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Authors: David Booth
Group: King Lab, Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: David Booth 2018. Protocol A: Preparation of endotoxin-free carrier DNA for transfection. protocols.io https://dx.doi.org/10.17504/protocols.io.h8cb9sw Copy   

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Authors: Angela Piersanti
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Angela Piersanti 2017. PCR on Euplotes whole cells. protocols.io https://dx.doi.org/10.17504/protocols.io.hhvb366 Copy   

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Authors: Morgan Colp
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Transformation of Acanthamoeba castellanii Neff (ATCC-30010) with plasmid DNA can be achieved with a modified version of the protocol described in Peng, Omaruddin, & Bateman (2005) using constructs from Bateman (2010). 

Proper citation: Morgan Colp 2018. Transformation of Acanthamoeba castellanii with Qiagen SuperFect reagent. protocols.io https://dx.doi.org/10.17504/protocols.io.s4regv6 Copy   

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Authors: April Woods, G Jason Smith
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab
Summary: A modified TOTO-1 iodide labeling protocol to enable visualization of nucleic acid loading efficiency, as a means to verify that nucleic acid constructs are entering the cells. With additional cleanup steps to decrease background staining. A convenient assay to use in conjunction with transfection optimization. (Modified from Golzio, M., Teissie, J. & Rols, M.P. 2002. Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci USA 99, 1292–1297.)

Proper citation: April Woods, G Jason Smith 2018. TOTO-1 nucleic acid labeling protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.nkfdctn Copy   

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Authors: Pia Elustondo
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Slamovits Lab

Proper citation: Pia Elustondo 2017. Transfection of Alexa488-labelled DNA into Oxyrrhis marina with polyethylenimine (PEI). protocols.io https://dx.doi.org/10.17504/protocols.io.ha8b2hw Copy   

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Authors: Brittany Sprecher, Huan Zhang, Senjie Lin
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Introducing CRISPR/Cas9 machinery into the cell as RNP complexes reduces the number of steps necessary to edit the genome, does not require codon optimization or a strong promoter, can result in higher editing efficiencies with fewer off target effects, and has lower risks of cell toxicity (Woo et al., 2015; Liang et al., 2015; Liang et al., 2017; Farboud et al., 2018). This protocol utilizes IDTs fluorescently labeled tracrRNAs to visually check to see whether the CRISPR/Cas9 RNP molecule can make it into non photosynthetic dinoflagellates.  This protocol serves as a good starting point to evaluate the CRISPR/Cas9 RNP method, the relative transformation efficiency, and can be used to help sort cells that contain the CRISPR/Cas9 RNP molecule from cells that do not.

Proper citation: Brittany Sprecher, Huan Zhang, Senjie Lin 2020. Protocol for introducing fluorescently labeled CRISPR/Cas9 RNP complex into heterotrophic dinoflagellates. protocols.io https://dx.doi.org/10.17504/protocols.io.bei2jcge Copy   

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Authors: Vincent A. Bielinski and Phillip D. Weyman
Group: Protist Research to Optimize Tools in Genetics (PROT-G), JCVI West Protocols
Summary: This protocol describes a method for screening transgenic lines of Phaeodactylum, specifically for those obtained via conjugation with episomal plasmids.  The assay can also be used for screening genomic integrants obtained through biolistics.  The assay uses patched colonies of diatom grown on agar plates for biomass and does not require the use of kits or any pre-processing (boiling, etc) of the samples prior to analysis.  This allows for relatively high-throughput screening of transgenic lines in a single day.

Proper citation: Vincent A. Bielinski and Phillip D. Weyman 2017. Patch-based Colony PCR Protocol for Screening Phaeodactylum tricornutum. protocols.io https://dx.doi.org/10.17504/protocols.io.hnzb5f6 Copy   

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Authors: Lawrence A. Klobutcher, Larry Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Numerous unpublished studies indicate that Euplotes crassus is resistant to extremely high concentrations of commonly used selective agents when grow in artificial seawater. We have found that when E. crassus is grown in 1 part artificial seawater + 9 parts 0.3 M glucose, they are much more sensitive to a number of selective agents. For example, 1080 ug/ml paromomycin in artificial seawater only inhibited growth, as little as 120 ug/ml was effective in killing cells when grown with 0.3 M glucose. Similarly, G418 at 400 ug/ml had little effect in seawater, but as little as 100 ug/ml blocked growth in 0.3 M glucose. This may not prove true for all selective agents, as we saw no effect with paclitaxel up to 100 uM under either growth condition.

