Strains

From Yingjun Wang, Spalding lab, GDCB department, Iowa State University, August 2015

This mutant is a high CO2 requiring mutant and grows slow autotrophically in air, but can grow on acetate plates. HCR209 is similar to CC-4428 cia5-2 mt+, which was generated from CC-425 background (mt+).

Van K, Wang Y, Nakamura Y, Spalding MH (2001) Insertional Mutants of Chlamydomonas reinhardtii That Require Elevated CO2 for Survival. Plant Physiol. 127:607-614

Spalding MH, Van K, Wang Y, Nakamura Y (2002) Acclimation of Chlamydomonas to changing carbon availability. Funct Plant Biol 29:221-230

  • Locus:
  • CIA5 [CCM1]
  • Chromosome:
  • 2

From Susan Dutcher, Washington University in St. Louis, September 2015

Phenotype: short flagella

This strain was generated from a cross between pf7 and pf8. It has short, stumpy flagella in N-free medium and is aflagellate in rich medium.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • PF7 [CCDC40], PF8 [CCDC39]
  • Chromosome:
  • 17,17

From Susan Dutcher, Washington University in St. Louis, September 2015

Phenotype: short flagella

This strain was generated from a cross between pf7 and tpg1.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • PF7 [CCDC40], TPG1 [TTLL9]
  • Chromosome:
  • 17,17

From Susan Dutcher, Washington University in St. Louis, September 2015

Phenotype: short flagella

This strain was generated from a cross between pf2 and pf7 pf8. It has short, stumpy flagella in N-free medium and is aflagellate in rich medium.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • PF2, PF7 [CCDC40], PF8 [CCDC39]
  • Chromosome:
  • 11,17,17

From Susan Dutcher, Washington University in St. Louis, September 2015

This strain was generated by UV mutagenesis and is able to generate slightly longer flagella than pf7 pf8 in N-free medium. Cannot suppress the motility defect.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • PF7 [CCDC40], PF8 [CCDC39], CNK11
  • Chromosome:
  • 17,17,7

From Susan Dutcher, Washington University in St. Louis, September 2015

This strain was generated by UV mutagenesis and is able to generate slightly longer flagella than pf7 pf8 in N-free medium. Cannot suppress the motility defect. Same allele as cnk11-5.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • PF7 [CCDC40], PF8 [CCDC39], CNK11
  • Chromosome:
  • 17,17,7

From Susan Dutcher, Washington University in St. Louis, September 2015

This strain was generated by UV mutagenesis and is able to generate slightly longer flagella than pf7 pf8 in N-free medium. Cannot suppress the motility defect. Same allele as cnk11-3.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • PF7 [CCDC40], PF8 [CCDC39], CNK11
  • Chromosome:
  • 17,17,7

From Susan Dutcher, Washington University in St. Louis, September 2015

This strain was generated from a cross between pf2 and cnk11-1. It has slightly longer flagella than pf2.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • PF2, CNK11
  • Chromosome:
  • 11,7

From Susan Dutcher, Washington University in St. Louis, September 2015

This strain was generated from a cross between CC-1026 pf3 mt+ and CC-124 wild type mt- 137c.

It has been been reported by Susan Dutcher that the Chlamy Center copy of CC-1026 pf3 mt+ actually carries a cnk11-6 mutation and the strain submitted here removes it.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • PF3
  • Chromosome:
  • 8

From Susan Dutcher, Washington University in St. Louis, September 2015

Mutant strain tpg1 cnk11-1 expressing HA-tubulin.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • TPG1 [TTLL9], CNK11
  • Chromosome:
  • 17,7

From Susan Dutcher, Washington University in St. Louis, September 2015

Mutant strain tpg1 cnk11-1 expressing HA-tubulin.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • TPG1 [TTLL9], CNK11
  • Chromosome:
  • 17,7

From Susan Dutcher, Washington University in St. Louis, September 2015

Spontaneous missense mutation of CPC1 raised from the CC-125 background. This mutation prevents the CPC1 protein from getting into the flagella.

  • Locus:
  • CPC1
  • Chromosome:
  • 3

From Susan Dutcher, Washington University in St. Louis, September 2015

The mating defect of imp3 is rescued by transformation of the HA-tagged wild-type PP2A catalytic subunit.

Lin H, Miller ML, Granas DM, Dutcher SK (2013) Whole genome sequencing identifies a deletion in protein phosphatase 2A that affects its stability and localization in Chlamydomonas reinhardtii. PLoS Genet. 9(9):e1003841

  • Locus:
  • IMP3
  • Chromosome:
  • 2

From Susan Dutcher, Washington University in St. Louis, September 2015

This strain was generated from a cross between CC-1037 pf19 mt+ and CC-124 wild type mt- 137c.

It has been reported by Susan Dutcher that the Chlamy Center copy of CC-1037 pf19 mt+ actually carries an imp3-2 mutation, which is different from the original imp3 mutation, but results in deletion in the same PP2A catalytic subunit. The strain submitted here removes it.

