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Authors: Philip D. Weyman
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: An easy to follow template for adding Golden Gate adapters to PCR primers. 

Proper citation: Philip D. Weyman 2017. Golden Gate Primer Design. protocols.io https://dx.doi.org/10.17504/protocols.io.heyb3fw 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 through microinjection into the macronucleus. protocols.io https://dx.doi.org/10.17504/protocols.io.hi7b4hn Copy   

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Authors: Jeric Harper
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab

Proper citation: Jeric Harper 2016. Diatom Transformation via Bacterial Conjugation. protocols.io https://dx.doi.org/10.17504/protocols.io.fp7bmrn Copy   

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Authors: Adrian Clarke
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Adrian Clarke 2017. Skeletonema DNA extraction by Plant DNAzol. protocols.io https://dx.doi.org/10.17504/protocols.io.g6bbzan Copy   

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Authors: Fatma Gomaa, Zhuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Bodo saltans kill curve protocol using G418 (Gentamicin)Gentamycin exhibits toxicity toward both eukaryotic and prokaryotic cells by disrupting ribosome function, thereby blocking the elongation step in protein synthesis.G418 is most commonly used as a selection agent for eukaryotic cells genetically engineered to express a neomycin resistance gene (NeoR), which is encoded by either transposon Tn601 (903) or Tn5.The resistant cells grow in medium containing G418, and may be used to establish stably transfected cell lines as all the non-resistant cells die due to G418 toxicity, typically within 6 – 14 days.To establish kill curve assay for saltans various concentrations of G418 were tested. The concentration range from 1 to 50 μg/mL for selection of eukaryotic, and then higher concentrations can be used for maintaining stable transfected cell line.Kill Curve Assay Harvest the B. saltans cells from a culture that is at peak density (1 – 3.0 x 105) by centrifugation at 800 x g for 5 min., discard the supernatant (medium). Replace the growth medium with fresh medium containing 0 – 50 μg/mL. For each concentration, test in triplicate. Using 6 wells plates, replace the medium in the wells every 3 – 4 days using fresh medium with the appropriate G418 concentration. Perform a daily visual inspection for evidence of toxicity, also cell count using hemocytometer. Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection. 5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection. G418 Sensitivity for B. saltans Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.Replace the growth medium with fresh medium containing 0 – 50 μg/mL. For each concentration, test in triplicate. Using 6 wells plates, replace the medium in the wells every 3 – 4 days using fresh medium with the appropriate G418 concentration. Perform a daily visual inspection for evidence of toxicity, also cell count using hemocytometer. Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection. 5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection. G418 Sensitivity for B. saltans Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.Using 6 wells plates, replace the medium in the wells every 3 – 4 days using fresh medium with the appropriate G418 concentration. Perform a daily visual inspection for evidence of toxicity, also cell count using hemocytometer. Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection. 5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection. G418 Sensitivity for B. saltans Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection. 5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection. G418 Sensitivity for B. saltans Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection.G418 Sensitivity for B. saltans AB1G418 (μg/ml) Cells survival (days) 250 2 320 2 415 3 510 4 65 6 73 8 82 12 91 >16 100 >16 A1G418 (μg/ml) A1Cell survival (days) Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.

Proper citation: Fatma Gomaa, Zhuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb 2018. G418 Kill curve protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.sh4eb8w Copy   

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

Proper citation: Pia Elustondo, Claudio Slamovits 2018. Transfection of O. marina using particle bombardment. protocols.io https://dx.doi.org/10.17504/protocols.io.ui7euhn Copy   

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

Proper citation: Francesca Papi, Angela Piersanti 2019. RNAi by feeding in Euplotes focardii (povisional). protocols.io https://dx.doi.org/10.17504/protocols.io.zk8f4zw Copy   

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Authors: Binnypreet Kaur1, 2, Drahomíra Faktorová1, 2 and Julius Lukeš1, 2, 3
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes
Summary: The process of NHEJ is completed when the DNA ligase complex ligates the broken ends of the DNA. Ku70-Ku80 are the DNA-dependent protein kinase heterodimeric regulatory factor that forms a link between two broken DNA ends and structurally support, align and protect them from further degradation. W7 inhibitor (W7(N-(6-aminohexyl)-5-chloro1-naphthalenesulfonamide) inhibits the cofactor of inositol hexakisphophate which effects the regulatory factor of Ku heterodimers.

Proper citation: Binnypreet Kaur1, 2, Drahomíra Faktorová1, 2 and Julius Lukeš1, 2, 3 2019. Transfectionof constructs in diplonemids to block NHEJ pathway using the inhibitors of KU70/80 proteins.. protocols.io https://dx.doi.org/10.17504/protocols.io.x35fqq6 Copy   

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Authors: This protocol was modified by Sabrina Geraci-Yee and is from: Pozarowski P, Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol Bio. 281:301-311.
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: Sabrina Geraci-Yee modified this protocol from: Pozarowski P., Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol. Bio. 281:301-311.

