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Freezing and unfreezing unicellular protists using Mr Frosty container Resource Report Resource Website |
Maria Rubio-Brotons | DOI:10.17504/protocols.io.gg3btyn | Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab | IBE CSIC-UPF | 2 | 2016 | Maria Rubio-Brotons 2016. Freezing and unfreezing unicellular protists using Mr Frosty container. protocols.io https://dx.doi.org/10.17504/protocols.io.gg3btyn | 2021-04-15 09:14:21 | ||||
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Acriflavine Centrifuge Stain for Labyrinthulomycetes Resource Report Resource Website |
Modified by Sabrina Geraci-Yee FROM: RAGHUKUMAR S, SCHAUMANN K. 1993. AN EPIFLUORESCENCE MICROSCOPY METHOD FOR DIRECT DETECTION AND ENUMERATION OF THE, THE THRAUSTOCHYTRIDS. LIMNOL. OCEANOGR. | DOI:10.17504/protocols.io.hgib3ue | Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab | , , | 1 | 2017 | Modified by Sabrina Geraci-Yee FROM: RAGHUKUMAR S, SCHAUMANN K. 1993. AN EPIFLUORESCENCE MICROSCOPY METHOD FOR DIRECT DETECTION AND ENUMERATION OF THE, THE THRAUSTOCHYTRIDS. LIMNOL. OCEANOGR. 2017. Acriflavine Centrifuge Stain for Labyrinthulomycetes. protocols.io https://dx.doi.org/10.17504/protocols.io.hgib3ue | 2021-04-15 09:14:22 | ||||
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Electroporation transformation of FITC-dextran into Oxyrrhis marina - an early branching dinoflagellate. Resource Report Resource Website |
Nick Irwin, Elisabeth Hehenberger, Liz Cooney, Yoshihisa Hirakawa, Patrick Keeling | DOI:10.17504/protocols.io.3cmgiu6 | Protist Research to Optimize Tools in Genetics (PROT-G) | INTRODUCTIONHere 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 1%.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). NOTE ON AUTOFLUORESCENCEAlthough Oxyrrhis is a non-photosynthetic dinoflagellate and therefore lacks plastid autofluorescence, we have observed a small amount of green autofluorescence that is typically isolated in the anterior of the cell (See top panel of Figure 1).The autofluorescence was observed in wildtype cells, cells electroporated without FITC-dextran, and cells that were incubated with FITC-dextran but not electroporated. The autofluorescence was observed in cultures with both Dunaliella tertiolecta or heat-killed E. coli. The brightness and frequency of the autofluorescence also seemed to be dependent on culture conditions. Not all cells had the autofluorescence.ADDITIONAL INFORMATIONAs 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. | Faktorová D, Nisbet RER, Robledo JAF, Casacuberta E, Sudek L, Allen AE, Ares M, Aresté C, Balestreri C, Barbrook AC, Beardslee P, Bender S, Booth DS, Bouget F, Bowler C, Breglia SA, Brownlee C, Burger G, Cerutti H, Cesaroni R, Chiurillo MA, Clemente T, Coles DB, Collier JL, Cooney EC, Coyne K, Docampo R, Dupont CL, Edgcomb V, Einarsson E, Elustondo PA, Federici F, Freire-Beneitez V, Freyria NJ, Fukuda K, García PA, Girguis PR, Gomaa F, Gornik SG, Guo J, Hampl V, Hanawa Y, Haro-Contreras ER, Hehenberger E, Highfield A, Hirakawa Y, Hopes A, Howe CJ, Hu I, Ibañez J, Irwin NAT, Ishii Y, Janowicz NE, Jones AC, Kachale A, Fujimura-Kamada K, Kaur B, Kaye JZ, Kazana E, Keeling PJ, King N, Klobutcher LA, Lander N, Lassadi I, Li Z, Lin S, Lozano J, Luan F, Maruyama S, Matute T, Miceli C, Minagawa J, Moosburner M, Najle SR, Nanjappa D, Nimmo IC, Noble L, Vanclová AMGN, Nowacki M, Nuñez I, Pain A, Piersanti A, Pucciarelli S, Pyrih J, Rest JS, Rius M, Robertson D, Ruaud A, Ruiz-Trillo I, Sigg MA, Silver PA, Slamovits CH, Smith GJ, Sprecher BN, Stern R, Swart EC, Tsaousis AD, Tsypin L, Turkewitz A, Turnšek J, Valach M, Vergé V, Dassow