID: 31116684; PMCID: PMC6727744.

From Erin Dymek, Elizabeth Smith lab, Dartmouth College, November 2019

FAP20 genomic clone in pCR2.1

Dymek EE, Lin J, Fu G, Porter ME, Nicastro D, Smith EF. PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility. Mol Biol Cell. 2019 Jul 15;30(15):1805-1816. doi: 10.1091/mbc.E19-01-0063. Epub 2019 May 22. PMID: 31116684; PMCID: PMC6727744.

From Erin Dymek, Elizabeth Smith lab, Dartmouth College, November 2019

PACRG cDNA clone in pET30a

Dymek EE, Lin J, Fu G, Porter ME, Nicastro D, Smith EF. PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility. Mol Biol Cell. 2019 Jul 15;30(15):1805-1816. doi: 10.1091/mbc.E19-01-0063. Epub 2019 May 22. PMID: 31116684; PMCID: PMC6727744.

From Erin Dymek, Elizabeth Smith lab, Dartmouth College, November 2019

FAP20 cDNA clone in pET30a

Dymek EE, Lin J, Fu G, Porter ME, Nicastro D, Smith EF. PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility. Mol Biol Cell. 2019 Jul 15;30(15):1805-1816. doi: 10.1091/mbc.E19-01-0063. Epub 2019 May 22. PMID: 31116684; PMCID: PMC6727744.

From Shan He, Martin Jonikas lab, Princeton University, February 2020

Host strain: DH5α
Selection in E. coli: ampicillin/carbenicillin

Sequence and map

From Shan He, Martin Jonikas lab, Princeton University, February 2020

Host strain: DH5α
Selection in E. coli: ampicillin/carbenicillin

Sequence and map

From Gary Yates, Luke Mackinder lab, University of York-UK, July 2020

RBMP1 (Cre06.g261750) coding sequence plus 3672 bp upstream of the start codon was cloned into pLM099 by recombineering to produce a C-terminal CrVenus-3xFLAG tag. Transformed into C. reinhardtii strain CMJ030 to produce CC-5646.

Host strain: DH10B
Selection in E. coli: kanamycin
Selection in C. reinhardtii: paromomycin

Sequence

https://www.biorxiv.org/content/10.1101/2020.08.16.252858v1.full

From Gary Yates, Luke Mackinder lab, University of York-UK, July 2020

RBMP2 (Cre09.g416850) coding sequence plus 2250 bp upstream of the start codon was cloned into pLM099 by recombineering to produce a C-terminal CrVenus-3xFLAG tag. Transformed into C. reinhardtii strain CMJ030 to produce CC-5647.

Host strain: DH10B
Selection in E. coli: kanamycin
Selection in C. reinhardtii: paromomycin

Sequence

https://www.biorxiv.org/content/10.1101/2020.08.16.252858v1.full

From Moritz Meyer, Martin Jonikas Lab, Princeton University, August 2020

FDX1 (Cre14.g626700) gDNA coding sequence, spanning the start codon to the triplet just before the stop codon, had been previously cloned in frame into pLM005 by Gibson assembly (as described in https://doi.org/10.1073/pnas.1522866113; CSI_FC1F06 = pLM005-Cre14.g626700-Venus-3xFLAG). That plasmid was then reengineered by restriction digestion and T4 ligation to add in-frame immediately after the 3xFLAG a synthetic fragment containing three copies of the sequence coding for the 15 C-terminal amino acids of SAGA2 (Cre 09.g394621), interspersed with a short flexible linker (GGGGGS). Transformed into C. reinhardtii strain cMJ030 with carrier DNA to produce CC-5648.

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin

Sequence

From Moritz Meyer, Martin Jonikas Lab, Princeton University, August 2020

MPM1 (Cre12.g498550) gDNA coding sequence, spanning the start codon to the triplet just before the stop codon, was cloned in frame into pLM005 by Gibson assembly (as described in https://doi.org/10.1073/pnas.1522866113. Transformed into C. reinhardtii strain cMJ030 to produce CC-5649.

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin

Sequence

From Moritz Meyer, Martin Jonikas Lab, Princeton University, August 2020

pMJ504, containing the gDNA of Cre12.g498550, was reengineered by restriction digestion and T4 ligation to add in-frame immediately after the 3xFLAG a synthetic fragment containing three copies of the sequence coding for the 15 C-terminal amino acids of SAGA2 (Cre 09.g394621), interspersed with a short flexible linker (GGGGGS). Transformed into C. reinhardtii strain cMJ030 to produce CC-5650.

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin

Sequence

From Moritz Meyer, Martin Jonikas Lab, Princeton University, August 2020

PSAK (Cre17.g724300) gDNA coding sequence, spanning the start codon to the triplet just before the stop codon, had been previously cloned in frame into pLM005 by Gibson assembly (as described in https://doi.org/10.1073/pnas.1522866113; pMJ280 = pLM005-Cre17.g724300-Venus-3xFLAG). That plasmid was then reengineered by restriction digestion and T4 ligation to add in-frame immediately after the 3xFLAG a synthetic fragment containing three copies of the sequence coding for the 15 C-terminal amino acids of SAGA2 (Cre 09.g394621), interspersed with a short flexible linker (GGGGGS). Transformed into C. reinhardtii strain cMJ030 with carrier DNA to produce CC-5651.

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin

Sequence

From Moritz Meyer, Martin Jonikas Lab, Princeton University, August 2020

Cre10.g430350 gDNA coding sequence, spanning the start codon to the triplet just before the stop codon, was cloned in frame into pLM005 by Gibson assembly (as described in https://doi.org/10.1073/pnas.1522866113). Transformed into C. reinhardtii strain cMJ030 to produce CC-5652.

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin

Sequence

From Moritz Meyer, Martin Jonikas Lab, Princeton University, August 2020

Previously synthesized pMJ507 (CC-5652) was modified by side-directed mutagenesis. Triplet coding for Tryptophan in position 51 of the mature protein now codes for an Alanine. Triplet coding for Arginine in position 52 of the mature protein now codes for an Alanine. Transformed into C. reinhardtii strain cMJ030 to produce CC-5653.

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin

Sequence

From Moritz Meyer, Martin Jonikas Lab, Princeton University, August 2020

Previously synthesized pMJ507 (CC-5652) was modified by side-directed mutagenesis. All four triplet pairs coding for a Tryptophan-Arginine dipeptide were mutagenized to code for a double Alanine (W47A/R48A/W51A/R52A/W110A/R111A/W148A/R149A). Transformed into C. reinhardtii strain cMJ030 to produce CC-5654.

Host strain: DH5α
Selection in E. coli: ampicillin
Selection in C. reinhardtii: paromomycin

Sequence