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Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Rachele Cesaroni 2017. Culturing Euplotes crassus to high densities using E. coli as the only food source. protocols.io https://dx.doi.org/10.17504/protocols.io.g46byze Copy   

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Authors: Elena Casacuberta, Cristina Aresté, Omaya Dudin , Elena Casacuberta
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Summary: Testing electroporation on Spaheroforma artica with two different electroporatorsNeon (invitrogen)Lonza (nucleofector)

Proper citation: Elena Casacuberta, Cristina Aresté, Omaya Dudin , Elena Casacuberta 2019. Unsucessful atemps on transfromation of the Icthyosporean Sphaeroforma artica. protocols.io https://dx.doi.org/10.17504/protocols.io.z6ef9be Copy   

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Authors: Fatma Gomaa, Zuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: B. saltans cells were electroporated using a square-wave electroporator (Nepa21, Bulldog Bio, Inc.) using one poring pulse of 200 volts with a pulse duration of 25 ms and five transfer pulses of 60 volts with a pulse duration of 99 ms, with plasmid targeting the 18S region (18S-GFP). A schematic representation of the plasmid, the target locus and the expected site of integration into the B. saltans genome is shown in Figure 1. Electroporated cells were selected with 1 µg/ml of G418, added 24 hours after electroporation. Cells were washed and subcultured into fresh selection medium every 3-4 days. G418 resistant cells started to emerge 7-9 days post-electroporation. Cells were processed for genotyping analysis to confirm plasmid integration 3 weeks post-electroporation. DNA was extracted from pools of transfected and wild cells using the Qiagen DNeasy Blood & Tissue kit. PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C. Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Electroporated cells were selected with 1 µg/ml of G418, added 24 hours after electroporation. Cells were washed and subcultured into fresh selection medium every 3-4 days. G418 resistant cells started to emerge 7-9 days post-electroporation. Cells were processed for genotyping analysis to confirm plasmid integration 3 weeks post-electroporation. DNA was extracted from pools of transfected and wild cells using the Qiagen DNeasy Blood & Tissue kit. PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C. Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Cells were processed for genotyping analysis to confirm plasmid integration 3 weeks post-electroporation. DNA was extracted from pools of transfected and wild cells using the Qiagen DNeasy Blood & Tissue kit. PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C. Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C. Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Figure 1: Schematic representation of the (A); the 18S- GFP plasmid; (B) Ribosomal operon in B. saltans genome (C); and the expected site of plasmid integration in B. saltans genome through homologous regions 1 and 2 (HR1, HR2).Figure 2: Agarose gel electrophoresis image of the amplified PCR products for B. saltans cells transfected with 18S-GFP Cassette (S1 and S2) and the wild type cells (C). The primers sets and the expected product sizes are mentioned above in the text.Figure 3: B. saltans cells transfected with 18S-GFP tagging cassette. On the left: a light microscopy image showing the B. saltans cells (around 4 cells at different level). On the right: GFP fluorescent signal detected in the transfected cell. Scale bar 10 μm.

Proper citation: Fatma Gomaa, Zuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb 2018. Generating Stable Transfection in Bodo saltans. protocols.io https://dx.doi.org/10.17504/protocols.io.sh3eb8n Copy   

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Authors: Oskar Johansson, Adrian Clarke
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Mapping of transformed DNA within mutated diatoms. A stepwise specific PCR reaction that allow for identification of genome location of transformed fragments such as antibiotic resistance genes or GFP constructs. Degenerate primer 1: 5' - GCCGGACTTAGCGAGCA TGTGTAGTACCTTGCCG ATCCTNASATANSATANT TC - 3'Degenerate primer 2: 5' - GCCGGACTTAGCGAGCA TGTGTAGTACCTTGCCG ATCCCNTSABGNACYTN CTG - 3'Degenerate primer 3: 5' - GCCGGACTTAGCGAGCA TGTGTAGTACCTTGCCG ATCCNGACGARWGANA WGAC - 3'Degenerate primer 4: 5' - GCCGGACTTAGCGAGCA TGTGTAGTACCTTGCCG ATCCTAHATGDAGKACN TAC - 3'Specific primer 1: 5' - GCCGGACTTAGCGAGCA TGTGTAG - 3'Specific primer 2: 5' - CATGTGTAGTACCTTGC CGATCC - 3'Internal primers must be designed with regard to each construct that are to be mapped. They should not overlap but instead cover consecutive stretches of known DNA sequence of the transformed construct.

