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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 dx.doi.org/10.17504/protocols.io.gg6btze Copy
Authors: Alle Lie
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology
Proper citation: Alle Lie 2016. The culturing of axenic Ochromonas sp. strain CCMP1393. protocols.io dx.doi.org/10.17504/protocols.io.ey8bfzw Copy
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 dx.doi.org/10.17504/protocols.io.ewjbfcn Copy
Authors: 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), CyanoHABs
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.Adapted from Nanodrop ND-1000 User Manual
Proper citation: Steven Wilhelm 2017. Nanodrop Spectrophotometer (ND-1000) for Nucleic Acid. protocols.io dx.doi.org/10.17504/protocols.io.id2ca8e Copy
Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Map and nucleotide sequence of the conjugation episome used to generate a TpSIl3p-EGFP expressing T. pseudonana strain reported in Faktorová D. et al.
Proper citation: Jernej Turnsek 2019. pTpPuc3_TpSil3p-eGFP. protocols.io dx.doi.org/10.17504/protocols.io.7ghhjt6 Copy
Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni 2019. Quick staining procedure of nuclei in Euplotes using DAPI. protocols.io dx.doi.org/10.17504/protocols.io.2ajgacn Copy
Authors: This protocol is modified 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
Summary: This protocol is modified from: Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct
detection and enumeration of the fungilike marine protists, the thraustochytrids. Limnol.
Oceanogr. 38(1): 182-187.
Proper citation: This protocol is modified from: Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct detection and enumeration of the, the thraustochytrids. Limnol. Oceanogr. 2017. Acriflavine Direct Detection Technique for Labyrinthulomycetes. protocols.io dx.doi.org/10.17504/protocols.io.hfib3ke Copy
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 dx.doi.org/10.17504/protocols.io.g5aby2e Copy
Authors: Paulo A. Garcia, Zhifei Ge, Laura Kelley, Steven Holcomb, Cullen R. Buie
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Synthetic biology holds great potential for addressing pressing challenges for mankind and our planet. One technical challenge in tapping into the full potential of synthetic biology is the low efficiency and low throughput of genetic transformation for many types of cells. In this paper, we discuss a novel microfluidic system for improving bacterial electrotransformation efficiency and throughput. Our microfluidic system is comprised of non-uniform constrictions in microchannels to facilitate high electric fields with relatively small applied voltages to induce electroporation. Additionally, the microfluidic device has regions of low electric field to assist in electrophoretic transport of nucleic acids into the cells. The device features hydro- dynamically controlled electric fields that allow cells to experience a time dependent electric field that is otherwise difficult to achieve using standard electronics. Results suggest that transformation efficiency can be increased by ∼4×, while throughput can increase by 100–1000× compared to traditional electroporation cuvettes. This work will enable high-throughput and high efficiency genetic transformation of microbes, fa- cilitating accelerated development of genetically engineered organisms.
Proper citation: Paulo A. Garcia, Zhifei Ge, Laura Kelley, Steven Holcomb, Cullen R. Buie 2017. High Efficiency Microfluidic Electrotransformation. protocols.io dx.doi.org/10.17504/protocols.io.hapb2dn Copy
Authors: RACHELE CESARONI, Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: RACHELE CESARONI, Rachele Cesaroni 2019. Dot blot to verify microinjection efficacy in Euplotes crassus. protocols.io dx.doi.org/10.17504/protocols.io.2aqgadw Copy
Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni 2019. Small-scale silencing experiment in vegetative Euplotes crassus (provisional). protocols.io dx.doi.org/10.17504/protocols.io.2asgaee Copy
Authors: RACHELE CESARONI
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: RACHELE CESARONI 2017. Euplotes crassus transformation using FuGene HD Transfection Reagent as vehicle. protocols.io dx.doi.org/10.17504/protocols.io.g43byyn Copy
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 dx.doi.org/10.17504/protocols.io.hinb4de Copy
Authors: Fatma Gomaa, Roberto Docampo, Zhuhong Li, Peter Girguis, Virginia Edgcomb
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Stable transfection of B. saltans has been achieved using plasmid for EF 1 alpha C terminal tagging.
Proper citation: Fatma Gomaa, Roberto Docampo, Zhuhong Li, Peter Girguis, Virginia Edgcomb 2019. Bodo saltans transfection with plasmid for tagging EF1alpha gene__IG BsTub. protocols.io dx.doi.org/10.17504/protocols.io.7fchjiw 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 corallochytrean Corallochytrium limacisporum, a unicellular relative to animals. protocols.io dx.doi.org/10.17504/protocols.io.gqmbvu6 Copy
Authors: Andrés Ramírez, Albane Ruaud, Fernan Federici, Peter von Dassow
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol has been optimized for electroporation of natural communities in coastal surface waters. As natural communities may vary in different places we recomend to use this protocol as a starting point and re-optimize according to the results. Guidelines are provided.
Proper citation: Andrés Ramírez, Albane Ruaud, Fernan Federici, Peter von Dassow 2017. Electroporation of natural communities in sea water. protocols.io dx.doi.org/10.17504/protocols.io.hpnb5me Copy
Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Daniel Richter, Nov 29, 2011based on RNAqueous May 29, 2008 protocol revision C, TURBO DNA-free June 9, 2009 protocol 1907M revision F
Proper citation: Daniel Richter 2017. RNAqueous with TURBO DNA-free and SUPERase-In. protocols.io dx.doi.org/10.17504/protocols.io.iqzcdx6 Copy
Authors: Lawrence A. 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 2017. Increased sensitivity of Euplotes crassus to selective agents using 0.3 M glucose-based culture conditions.. protocols.io dx.doi.org/10.17504/protocols.io.gwnbxde Copy
Authors: Erin Garza, Vincent Bielinski
Group: Protist Research to Optimize Tools in Genetics (PROT-G), JCVI West Protocols
Summary: A high-throughput method for measuring β-glucuronidase (GUS) activity in the diatom Phaeodactylum tricornutum. This protocol has been optimized for 250 μl volumes. For larger volumes see the following protocol dx.doi.org/10.17504/protocols.io.hefb3bn, which this protocol was based off of.
Proper citation: Erin Garza, Vincent Bielinski 2020. High-Throughput Beta-glucuronidase (GUS) assay for Phaeodactylum tricornutum. protocols.io dx.doi.org/10.17504/protocols.io.bbexijfn Copy
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 dx.doi.org/10.17504/protocols.io.sh3eb8n Copy
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