Proper citation: Lawrence A. Klobutcher, Larry Klobutcher 2019. Increased sensitivity of Euplotes crassus to selective agents using 0.3 M glucose-based culture conditions.. protocols.io https://dx.doi.org/10.17504/protocols.io.2ahgab6 Copy   

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Authors: Binnypreet Kaur, Drahomíra Faktorová, Julius Lukeš, ,
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes
Summary: This protocol describes transfection of ribonucleoprotein (RNP) complexes that consist of purified Cas9 nuclease duplexed with synthetic guide RNA in cultured cells using the AMAXA Nucleo AMAXA Nucleofector II Electroporation system using Program X-001.RNPs complex have only transient expression inside the cell, this allows for the highest levels of editing efficiency and greatly reduces the chances of possible off target and toxic effects of integration of foreignDNA in host genome

Proper citation: Binnypreet Kaur, Drahomíra Faktorová, Julius Lukeš, , 2019. Tranfection of sgRNA using SpCas9 containing plasmids to generate cell lines with Cas9. protocols.io https://dx.doi.org/10.17504/protocols.io.x39fqr6 Copy   

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Authors: Monica Berjon-Otero, Sarah Duponchel, Matthias Fischer
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol allows to remove bacteria from cultures of the marine heterotrophic nanoflagellate Cafeteria roenbergensis without affecting flagellate viability. Bacterial removal is essential to reduce contamination and background signal in subsequent microscopy analysis, protein extraction, DNA extraction or other applications. With some changes, this protocol could be adapted to different types of protists.

Proper citation: Monica Berjon-Otero, Sarah Duponchel, Matthias Fischer 2019. Lysozyme-based removal of bacteria from cultures of the marine heterotrophic flagellate Cafeteria roenbergensis. protocols.io https://dx.doi.org/10.17504/protocols.io.88ihzue 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. Modified Qiagen gel extraction with MinElute spin column.. protocols.io https://dx.doi.org/10.17504/protocols.io.ivdce26 Copy   

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Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Simple and rapid vacuole staining protocol for Phaeodactylum tricornutum. Note that RatioWorksTM PDMPO and LysoSensorTM Yellow/Blue DND-160 are the same thing and can thus be used interchangeably. RatioWorksTM PDMPO  was used in this protocol. 

Proper citation: Jernej Turnsek 2018. Vacuole staining in Phaeodactylum tricornutum using RatioWorks™ PDMPO. protocols.io https://dx.doi.org/10.17504/protocols.io.nbxdapn Copy   

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Authors: Felix Weber, Tatiana Zaliznyak, Gordon Taylor
Group: Protist Research to Optimize Tools in Genetics (PROT-G), TaylorLab
Summary: Sample preparation for Raman Microspectroscopic analysis of microbial cells mostly requires mounting of cells to Raman-inactive substrata such as mirror-finished stainless steel microscope slides. This can be achieved either by drying cell suspensions on stainless steel slides or by applying the Filter-Transfer-Freeze ("FTF") Technique (Taylor et al. 2017 protocols.io; dx.doi.org/10.17504/protocols.io.ikqccvw) when microbial cells have been collected on polycarbonate membrane filters before. Both techniques work very well for small and rigid prokaryotes. However, the compression forces and dehydration caused by the FTF-technique or by dry mounting results in considerable morphological distortions and collapsing of cells when applied to specimens of larger protists (e.g. ciliates). Therefore, we developed a sample preparation method that accommodates the delicateness of ciliates and allows Raman analysis of fully intact and hydrated cells under water through a water immersion objective lens. The protocol was established with the ciliate species Tetrahymena pyriformis and and enabled 3D-mapping of chemical fingerprints on a subcellular level during Raman scans of 12 hours and longer. This method might be applicable not only to protists, but with slight alterations to larger specimens or delicate tissues in general.

Proper citation: Felix Weber, Tatiana Zaliznyak, Gordon Taylor 2019. Sample Preparation for 3D-Raman Microspectroscopic Mapping of Fully Hydrated Protist Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.ztyf6pw Copy   

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Authors: David Booth
Group: King Lab, Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: David Booth 2018. Protocol C: Preparation of heparin for transfection. protocols.io https://dx.doi.org/10.17504/protocols.io.h8vb9w6 Copy   

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Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This is an effective protocol for genotyping diatom exconjugants which we believe is readily applicable to any other genetically manipulated marine microeukaryote.