Proper citation: Oskar Johansson, Adrian Clarke 2019. Genomic mapping of transformed DNA fragments. protocols.io https://dx.doi.org/10.17504/protocols.io.yq2fvye 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 D: Preparation of BSA for quenching pretreatment. protocols.io https://dx.doi.org/10.17504/protocols.io.h8wb9xe 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 G: Preparation of reporter DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.h8yb9xw Copy   

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

Proper citation: Angela Piersanti 2019. Transfection by Electroporation in Euplotes crassus. protocols.io https://dx.doi.org/10.17504/protocols.io.2a9gah6 Copy   

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Authors: G Jason Smith
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab
Summary: This is a modification of the origianl Chelex 100 extraction described by Walsh, Metzger and Higuchi (1991 BioTechniques 10(4):506). Adding PVPP facilitates working with environmental samples as well as pure cultures hi in phenolics or other contaminants.

Proper citation: G Jason Smith 2017. Simple DNA Extraction for Phytoplankton Using Chelex 100. protocols.io https://dx.doi.org/10.17504/protocols.io.h58b89w Copy   

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Authors: Alle Lie
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology

Proper citation: Alle Lie 2016. Algal culture harvest and RNA extraction for RNA-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.ewjbfcn Copy   

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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. Super simple In vivo Hoechst staining of unicellular protists. protocols.io https://dx.doi.org/10.17504/protocols.io.gg6btze Copy   

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Authors: Senjie Lin, Huan Zhang, Brittany Sprecher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Senjie Lin, Huan Zhang, Brittany Sprecher 2018. Nucleofector Protocol for Dinoflagellates using Lonza’s 4D-Nucleofector X Unit. protocols.io https://dx.doi.org/10.17504/protocols.io.qm8du9w Copy   

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Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: RACHELE CESARONI 2017. Euplotes crassus transfection using FuGene HD Transfection Reagent as vehicle (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.hjdb4i6 Copy   

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Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: RACHELE CESARONI 2017. Euplotes crassus transformation using Lipofectamine 2000 as vehicle. protocols.io https://dx.doi.org/10.17504/protocols.io.g5aby2e Copy   

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Authors: Sarah Duponchel, Monica Berjon-Otero, Matthias Fischer
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Heterotrophic flagellates require co-culture with bacteria that serve as a food source. This protocol explains how to reduce the bacterial background of cultures of Cafeteria roenbergensis.

Proper citation: Sarah Duponchel, Monica Berjon-Otero, Matthias Fischer 2019. Density-based removal of bacteria from a culture of the marine heterotrophic flagellate Cafeteria roenbergensis . protocols.io https://dx.doi.org/10.17504/protocols.io.qiiduce Copy   