Proper citation: This protocol was modified by Sabrina Geraci-Yee and is from: Pozarowski P, Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol Bio. 281:301-311. 2017. Staining Labyrinthulomycetes with Propidium Iodide. protocols.io https://dx.doi.org/10.17504/protocols.io.hfjb3kn Copy   

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Authors: Erin Garza, Vincent Bielinski
Group: Protist Research to Optimize Tools in Genetics (PROT-G), JCVI West Protocols
Summary: This method can be used to increase the efficiency of Gibson Assemblies containing many pieces and/or difficult to assemble DNA fragments.

Proper citation: Erin Garza, Vincent Bielinski 2020. Nested Gibson Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.bbikikcw Copy   

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Authors: Jian Guo, Alexandra Worden, Grant Hartzog, and Manuel Ares
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: We have available several plasmids designed for expression of Cas9, guide RNA, chloramphenicol acetyl-transferase, GFP, and beta-lactamase in Micromonas CCMP1545. For protein expression we used the promotor and 3' end elements from the endogenous RPS9 gene, and codon optimized the coding region.  For expression of guide RNAs we used the Micromonas U6 snRNA promoter.  DNA is available by contacting M. Ares .We have constructed and sequence verified 9 plasmids which we would like to make available to others attempting to detect transformation of DNA into Micromonas. Using the CCMP1545 genome as a source for the U6 promoter sequence, and for the promoter and 3' UTR sequences of ribosomal protein RPS9, we built the following plasmids:CRISPR/Cas9 plasmids for Micromonas1. Mp U6 promoter driving Bae cassette for guide RNA expression in pUC132. Mp RPS9-Cas9SV40-RPS9 in pUC133. Both Mp U6 promoter driving Bae cassette for guide RNA expression and Mp RPS9-Cas9SV40-RPS9 in pUC13These first three plasmids were anticipated to enable stable incorporation of transgenes at specific genomic locations. BaeI is a type IIS restriction enzyme that leaves noncompatible sticky ends. In the context of the gRNA cassette, a pair of 24 nt oligos designed to have the sticky ends compatible with BaeI cleaved plasmid are annealed and cloned into the plasmid, replacing the BaeI cassette while adding the 20 nt target complementary sequence of the desired guide RNA.Selectable/Detectable Marker genes4. Mp RPS9-codon optimized GFPsv40-RPS9 in pUC135. Mp RPS9-codon optimized chloramphenicol acetyltransferase-RPS9 in pUC13 6. Mp RPS9-codon optimized beta-lactamase-RPS9 in pUC13 These three plasmids have the indicated coding regions codon optimized for Micromonas flanked by RPS9 promoter and 3' end sequences. In the case of GFP, a nuclear localization signal from SV40 has been added to the C-terminus. Plasmids for Agrobacterium-mediated gene transfer 7. Mp RPS9-codon optimized GFPsv40-RPS9 in pOSCAR 8. Mp RPS9-codon optimized chloramphenicol acetyltransferase-RPS9 in pOSCAR 9. Mp RPS9-codon optimized beta-lactamase-RPS9 in pOSCAR

Proper citation: Jian Guo, Alexandra Worden, Grant Hartzog, and Manuel Ares 2018. Plasmid DNAs designed for expression in Micromonas CCMP1545. protocols.io https://dx.doi.org/10.17504/protocols.io.i9wch7e Copy   

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Authors: Daniel Vaulot
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Concentrate phytoplankton samples about 100-fold typically from 5L down to 20 mL. Takes about 1 hour per sample. Samples can be used for flow cytometry sorting or for cultures. Enrichment by TFF usually keep growing for a longer time than unconcentrate samples.ReferenceMarie, D., Shi, X.L., Rigaut-Jalabert, F. & Vaulot, D. (2010). Use of flow cytometric sorting to better assess the diversity of small photosynthetic eukaryotes in the English Channel. FEMS Microbiology Ecology. 72. p.pp. 165–178.

Proper citation: Daniel Vaulot 2016. Tangential flow filtration (TFF) concentration of phytoplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.gpybvpw Copy   