Pv, Haar Tvd, Waller RF, Wang L, Wen X, Wheeler G, Woods A, Zhang H, Mock T, Worden AZ, Lukeš J, Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nature Methods 17(5). doi: 10.1038/s41592-020-0796-x | University of British Columbia, University of British Columbia, University of British Columbia, University of Tsukuba, University of British Columbia | https://doi.org/10.1038/s41592-020-0796-x | 3 | 2019 | Nick Irwin, Elisabeth Hehenberger, Liz Cooney, Yoshihisa Hirakawa, Patrick Keeling 2019. Electroporation transformation of FITC-dextran into Oxyrrhis marina - an early branching dinoflagellate.. protocols.io https://dx.doi.org/10.17504/protocols.io.3cmgiu6 | 2021-04-15 09:14:22 | |
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Genome-wide Kozak Sequence Free Energy Analysis Resource Report Resource Website |
Mariana Rius, Joshua Rest | DOI:10.17504/protocols.io.h9mb946 | Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab | The purpose of this protocol is to evaluate the significance of delta G values, as a strategy to determine the significance of secondary structure. Sequences are shuffled randomly 100x, maintaining base composition but not base order. The percentile of the delta G vlaue of the non-scrambled (original) sequence is obtained from the distribution created by the delta G values of the 100X scrambled sequences. | Stony Brook University, Stony Brook University | 1 | 2017 | Mariana Rius, Joshua Rest 2017. Genome-wide Kozak Sequence Free Energy Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.h9mb946 | 2021-04-15 09:14:25 | |||
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Pour plating of Thalassiosira pseudonana (Tp) Resource Report Resource Website |
Jernej Turnsek, Chris Dupont | DOI:10.17504/protocols.io.jfncjme | Protist Research to Optimize Tools in Genetics (PROT-G) | Here we present an efficient way to grow Tp colonies inside a 0.25% superclean agar matrix. This protocol could possibly be applicable to other marine microeukaryotes that are problematic to grow on fully solid support. | University of California, Berkeley, J. Craig Venter Institute | 3 | 2017 | Jernej Turnsek, Chris Dupont 2017. Pour plating of Thalassiosira pseudonana (Tp). protocols.io https://dx.doi.org/10.17504/protocols.io.jfncjme | 2021-04-15 09:14:30 | |||
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Phenol/chloroform genomic DNA extraction-1.5 ml Eppendorf tube. Resource Report Resource Website |
Daniel Richter | DOI:10.17504/protocols.io.inzcdf6 | Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G) | Richter, Daniel J and Fozouni, Parinaz and Eisen, Michael and King, Nicole. Gene family innovation, conservation and loss on the animal stem lineage. 2018;7:e34226 https://doi.org/10.7554/eLife.34226 | King Lab, University of California Berkeley. | https://elifesciences.org/articles/34226 | 1 | 2017 | Daniel Richter 2017. Phenol/chloroform genomic DNA extraction-1.5 ml Eppendorf tube.. protocols.io https://dx.doi.org/10.17504/protocols.io.inzcdf6 | 2021-04-15 09:14:26 | ||
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Seminavis robusta protocol collection Resource Report Resource Website |
Lev Tsypin, Aaron Turkewitz | DOI:10.17504/protocols.io.4p8gvrw | Protist Research to Optimize Tools in Genetics (PROT-G) | This is a collection of media, reagents, and protocols for the cultivation and manipulation of the diatom Seminavis robusta. Our hope is that these documents will help future researchers develop genetic tools for studying this organism. | California Institute of Technology, University of Chicago | 2 | 2019 | Lev Tsypin, Aaron Turkewitz 2019. Seminavis robusta protocol collection. protocols.io https://dx.doi.org/10.17504/protocols.io.4p8gvrw | 2021-04-15 09:14:34 | |||