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Authors: Zhu-Hong Li, Roberto Docampo
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Using FACS to sort fluorescent Bodo cellsIntroduction: Fluorescence-activated cell sorting (FACS) is a powerful method to isolate cells expressing fluorescent proteins. It is a routine technique in our lab to enrich, and subclone cells that express exogenously introduced fluorescent reporter proteins. We developed this protocol to isolate Bodo saltans that express a fluorescent protein.Unlike our previous work to isolate T. gondii, or T cruzi, there are two major obstacles to the application of this technology to B. saltans. The first obstacle is that B. saltans needs bacteria as food in the culture so there are multiple organisms in the medium. We have to determine which cells are B. saltans, and which are feeder bacteria. The second problem is that the osmolarity of the solution used for standard FACS is not appropriate for B. saltans. B. saltans lives in low osmolarity solutions. We needed to determine a proper solution in which B. saltans can survive, and if it works with the FACS system.Step 1. Determine a FACS solution appropriate for B. saltans.The solution that runs in a flow cytometer is called sheath fluid. Our FACS facility has its standard sheath fluid. In order to choose a proper sheath fluid for B. saltans we needed to make sure that: 1) the cells can survive in this sheath fluid for a significant time, 2) the sheath fluid can still support FACS since there is a requirement for the presence of ions for FACS to work. We tested serial dilutions of the standard sheath fluid. We found that B. saltans dies almost instantly in standard sheath fluid, but can live for at least 20 minutes in 50% sheath fluid. We also determined that the minimal sheath fluid needed for FACS is 40% of the standard sheath fluid. So we decided to use this diluted sheath fluid.Step 2. Determine which is the population of B. saltans. Wild type cultures are passed through a 10 nm Nylon filter to remove big residues in the culture medium, and filtered cells are collected by centrifugation at 1,200 x g for 5 minutes. The cells are further washed three times by centrifugation at 1,200 x g for 5 minutes, and twice at 1,200 x g for 5 minutes. The washing step is to get rid of most bacteria in the culture medium.After the last wash, the cells are resuspended in culture medium. We sort the cells by size and shape. There are usually two major distinct populations. We sort each of the populations into a collection tube. After the sorting, the cells are checked by microscopy. We confirm that the population 1 is B. saltans, and that the cells can survive the FACS sorting when we use 40% of standard sheath fluid as our sorting solution.Fig 1: R1 is B. saltans population. We previously used MoFlo XDP Sorter From Beckman Coulter, now we are upgrading to Beckman Coulter MoFlo Astrios EQ version. In the SSC Vs FSC dot plot, R1 represents B. saltans cells.Step 3. Sorting of B. saltans transfected with a reporter gene.1, 24 hours after electroporation, B. saltans (wild type and transfected) are filtered through a 10 nm Nylon membrane. The cells are collected by centrifugation at 1,000 x g for 5 minutes.2, Cells are washed twice with autoclaved distilled water.3, After the final wash, cells are resuspended in ~1-1.5 ml medium. We also prepare a collection tube with 2 ml fresh culture medium.4, After the sorter has been set up, we first run the wild type control cells to set up the parameters for sorting. In the SSC Vc FSC dot plot, the B. saltans population is chosen. Then in the fluorescence plot, we use DsRed Vs GFP dot plot to set the gate. There are no cells in the gated area for wild type cells.5, After the parameters are set, we run the transfected B. saltans. A small population of fluorescent cells should appear in the fluorescence plot. This is the population we want to sort into a collection tube.6, After the sorting is done, cells are transferred to the collection tube and then to a T25 flask, adding 4 ml culture medium and a small colony of bacteria taken from a plate.7, Cells are cultured for further analysis.Fig. 2. Representative sorting of B. saltans

Proper citation: Zhu-Hong Li, Roberto Docampo 2018. Using FACS to sort fluorescent Bodo cells. protocols.io https://dx.doi.org/10.17504/protocols.io.skpecvn Copy   

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

Proper citation: Angela Piersanti 2017. Euplotes focardii RNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.hhwb37e Copy   

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Authors: Lawrence A. Klobutcher, Larry Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: The electroporation procedure and conditions described allow for the entry of DAPI into the cell, resulting in nuclear staining. We have not yet been successful in using these conditions for transformation of Euplotes crassus, but only a couple of constructs have been tried to date.

Proper citation: Lawrence A. Klobutcher, Larry Klobutcher 2019. Electroporation of Euplotes crassus: conditions that allow for the uptake of DAPI.. protocols.io https://dx.doi.org/10.17504/protocols.io.2angade Copy   

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Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Rachele Cesaroni 2017. Culturing Euplotes crassus to high densities using E. coli as the only food source. protocols.io https://dx.doi.org/10.17504/protocols.io.hjbb4in Copy   

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Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: RACHELE CESARONI 2017. Dot blot to verify microinjection efficacy in Euplotes crassus. protocols.io https://dx.doi.org/10.17504/protocols.io.hi6b4he 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: RS-1 (RAD51-stimulatory compound 1) is a stimulator of homologous recombination (HR) protein RAD51 .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Binnypreet Kaur, Drahomíra Faktorová , Julius Lukeš 2019. Transfection of constructs in diplonemids to enhance the HR pathway, using RS-1 (3-(benzylamino) sulfonyl)-4-bromo-N-(4-bromophenyl) benzamide).. protocols.io https://dx.doi.org/10.17504/protocols.io.x34fqqw Copy   

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