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Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab

Proper citation: Maria Rubio-Brotons 2016. Freezing and unfreezing unicellular protists using Mr Frosty container. protocols.io https://dx.doi.org/10.17504/protocols.io.gg3btyn Copy   

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Authors: 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.
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab

Proper citation: 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 Copy   

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Authors: Nick Irwin, Elisabeth Hehenberger, Liz Cooney, Yoshihisa Hirakawa, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: 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.

Proper citation: 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 Copy   

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Authors: Mariana Rius, Joshua Rest
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: 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.

Proper citation: Mariana Rius, Joshua Rest 2017. Genome-wide Kozak Sequence Free Energy Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.h9mb946 Copy   

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Authors: Jernej Turnsek, Chris Dupont
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: 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.

Proper citation: Jernej Turnsek, Chris Dupont 2017. Pour plating of Thalassiosira pseudonana (Tp). protocols.io https://dx.doi.org/10.17504/protocols.io.jfncjme Copy   

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Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Daniel Richter 2017. Phenol/chloroform genomic DNA extraction-1.5 ml Eppendorf tube.. protocols.io https://dx.doi.org/10.17504/protocols.io.inzcdf6 Copy   

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Authors: Lev Tsypin, Aaron Turkewitz
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: 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.

Proper citation: Lev Tsypin, Aaron Turkewitz 2019. Seminavis robusta protocol collection. protocols.io https://dx.doi.org/10.17504/protocols.io.4p8gvrw Copy   

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Authors: Vincent A. Bielinski and Philip D. Weyman
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: A conventient extraction and measurement protocol for GUS activity from Phaeodactylum tricornutum.

Proper citation: 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 Copy   

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Authors: Morgan Colp
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: 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.

Proper citation: 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 Copy   

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Authors: Mariana Rius
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: 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 Copy   

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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: Trace metals solution to be used in ESAW Media for Marine Phytoplankton 

Proper citation: Ashley Humphrey 2016. Trace Metal Solution for ESAW Media. protocols.io https://dx.doi.org/10.17504/protocols.io.ge8bthw Copy   

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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), CyanoHABs

Proper citation: Ashley Humphrey 2017. Phenol Chloroform DNA extraction from polycarbonate filters. protocols.io https://dx.doi.org/10.17504/protocols.io.ibbcain Copy   

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Authors: Anna Vanclová and Natalia Wandyszewska
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol was used to check, whether the DNA can be delivered to Eutreptiella gymnastica.

Proper citation: 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 Copy   

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Authors: Dr. Steven Wilhelm, Samantha Coy
Group: 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: 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.

Proper citation: Dr. Steven Wilhelm, Samantha Coy 2017. MBBM Stock Solutions. protocols.io https://dx.doi.org/10.17504/protocols.io.hgtb3wn Copy   

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Authors: Jeff Lewis
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Lewis Lab
Summary: 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.

Proper citation: Jeff Lewis 2017. Acquired Stress Resistance Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.g7sbzne Copy   

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Authors: Eric Gann
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)

Proper citation: Eric Gann 2017. Transformation of Aureococcus anophagefferens by Electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.g2vbye6 Copy   

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Authors: Philip D. Weyman
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: 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.

Proper citation: Philip D. Weyman 2017. Cloned Genomic Library using Illumina Adapters. protocols.io https://dx.doi.org/10.17504/protocols.io.hfbb3in Copy   

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Authors: Fulei Luan, Xiaoxue Wen, Patrick Beardslee and Heriberto Cerutti
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Procedure for the transformation of the nuclear genome of Tetraselmis striata by microprojectile bombardment.

Proper citation: 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 Copy   

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Authors: Lawrence A. Klobutcher, Larry Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: 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

Proper citation: 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 Copy   

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Authors: Angela Piersanti, Rachele Cesaroni, Larry Klobutcher, Cristina Miceli, Sandra Pucciarelli
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: 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 Copy   

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