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Beta-glucuronidase (GUS) assay (adapted for Phaeodactylum tricornutum) Resource Report Resource Website |
Vincent A. Bielinski and Philip D. Weyman | DOI:10.17504/protocols.io.hefb3bn | Protist Research to Optimize Tools in Genetics (PROT-G) | A conventient extraction and measurement protocol for GUS activity from Phaeodactylum tricornutum. | J. Craig Venter Institute | 1 | 2017 | Vincent A. Bielinski and Philip D. Weyman 2017. Beta-glucuronidase (GUS) assay (adapted for Phaeodactylum tricornutum). protocols.io https://dx.doi.org/10.17504/protocols.io.hefb3bn | 2021-04-15 09:14:27 | |||
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Single cell isolation and monoclonal culture establishment of Acanthamoeba castellanii using migration on agar plates Resource Report Resource Website |
Morgan Colp | DOI:10.17504/protocols.io.bc3eiyje | Protist Research to Optimize Tools in Genetics (PROT-G) | Isolation of single Acanthamoeba castellanii cells from agar plates for establishment of monoclonal cultures. This protocol can be useful for isolating clones of a mixed wild-type population or of transformants. After isolation, growth can be re-established in typical Neff liquid medium. | Dalhousie University | 1 | 2020 | Morgan Colp 2020. Single cell isolation and monoclonal culture establishment of Acanthamoeba castellanii using migration on agar plates. protocols.io https://dx.doi.org/10.17504/protocols.io.bc3eiyje | 2021-04-15 09:14:42 | |||
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Cell Preparation for Electroporation of Aurantiochytrium using Salt and Sucrose solutions Resource Report Resource Website |
Mariana Rius | DOI:10.17504/protocols.io.h65b9g6 | Protist Research to Optimize Tools in Genetics (PROT-G) | Stony B | 1 | 2017 | Mariana Rius 2017. Cell Preparation for Electroporation of Aurantiochytrium using Salt and Sucrose solutions. protocols.io https://dx.doi.org/10.17504/protocols.io.h65b9g6 | 2021-04-15 09:14:44 | ||||
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Trace Metal Solution for ESAW Media Resource Report Resource Website |
Ashley Humphrey | DOI:10.17504/protocols.io.ge8bthw | Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs) | Trace metals solution to be used in ESAW Media for Marine Phytoplankton | University of Tennessee, Knoxville | 2 | 2016 | Ashley Humphrey 2016. Trace Metal Solution for ESAW Media. protocols.io https://dx.doi.org/10.17504/protocols.io.ge8bthw | 2021-04-15 09:14:44 | |||
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Phenol Chloroform DNA extraction from polycarbonate filters Resource Report Resource Website |
Ashley Humphrey | DOI:10.17504/protocols.io.ibbcain | Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), CyanoHABs | University of Tennessee, Knoxville | 1 | 2017 | Ashley Humphrey 2017. Phenol Chloroform DNA extraction from polycarbonate filters. protocols.io https://dx.doi.org/10.17504/protocols.io.ibbcain | 2021-04-15 09:14:46 | ||||
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Experimental check of DNA delivery into E. gymnastica by electroporation Resource Report Resource Website |
Anna Vanclová and Natalia Wandyszewska | DOI:10.17504/protocols.io.hh5b386 | Protist Research to Optimize Tools in Genetics (PROT-G) | This protocol was used to check, whether the DNA can be delivered to Eutreptiella gymnastica. | Charles University in Prague | 1 | 2017 | Anna Vanclová and Natalia Wandyszewska 2017. Experimental check of DNA delivery into E. gymnastica by electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.hh5b386 | 2021-04-15 09:14:46 | |||