  • Locus:
  • PF19
  • Chromosome:
  • 10

From Susan Dutcher, Washington University in St. Louis, September 2015

This strain was generated from a cross between fla12 and cnk11-3. It partially rescues the temperature sensitive phenotype of fla12 at 32 °C. It cannot suppress the motility defect.

Lin H, Zhang Z, Guo S, Chen F, Kessler JM, Wang YM, Dutcher SK (2015) A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures. PLoS Genet. Sep 8;11(9):e1005508

  • Locus:
  • FLA12, CNK11
  • Chromosome:
  • 17,7

From James Umen, Donald Danforth Plant Science Center, Februaly 2017

This strain is a mt- progeny from a cross between wild-type parental strains CC-1690 (21gr) and CC-2895 (6145c).

Zones JM, Blaby IK, Merchant SS, Umen JG (2015) High-Resolution Profiling of a Synchronized Diurnal Transcriptome from Chlamydomonas reinhardtii Reveals Continuous Cell and Metabolic Differentiation. The Plant Cell. 27: 2743–2769

From Pete Lefebvre, University of Minnesota, October 2015

To facilitate further genetic studies using mutants from the CLiP library (https://www.chlamylibrary.org), CC-4533 cw15 mt- [Jonikas CMJ030] was crossed with CC-125 wild type mt+ [137c] and then backcrossed a mt+ progeny to CC-4533 five times. This procedure yielded a mt+ progeny strain designated CC-5155, which should be close to isogenic to CC-4533.

From Jason Brown, George Witman lab, University of Massachusetts Medical School, October 2015

This strain was generated by transformation of fap46-1 with the plasmid pFAP46 containing wild-type FAP46 genomic sequence and a phleomycin-resistance cassette (Stevens et al., 1996, Mol. Gen. Genet.).

Brown JM, Dipetrillo CG, Smith EF, Witman GB (2012) A FAP46 mutant provides new insights into the function and assembly of the C1d complex of the ciliary central apparatus. J Cell Sci. 125: 3904-3913

  • Locus:
  • FAP46
  • Chromosome:
  • 10

From Jason Brown, George Witman lab, University of Massachusetts Medical School, October 2015

This strain was generated by transformation of fap46-1 with the plasmid pFAP46-HA containing wild-type FAP46 genomic sequence with a 3x-HA tag inserted immediately 5’ to the stop codon; this plasmid also contains a phleomycin-resistance cassette (Stevens et al., 1996, Mol. Gen. Genet.).

Brown JM, Dipetrillo CG, Smith EF, Witman GB (2012) A FAP46 mutant provides new insights into the function and assembly of the C1d complex of the ciliary central apparatus. J Cell Sci. 125: 3904-3913

  • Locus:
  • FAP46
  • Chromosome:
  • 10

From Jason Brown, George Witman lab, University of Massachusetts Medical School, October 2015

The ift74-1 allele was identified in strain BB12 generated by insertional mutagenesis of strain D10 (CC-124 background) with the 1.7 kb HindIII fragment of pHyg3 (Berthold et al., 2002, Protist). Strain ift74-1 is a product of two backcrosses of BB12 to CC-125. A more complete description of the parental strain is in Brown et al., 2015.

ift74-1 expresses a truncated IFT74 protein lacking 196 aa from its N terminus. It assembles short flagella when maintained in M medium without aeration.

Brown JM, Cochran DA, Craige B, Kubo T, Witman GB (2015) Assembly of IFT trains at the ciliary base depends on IFT74. Curr Biol. 25: 1583-1593

  • Locus:
  • IFT74
  • Chromosome:
  • 1

From Jason Brown, George Witman lab, University of Massachusetts Medical School, October 2015

This strain was generated by transformation of ift74-1 with the plasmid pIFT74aphVIII containing wild-type IFT74 genomic sequence and the paromomycin-resistance cassette from pKS-aphVIII-lox (Heitzer and Zschoernig, 2007, BioTechniques).

This strain has restored wild-type flagella assembly.

Brown JM, Cochran DA, Craige B, Kubo T, Witman GB (2015) Assembly of IFT trains at the ciliary base depends on IFT74. Curr Biol. 25: 1583-1593

  • Locus:
  • IFT74
  • Chromosome:
  • 1

From Jason Brown, George Witman lab, University of Massachusetts Medical School, October 2015

The ift74-2 allele was identified in strain 11C#3 generated by insertional mutagenesis of strain oda2 (Kamiya, 1988, J. Cell. Biol.) with the 1.7 kb HindIII fragment of pHyg3 (Berthold et al., 2002, Protist). The ift74-2 strain is the result of a backcross to CC-124 (137c mt-) that replaced the oda2 allele with a wild-type ODA2 allele.

This strain almost completely fails to assemble flagella.

Brown JM, Cochran DA, Craige B, Kubo T, Witman GB (2015) Assembly of IFT trains at the ciliary base depends on IFT74. Curr Biol. 25: 1583-1593

  • Locus:
  • IFT74
  • Chromosome:
  • 1

From Jason Brown, George Witman lab, University of Massachusetts Medical School, October 2015

This strain is a transformant of ift74-2 with the plasmid pIFT74aphVIII containing wild-type IFT74 genomic sequence and the paromomycin-resistance cassette from pKS-aphVIII-lox (Heitzer and Zschoernig, 2007, BioTechniques).

