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Authors: David Booth
Group: King Lab, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol details the preparation and execution of CRISPR/Cas9 genome editing in S. rosetta. The protocol builds on a method to transfect macromolecules into S. rosetta for delivering a purified Cas9 ribonucleoprotein from Streptomyces pyogenes (SpCas9 RNP) into S. rosetta. Upon cleaving the S. rosetta genome at locations specified by the guide RNA (gRNA) of the SpCas9 RNP, S. rosetta can use DNA oligonucleotides as templates to repair the double-stranded break. Those repair templates can encode foreign sequences and mutations for editing the S. rosetta genome, so long as DNA oligonucleotides have >30 bases of sequence that is homologous to both sides of the Cas9 cleavage site.
Proper citation: David Booth 2020. Genome editing in the choanoflagellate Salpingoeca rosetta. protocols.io https://dx.doi.org/10.17504/protocols.io.89fhz3n Copy
Authors: Jeric Harper
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab
Summary: Preparation of BG-11 growth medium.
Proper citation: Jeric Harper 2017. Saltwater BG-11 recipe. protocols.io https://dx.doi.org/10.17504/protocols.io.f58bq9w Copy
Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab
Proper citation: Maria Rubio-Brotons 2016. How to culture ichthyosporean Abeoforma whisleri, a unicellular relative to animals. protocols.io https://dx.doi.org/10.17504/protocols.io.gqnbvve Copy
Authors: Dr. Steven Wilhelm, Samantha Coy
Group: VERVE Net, Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Samantha R Coy's Protocols
Summary: Adapted from: Van Etten, J. (n.d.). Titering of Chlorella Viruses. Retrieved from http://ncv.unl.edu/vanettenlab/
Contact Dr. Steven Wilhelm ([email protected]) or Samantha Coy ([email protected]) for additional information regarding this protocol.
Proper citation: Dr. Steven Wilhelm, Samantha Coy 2017. Chlorella Virus Plaque Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.hgqb3vw Copy
Authors: Dr. Steven Wilhelm
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.Modified from Wilhelm SW and CG Trick. 1995. Physiological profiles of Synechococcus (Cyanophyceae) in iron-limiting continuous cultures. Journal of Phycology, 31:79-85.
Proper citation: Dr. Steven Wilhelm 2017. A+ Media for Marine Phytoplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.ibncame Copy
Authors: Matus Valach
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Variant protocol for transformation of Diplonema papillatum by electroporation using a "home-made" transformation buffer. The procedure was devised based on previously published protocols by Kaur et al. (DOI: 10.1111/1462-2920.14041) and Dyer et al. (DOI: 10.3791/54342). For additional details, see also Faktorová et al. (DOI: 10.1111/1462-2920.15130).
Proper citation: Matus Valach 2020. Transformation of Diplonema papillatum by electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.bq3nmyme Copy
Authors: Nicholas Irwin, Elisabeth Hehenberger, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Here we describe a protocol for transforming Oxyrrhis marina with FITC-labelled dextran (a DNA analog) by electroporation. This protocol is reproducible, and results in a transformation efficiency of around 50%.We have confirmed the viability of transformed cells by allowing them to recover in the dark (and thus maintain fluorescence intensity) and then by observing fluorescent cells swimming normally, days after transformation (see the video here). As of yet we have been unable to demonstrate the actual uptake of plasmid DNA. We have tried using rhodamine labelled plasmids to demonstrate this but the results were inconclusive due to issues with cellular autofluorescence. We have also tried this method with a variety of expression plasmids encoding different promoters. For example, we tried the MOE promoter from Perkinsus marinus (a closely related species) and the generic eukaryotic CMV promoter. However no GFP fluorescence was detected in these samples. We have also tried transforming in vitro synthesized GFP encoding mRNA using this protocol. As of yet, no GFP production has been detected, but this may reflect non-cannonical translational systems in dinoflagellates as the same mRNA could be translated in starfish embryos.Although the development of expression systems in dinoflagellates remains a challenge, our results suggest that O. marina may be amenable to genetic transformation.