Proper citation: Jernej Turnsek 2017. Simple & rapid genotyping of marine microeukaryotes. protocols.io https://dx.doi.org/10.17504/protocols.io.jcdcis6 Copy   

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Authors: David Booth
Group: King Lab, Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: David Booth 2018. Protocol F: Preparation of recovery buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.h82b9ye Copy   

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Authors: Fatma Gomaa, Paulo A. Garcia, Jennifer Delaney, Peter R. Girgius, Cullen R. Buie, Virginia P. Edgcomb
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: We recently developed a continuous flow system to transform microorganisms in high throughput in a microfluidic device (Garcia et al., 2017). This system employs microfluidic channels that contain a bilateral constriction between the inlet and outlet electrode connections (length = 3.0 mm, widthmin = 50 μm, widthmax = 2.0 mm, and height = 100 μm). The constriction amplifies the electric field under an applied voltage between the inlet and outlet electrodes to levels sufficiently high to induce electroporation. During P. caudutus transformations, the cells were driven through the microfluidic device at flow rates of 50 μL/min and 500 μL/min, which correspond to residence times (i.e. pulse durations) of 20 ms and 2 ms, respectively. Square wave pulses with, for example, 5 ms ON and 5 ms OFF cycles (50 % duty cycle) are applied to the microchannel through the dispensing needle. Therefore the cell viability cannot be accurately evaluated since only 50 % of the cells experience the electric field. The pulses are delivered from electrodes with alternating polarity between the pulses to reduce electrolytic effects at the electrode-buffer interface (Fig. 1). After flowing through the microchannel, each 200 μL cell sample is added to a 1.5 ml Eppendorf tube containing 1 ml of fresh growth media for cell recovery. The applied voltages we evaluated had amplitudes of 250 V (Emax = 1,500 V/cm), 375 V (Emax = 2,250 V/cm), and 500 V (Emax = 3,000 V/cm) for each polarity. The non-uniform constriction in the microfluidic devices generates a variable electric field that is capable of transfecting cells while minimizing exposure to the highest electric field. Figure 1: Electric field waveforms employed for transient and stable transfection of Bodo caudatus. Three independent electroporation systems were used for reproducible transfection including a) our microfluidic electroporation platform (Garcia et al., 2017), the NEPA21 square-wave transfection system (BulldogBio), and the MicroPulserTM exponential decay electroporator (Bio-Rad). Additionally, the b) signature waveforms for the NEPA21 square wave transfection system of ‘poring’ and ‘transfer’ pulses for electroporation are shown. Note: The time scale in Fig. 1a is a zoomed-in version of the red-dashed box from Fig. 1b.Garcia, P.A., Ge, Z., Kelley, L.E., Holcomb, S.J., and Buie, C.R. (2017) High efficiency hydrodynamic bacterial electrotransformation. Lab Chip (17): 490-500.

Proper citation: Fatma Gomaa, Paulo A. Garcia, Jennifer Delaney, Peter R. Girgius, Cullen R. Buie, Virginia P. Edgcomb 2017. Microfluidic Transfection of Parabodo caudatus with plasmids. protocols.io https://dx.doi.org/10.17504/protocols.io.hmgb43w 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: Fe-EDTA Stock Solution for ESAW Media for Marine Phytoplankton

Proper citation: Ashley Humphrey 2016. Fe-EDTA Stock solution for ESAW Media for Marine Phytoplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.getbten Copy   

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Authors: Matthias Fischer
Group: VERVE Net, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: The concentration and purification of giant DNA viruses from bacteria-containing cultures can present problems because the particle size of giant viruses overlaps with that of smaller bacteria, hence filtration as the only separation method is not possible.In this protocol, a combination of centrifugation, tangential flow filtration, and density-gradient ultracentrifugation is used to concentrate and purify particles of the giant marine virus CroV that infects the heterotrophic flagellate Cafeteria roenbergensis.

Proper citation: Matthias Fischer 2018. Purification of Cafeteria roenbergensis virus particles. protocols.io https://dx.doi.org/10.17504/protocols.io.qz2dx8e Copy   

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Authors: Angela Piersanti
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Angela Piersanti 2017. Propidium Iodide Nucleic Acid Stain in Euplotes. protocols.io https://dx.doi.org/10.17504/protocols.io.hhrb356 Copy   

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