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MBBM Stock Solutions Resource Report Resource Website |
Dr. Steven Wilhelm, Samantha Coy | DOI:10.17504/protocols.io.hgtb3wn | 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 | Van Etten, J. (n.d.). Formulation of Modified Bold's Basal Medium (MBBM). Retrieved from http://ncv.unl.edu/vanettenlab/ Contact Dr. Steven Wilhelm ([email protected]) or Samantha Coy ([email protected]) for additional information regarding this protocol. | , | http://ncv.unl.edu/vanettenlab/ | 2 | 2017 | Dr. Steven Wilhelm, Samantha Coy 2017. MBBM Stock Solutions. protocols.io https://dx.doi.org/10.17504/protocols.io.hgtb3wn | 2021-04-15 09:14:31 | ||
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Acquired Stress Resistance Assay Resource Report Resource Website |
Jeff Lewis | DOI:10.17504/protocols.io.g7sbzne | Protist Research to Optimize Tools in Genetics (PROT-G), Lewis Lab | A simple 96-well plate assay for stress resistance across a range of stress doses. Originally designed for yeast, but easily modifiable for any colony-forming microbe. | McDaniel EA, Stuecker TN, Veluvolu M, Gasch AP, Lewis JA, Independent Mechanisms for Acquired Salt Tolerance versus Growth Resumption Induced by Mild Ethanol Pretreatment in . mSphere 3(6). doi: 10.1128/mSphere.00574-18 | University of Arkansas | https://doi.org/10.1371/journal.pgen.1007335 | 1 | 2017 | Jeff Lewis 2017. Acquired Stress Resistance Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.g7sbzne | 2021-04-15 09:14:36 | |
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Transformation of Aureococcus anophagefferens by Electroporation Resource Report Resource Website |
Eric Gann | DOI:10.17504/protocols.io.g2vbye6 | Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs) | 1 | 2017 | Eric Gann 2017. Transformation of Aureococcus anophagefferens by Electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.g2vbye6 | 2021-04-15 09:14:49 | |||||
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Cloned Genomic Library using Illumina Adapters Resource Report Resource Website |
Philip D. Weyman | DOI:10.17504/protocols.io.hfbb3in | Protist Research to Optimize Tools in Genetics (PROT-G) | A protocol to create a genomic library (2-5 kb insert size) to screen for genomic regions functioning as centromeres. Uses the NEBNext Illumina library prep kit from NEB to add adapters to fragments and then the fragments can be efficiently assembled into a vector containing homology to the adapter sequences using Gibson Assembly. | J. Craig Venter Institute | 1 | 2017 | Philip D. Weyman 2017. Cloned Genomic Library using Illumina Adapters. protocols.io https://dx.doi.org/10.17504/protocols.io.hfbb3in | 2021-04-15 09:14:48 | |||
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Tetraselmis transformation by microprojectile bombardment Resource Report Resource Website |
Fulei Luan, Xiaoxue Wen, Patrick Beardslee and Heriberto Cerutti | DOI:10.17504/protocols.io.hjtb4nn | Protist Research to Optimize Tools in Genetics (PROT-G) | Procedure for the transformation of the nuclear genome of Tetraselmis striata by microprojectile bombardment. | Faktorová D, Nisbet RER, Robledo JAF, Casacuberta E, Sudek L, Allen AE, Ares M, Aresté C, Balestreri C, Barbrook AC, Beardslee P, Bender S, Booth DS, Bouget F, Bowler C, Breglia SA, Brownlee C, Burger G, Cerutti H, Cesaroni R, Chiurillo MA, Clemente T, Coles DB, Collier JL, Cooney EC, Coyne K, Docampo R, Dupont CL, Edgcomb V, Einarsson E, Elustondo PA, Federici F, Freire-Beneitez V, Freyria NJ, Fukuda K, García PA, Girguis PR, Gomaa F, Gornik