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Authors: Sarah Hu
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology, SCOPE
Summary: This protocol describes steps and considerations for working in the field to collect samples to be used for molecular work targetting marine (or freshwater) microbial eukaryotes. See downstream protocols for DNA/RNA extraction and library prep (pending) for 18S rRNA gene tag sequencing.Many steps in this protocol can be modified to fit a users' specific needs or environment type. Here, we strive to make recommendations that consider the delicate nature of single-celled eukaryotes and collect samples in the field as cleanly as possible. We use this approach for RNA and DNA-based sequencing - both for tag sequencing and metatranscriptomics.This protocol is based off of the field work we conduct at the San Pedro Ocean Time-series (SPOT) station. We go out to the SPOT station once a month. Once we arrive at the SPOT station, we try to sample at the same time of day each month. Timelapse video for sampling SPOT: https://youtu.be/1US3h_qD00wMaterials required:(1) 20 L carboys (volume amount flexible)(2) Covers for carboys (we use pizza bags, black trash bags, or coolers)(3) Ice packs(4) Tubing for Niskin (5) Vacuum pump filtration system (see time-lapse video)    - trap flask    - pump    - extra tubing & rubber/silicon stopper (to fit filter towers)(6) Filter towers that fit in vacuum pump filtration system(7) In-line filters, 47 mm(8) Nitex mesh, cut for 47 mm - 200 um & 80 um(9) RNase-spray(10) Absorbent pads (for reducing water spillage & covering countertops)(11) Liquid nitrogen - make sure to check regulations on transporting liquid nitrogen and storing it on-site/shipboard(12) 15 mL falcon tubes - RNase/DNase-free(13) RNA later or Lysis buffer (e.g. RLT buffer from this protocol)(14) Filters for collecting material, we use 0.7um GF/F filters**Our reasoning to use this filter size is that the 0.7um nominal pore size will collect the protistan fraction of the community, but it is a woven filter style, so more water can be filtered through without clogging.

Proper citation: Sarah Hu 2017. Sample collection from the field for downstream molecular analysis - microbial eukaryote-focused. protocols.io https://dx.doi.org/10.17504/protocols.io.hisb4ee Copy   

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Authors: Francois Yves Bouget, Francois-Yves Bouget
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes the preparation of cells and introduction of DNA into the cells by electroporation.  For selection of stable transformants or measure of transient gene expression see related protocols.

Proper citation: Francois Yves Bouget, Francois-Yves Bouget 2019. Transient transformation of Ostreococcus species (OTTH595, RCC809 and RCC802) and Bathycoccus. protocols.io https://dx.doi.org/10.17504/protocols.io.83uhynw Copy   

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Authors: Fatma Gomaa, Zhou Zhuha, Roberto Docampo, Virginia Edgcomb, Peter Girguis
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Fatma Gomaa, Zhou Zhuha, Roberto Docampo, Virginia Edgcomb, Peter Girguis 2018. Bodo saltans Cassette for tagging EF1alpha gene__IG BsTub. protocols.io https://dx.doi.org/10.17504/protocols.io.s5jeg4n Copy   

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Authors: Francois-Yves bouget
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol  describes the selection and growth of stable transformants in semi solid agarose medium. Developped initially for Ostreococcus tauri, it also works for Ostreococcus lucimarinus and Bathycoccus.

Proper citation: Francois-Yves bouget 2019. Selection of stable transformants in Ostreococcus tauri, Ostreococcus lucimarinus and Bathycoccus prasinos. protocols.io https://dx.doi.org/10.17504/protocols.io.zj2f4qe Copy   

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Authors: Dan Needleman
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Dan Needleman 2017. Protocol for drug sensitivity assay with S. robusta. protocols.io https://dx.doi.org/10.17504/protocols.io.g9ebz3e Copy   

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Authors: Lisa Sudek, Alexandra Worden, Manny Ares
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Worden Lab
Summary: This protocol describes the method used to test various amounts (0, 0.3, 3, 10, 20 and 30 µg) of reporter (H3proMpo-LUC) DNA. The plasmid H3proMpo-LUC carries a codon optimized Nanoluc coding region, whose expression is driven by the histone H3 promoter and 3' mRNA end formation and termination sequences. Lumen per total number of cells (including transfected and non-transfected cells within a culture) is used as a proxy for relative transfection efficiencies. It is determined in each culture based on luminescence measurements performed 72 hrs after transfection using the Nano-Glo® Luciferase Assay System.

Proper citation: Lisa Sudek, Alexandra Worden, Manny Ares 2019. Reporter titrations for transfection of Micromonas commoda CCMP2709. protocols.io https://dx.doi.org/10.17504/protocols.io.57rg9m6 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.2amgac6 Copy   

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Authors: G Jason Smith, April Woods
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Environmental Biotechnology Lab
Summary: This is a modified version of  'Conjugation of Thalassiosira pseudonana', publishedby J. Turnsek dx.doi.org/10.17504/protocols.io.f55bq86 .The protocol was modified to enhance viability in the diatom Pseudo-nitzschia multiseries. This version was used to transform Pseudo-nitzschia multiseries isolate 15091C3 with an episomal plasmid derived from pPtPUC3 to express a egfp gene under control of the P. multiseries actin promoter and termination domains. PLASMID INFOpPmAGFPC10 EPI : A P. multiseries derived expression cassette under control of P. multiseries ca 1000 bp of actin promoter and termination domains. Pm actPs::eGFP::actTs

Proper citation: G Jason Smith, April Woods 2019. Modified Bacterial Conjugation Protocol For Pseudo-nitzschia multiseries. protocols.io https://dx.doi.org/10.17504/protocols.io.7vhhn36 Copy   

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