Proper citation: Nicholas Irwin, Elisabeth Hehenberger, Patrick Keeling 2016. Electroporation transformation of FITC-dextran into Oxyrrhis marina - an early branching dinoflagellate.. protocols.io https://dx.doi.org/10.17504/protocols.io.ginbude Copy
Authors: Aleksandra Kozyczkowska
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Proper citation: Aleksandra Kozyczkowska 2018. Transient tranfection of unicellular relative of animals, Creolimax fragrantissima, using Lonza Nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.r65d9g6 Copy
Authors: Glen Wheeler, Rowena Stern, Cecilia Balesteri
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Glen Wheeler, Rowena Stern, Cecilia Balesteri 2017. Method for electroporation of Isochrysis galbana CCMP1323. protocols.io https://dx.doi.org/10.17504/protocols.io.hmab42e Copy
Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Yeilds 20 mL of Lysis buffer to be used in Phenol/ chloroform genomic DNA extrations.
Proper citation: Daniel Richter 2017. Lysis Buffer for Phenol/ chloroform genomic DNA extraction (makes 20 mL of buffer). protocols.io https://dx.doi.org/10.17504/protocols.io.juicnue Copy
Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab
Proper citation: Maria Rubio-Brotons 2017. Stable transfection protocol of unicellular relative to animals Corallochytrium limacisporum. protocols.io https://dx.doi.org/10.17504/protocols.io.hmxb47n Copy
Authors: Tamara Matute, Isaac Nuñez, Peter Von Dassow, Fernan Federici
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Laboratorio de Tecnologias Libres
Summary: We used this protocol to build a series of codon optimized BFP expressing vectors in the context of the EMS initiative from Gordon and Betty Moore foundation. A complete list of files corresponding to the DNA parts and vectors used in this study can be found at our Drive and zenodo database.
Proper citation: Tamara Matute, Isaac Nuñez, Peter Von Dassow, Fernan Federici 2019. Fabrication of DNA constructs by Gibson Assembly and Golden Gate reactions. protocols.io https://dx.doi.org/10.17504/protocols.io.4fcgtiw Copy
Authors: Angela Piersanti, Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Euplotes crassus GFP artificial nanochromosome
Proper citation: Angela Piersanti, Rachele Cesaroni 2019. Euplotes crassus GFP artificial nanochromosome sequence. protocols.io https://dx.doi.org/10.17504/protocols.io.2awgafe Copy
Authors: Anastasios Tsaousis
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Anastasios Tsaousis 2017. Transfected Naegleria Fluorescence Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.hpvb5n6 Copy
Authors: Mariana Rius, Jackie Collier
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: Electroporation strategy used in the transformation of Aurantiochytrium limacinum (ATCC MYA-1381; Stramenopile/ Heterokont, Thraustochytrid). This protocol is following the guidance of Ono et al. 2011. US Patent # 7,888,123 B2.
Proper citation: Mariana Rius, Jackie Collier 2018. Electroporation of Aurantiochytrium limacinum (ATCC MYA-1381). protocols.io https://dx.doi.org/10.17504/protocols.io.qjcduiw Copy
Authors: Angela Piersanti
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Angela Piersanti 2017. Euplotes transfection using Lipofectamine 3000 (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.hhub36w Copy
Authors: Alle Lie
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology
Proper citation: Alle Lie 2016. Making heat-killed bacteria for feeding axenic phagotrophic protists. protocols.io https://dx.doi.org/10.17504/protocols.io.ewkbfcw Copy
Authors: Jeric Harper
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab
Proper citation: Jeric Harper 2016. Full Contact Microbiology (a.k.a Diatom Transformation via Bacterial Conjugation). protocols.io https://dx.doi.org/10.17504/protocols.io.fzrbp56 Copy
Authors: Ashley Humphrey
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Stock vitamin solution for ESAW Media for Marine Phytoplankton
Proper citation: Ashley Humphrey 2016. Vitamin Solution for ESAW Media for Marine Phytoplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.ge9bth6 Copy
Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Details of the conjugation episome used to generate the TpSil3p-EGFP expressing Thalassiosira pseudonana strain as reported in Faktorová et al (2019). The file contains:1/ a vector scheme summarizing all of its elements2/ expression cassette elements and their nucleotide sequences3/ fusion protein amino acid sequence4/ full vector DNA sequence with expression cassette elements annotated
Proper citation: Jernej Turnsek 2019. Faktorová_et_al_2019_pTpPuc3_TpSil3p-eGFP. protocols.io https://dx.doi.org/10.17504/protocols.io.8bdhsi6 Copy
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