SG, Guo J, Hampl V, Hanawa Y, Haro-Contreras ER, Hehenberger E, Highfield A, Hirakawa Y, Hopes A, Howe CJ, Hu I, Ibañez J, Irwin NAT, Ishii Y, Janowicz NE, Jones AC, Kachale A, Fujimura-Kamada K, Kaur B, Kaye JZ, Kazana E, Keeling PJ, King N, Klobutcher LA, Lander N, Lassadi I, Li Z, Lin S, Lozano J, Luan F, Maruyama S, Matute T, Miceli C, Minagawa J, Moosburner M, Najle SR, Nanjappa D, Nimmo IC, Noble L, Vanclová AMGN, Nowacki M, Nuñez I, Pain A, Piersanti A, Pucciarelli S, Pyrih J, Rest JS, Rius M, Robertson D, Ruaud A, Ruiz-Trillo I, Sigg MA, Silver PA, Slamovits CH, Smith GJ, Sprecher BN, Stern R, Swart EC, Tsaousis AD, Tsypin L, Turkewitz A, Turnšek J, Valach M, Vergé V, Dassow Pv, Haar Tvd, Waller RF, Wang L, Wen X, Wheeler G, Woods A, Zhang H, Mock T, Worden AZ, Lukeš J, Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nature Methods 17(5). doi: 10.1038/s41592-020-0796-x | University of Nebraska-Lincoln, University of Nebraska-Lincoln, University of Nebraska-Lincoln | https://doi.org/10.1038/s41592-020-0796-x | 1 | 2017 | Fulei Luan, Xiaoxue Wen, Patrick Beardslee and Heriberto Cerutti 2017. Tetraselmis transformation by microprojectile bombardment. protocols.io https://dx.doi.org/10.17504/protocols.io.hjtb4nn | 2021-04-15 09:14:57 | |
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Culturing Euplotes crassus to high densities using a combination of algae and bacteria as the food source. Resource Report Resource Website |
Lawrence A. Klobutcher, Larry Klobutcher | DOI:10.17504/protocols.io.2aggabw | Protist Research to Optimize Tools in Genetics (PROT-G) | This procedure was developed to investigate the possible use of RNAi through bacterial feeding as a means of knocking down expression of particular genes.While the utility of RNAi is still being investigated, the method is effective in growing Euplotes crassus to higher densities than is possible using algae alone. Using moderately dense cultures of algae, we typically achieve cell densities of 500-1,000 cells/ml, while with the E. coli supplemented cultures we describe here, Euplotes cell densities exceed 3,000 cells/ml. In fact, preliminary studies mixing a volume of a Euplotes culture with an equal volume of resuspended E. coli have produced a density of ~8,000 cells/ml.While the described procedure involves small cultures, it can likely be scaled up as required.For some species of Euplotes, long-term culture using bacteria as a food source has not been successful (C. Miceli, personal communication), suggesting that some essential nutrient is not being provided in sufficient quantitities | University of Connecticut Health Center, University of Connecticut, School of medicine | 1 | 2019 | Lawrence A. Klobutcher, Larry Klobutcher 2019. Culturing Euplotes crassus to high densities using a combination of algae and bacteria as the food source.. protocols.io https://dx.doi.org/10.17504/protocols.io.2aggabw | 2021-04-15 09:15:04 | |||
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Euplotes - Miceli Lab Resource Report Resource Website |
Angela Piersanti, Rachele Cesaroni, Larry Klobutcher, Cristina Miceli, Sandra Pucciarelli | DOI:10.17504/protocols.io.2a8gahw | Protist Research to Optimize Tools in Genetics (PROT-G) | University of Camerino, Universität Bern, University of Connecticut, School of medicine, University of Connecticut, School of medicine, University of Camerino | 1 | 2019 | Angela Piersanti, Rachele Cesaroni, Larry Klobutcher, Cristina Miceli, Sandra Pucciarelli 2019. Euplotes - Miceli Lab. protocols.io https://dx.doi.org/10.17504/protocols.io.2a8gahw | 2021-04-15 09:14:58 |
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