This strain is a phenotypic rescue of ift74-2.

Brown JM, Cochran DA, Craige B, Kubo T, Witman GB (2015) Assembly of IFT trains at the ciliary base depends on IFT74. Curr Biol. 25: 1583-1593

  • Locus:
  • IFT74
  • Chromosome:
  • 1

From Rasha Abdrabu, NYU Abu Dhabi, October 2015

UV mutagenesis of CC-503 yielding high lipid producing isolate.

CC-503 was grown using TAP liquid media. 3.25 x 10^8 cells/mL were transferred to TAP agar plates exposed to ultraviolet (UV) light at a distance of 30 cm (253.7 nm, 100 μW/cm2, 60 Hz, NuAire, http://www.nuaire.com) for 2 minutes under sterile conditions. The plates were subsequently kept in dark for one day to prevent photo reactivation of the DNA repair mechanism. The plates were then allowed to grow under light for approximately a week. The resulting colonies were suspended in TAP liquid media and stained by BODIPY 505/515 for visualizing cells containing neutral lipids, which were then sorted using BD FACSAria III instrument. Using this protocol in an iterative fashion, an increase in lipid accumulation after four rounds of mutagenesis was observed. The cells selected after the fourth round, were sorted again without mutagenesis to further enrich the lipid accumulating cells. The sorted cells from the forth round of mutagenesis were grown using a TAP liquid media. The resulting single colonies were picked and suspended in TAP liquid media and stained by BODIPY 505/515 to illuminate cells containing neutral lipids, which were then analyzed using a FACS instrument. H5-M1 produced the highest labeled signal.

Rasha Abdrabu, Sudhir Kumar Sharma, Basel Khraiwesh, Kenan Jijakli, David R. Nelson, Amnah Alzahmi, Joseph Koussa, Mehar Sultana, Sachin Khapli, Ramesh Jagannathan, Kourosh Salehi-Ashtiani. "Single-Cell Characterization of Microalgal Lipid Contents with Confocal Raman Microscopy". Essentials of Single-Cell Analysis; part of the series Series in BioEngineering pp 363-382: 22 January 2016

From Rasha Abdrabu, Salehi-Ashtiani lab, NYU Abu Dhabi, October 2015

A soil alga, strain 3222, was isolated from Abu Dhabi (Mussaffah area), UAE. 3222 grew as low as 20°C and as high as 45°C, with optimal growth between 35°C- 40°C.

S. Sharma, D. Nelson, R. Abdrabu, B. Khraiwesh, K. Jijakli, M. Arnoux, M. O'Connor, T. Bahmani, H.
Cai, S. Khapli, R. Jagannathan, K. Salehi-Ashtiani (2015) An integrative Raman microscopy-based workflow for rapid in situ analysis of microalgal lipid bodies, Biotechnology for Biofuels, 8 164

From Munevver Aksoy, Grossman Lab, Carnegie Institution for Science, November 2015

The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex.

Aksoy M, Pootakham W, Grossman AR (2014) Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation. Plant Cell 26:4214-29

  • Locus:
  • VTC1
  • Chromosome:
  • 12

From Munevver Aksoy, Grossman Lab, Carnegie Institution for Science, November 2015

Aksoy M, Pootakham W, Grossman AR (2014) Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation. Plant Cell 26:4214-29

  • Locus:
  • VTC1
  • Chromosome:
  • 12

From Saul Purton, University College London, February 2016

This mutant is a chloroplast transformant in which the aadA (SpcR) cassette has been inserted between the BstXI site in the middle of the psbH coding sequence and the MluI site in the psbH-trnE2 intergenic region, thereby deleting a 0.36 kb genomic region including a large part of psbH. The aadA gene is in the same orientation as psbH.

Transformant is PSII-minus (i.e. acetate-requiring) due to the loss of PsbH, and spectinomycin resistant due to the presence of AadA. TN72 serves as a recipient strain for chloroplast transformation using the pASapI, pSRSapI and pWUCA2 expression vectors developed in the Purton lab.

Young RE and Purton S (2014) Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression. Plant J 80:915–925

Wannathong et al. (2016). New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Applied Microbiology and Biotechnology. In press.

Young REB, Purton S (2016) Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J [Epub ahead of print]. doi: 10.1111/pbi.12490

  • Locus:
  • psbH
  • Chromosome:
  • chloroplast

From Boris Zorin, Hegemann lab, Humboldt University-Berlin, November 2015

Zorin B, Lu Y, Sizova I, Hegemann P (2008) Nuclear gene targeting in Chlamydomonas as exemplified by disruption of the PHOT gene. Gene 432:91-6

From Andre Greiner, Hegemann lab, Humboldt University-Berlin, November 2015

Trippens J, Greiner A, Schellwat J, Neukam M, Rottmann T, Lu Y, Kateriya S, Hegemann P, Kreimer G (2012) Phototropin influence on eyespot development and regulation of phototactic behavior in Chlamydomonas reinhardtii. Plant Cell 24:4687-702