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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.x45fqy6 Copy   

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Authors: Binnypreet Kaur1, 2 , Drahomíra Faktorová1, 2, Priscila Peña-Diaz1 and Julius Lukeš1, 2
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes

Proper citation: Binnypreet Kaur1, 2 , Drahomíra Faktorová1, 2, Priscila Peña-Diaz1 and Julius Lukeš1, 2 2019. Immunoflourescent assay. protocols.io https://dx.doi.org/10.17504/protocols.io.hf4b3qw 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: Describes novel low conductivity media and multipulse electroporation protocol using a NEPA21 Type II pulse generator with settings optimized to balance transfection efficiency with cell viability for this marine diatom.

Proper citation: April Woods, G. Jason Smith 2017. Electroporation of Pseudo-nitzschia multiseries. protocols.io https://dx.doi.org/10.17504/protocols.io.hntb5en Copy   

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Authors: Anna Vanclová and Vladimír Hampl
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: The protocol describes our attempt to transform three marine protists by biolistic.

Proper citation: Anna Vanclová and Vladimír Hampl 2017. Biolistic transformation experiment on Eutreptiella gymnastica, Pyramimonas parkeae and Bigelowiella natans- 1250 psi gene gun. protocols.io https://dx.doi.org/10.17504/protocols.io.ibvcan6 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 through microinjection into the macronucleus. protocols.io https://dx.doi.org/10.17504/protocols.io.g3fbyjn 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 2017. Transient transfection of unicellular relative to animals Abeoforma whisleri with electroporation using Neon System. protocols.io https://dx.doi.org/10.17504/protocols.io.hmvb466 Copy   

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Authors: Oskar N. Johansson., Adrian K. Clarke
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: The following transformation protocol is designed for the insertion of linear DNA constructs into the nuclear genome of Skeletonema marinoi by non-homologous recombination.

Proper citation: Oskar N. Johansson., Adrian K. Clarke 2017. Transformation of Skeletonema marinoi using Multipulse Electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.g6nbzde 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. Culture of unicellular protists in marine broth. protocols.io https://dx.doi.org/10.17504/protocols.io.gg2btye Copy   

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Authors: Sarah Duponchel, Monica Berjon-Otero, Matthias Fischer
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Sarah Duponchel, Monica Berjon-Otero, Matthias Fischer 2019. Electroporation of the protist Cafeteria roenbergensis cells using the Neon Transfection System. protocols.io https://dx.doi.org/10.17504/protocols.io.qvhdw36 Copy   

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Authors: Steven Wilhelm
Group: VERVE Net, Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Adapted from: Van Etten, J. (n.d.). Titering of Chlorella Viruses. Retrieved from http://ncv.unl.edu/vanettenlab/

Proper citation: Steven Wilhelm 2016. Chlorella Virus Plaque Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.ge2btge Copy   

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Authors: Dan Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: March, 2012, based on RNAqueous May 29, 2008 protocol revision C, phenol/chloroform protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5438.full), ethanol precipitation protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5440.full)

Proper citation: Dan Richter 2017. RNAqueous with No DNAse, DNA Removal by Acid Phenol:Chloroform. protocols.io https://dx.doi.org/10.17504/protocols.io.itbcein Copy   

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

Proper citation: Mariana Rius 2017. Electroporation of Aurantiochytrium limacinum (ATCC MYA-1381). protocols.io https://dx.doi.org/10.17504/protocols.io.hg6b3ze Copy   

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Authors: Tara Stuecker
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Lewis Lab
Summary: Protocol for obtaining high quality RNA from yeast using acid-phenol lysis at 65°C. Modifed from DOI: 10.1016/S0076-6879(02)50976-9 to use 10-15 ml of yeast culture. Expected yield of 100-200 μg of RNA. For qPCR or RNA-seq, we recommend DNase-treatment and further purification (e.g. Zymo RNA Purification Kit (R1057)).

Proper citation: Tara Stuecker 2017. RNA Isolation from Yeast. protocols.io https://dx.doi.org/10.17504/protocols.io.inwcdfe Copy   

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Authors: Ana Del Arco, Noemi Woltermann, Lutz Becks
Group: VERVE Net, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Chemostats and other flow through culture systems are a powerful tool for the study of microbial and plankton communities in experimental ecology and evolutionary studies. Commercially available chemostat systems allow the control of a large number of parameters (e.g., ph, pressure, CO2 concentration) but are often expensive and offer a high level of control that is often not needed for many experimental studies. Non-commercial chemostats are more cost efficient, easily set up and flexible in volumes used. Different from semi-continuous culture systems, the flow through conditions of chemostats allow a constant inflow of resources from a reservoir (medium bottle) and outflow of unused nutrients, waste products and organisms, which are all collected in a waste bottle. Nutrient levels in the reservoir and the flow rate of the chemostat system (often referred to as dilution rate and presented as the fraction of the volume of the chemostat that is replaced per day) determines population growth rates and dynamics (for the theory behind chemostats see Smith & Waltman, 1995 and Weitz, 2015). Chemostats have been used in a number of studies and with different organisms and combinations of organisms. For example, Boer et al. (2010) studied how growth-limiting intracellular metabolites control yeast growth under diverse nutrient availability (Saccharomyces cerevisiae growing under five different nutrient supply of nitrogen: carbon: phosphorus). Becks et al. (2005) used chemostat systems to show how changes in the flow rate of a chemostat system influences the population dynamics of a three species microbial system. Frickel et al. (2016) used chemostats to investigate eco- evolutionary dynamics in a coevolving host-virus system (algae Chlorella variabilis and Chlorovirus strain PBCV-1) for 90 days.We present here an instruction for a cost efficient and flexible chemostat systems. These chemostats are composed of four main parts: a syringe unit, a glass bottle (i.e. the chemostat), a medium bottle and a waste bottle, all connected by tubing. A peristaltic pump and a low overpressure in the system allow the flow of medium from the reservoir to the chemostat bottle and of unused nutrients, waste products and organisms to the waste bottle. Chemostats are put on stirring plates to create a homogenosus environment within.Becks, L., Hilker, F.M., Malchow, H., Jürgens, K., Arndt, H., 2005. Experimental demonstration of chaos in a microbial food web. Nature 435, 1226–1229. https://doi.org/10.1038/nature03627Boer, V.M., Crutchfield, C.A., Bradley, P.H., Botstein, D., Rabinowitz, J.D., 2010. Growth-limiting Intracellular Metabolites in Yeast Growing under Diverse Nutrient Limitations 21, 198–211. https://doi.org/10.1091/mbc.E09Frickel, J., Sieber, M., Becks, L., 2016. Eco-evolutionary dynamics in a coevolving host – virus system. Ecol. Lett. 19, 450–459. https://doi.org/10.1111/ele.12580Smith, H., Waltman, P., 1995.The Theory of the Chemostat: Dynamics of Microbial Competition (Cambridge Studies in Mathematical Biology). Cambridge: Cambridge University Press. doi:10.1017/CBO9780511530043Weitz, J.S. 2015. Quantitative Viral Ecology. Dynamics of viruses and their microbial host. Princeton University Press. ISBN: 9780691161549 .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Ana Del Arco, Noemi Woltermann, Lutz Becks 2020. Building up chemostats for experimental eco-evolutionary studies. protocols.io https://dx.doi.org/10.17504/protocols.io.tkxekxn 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. Staining protocol for unicellular protists: Mitotracker and Dapi. protocols.io https://dx.doi.org/10.17504/protocols.io.gg5bty6 Copy   

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Authors: Binnypreet Kaur, Drahomira Faktorova, Julius Lukes Group
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes
Summary: p57-V5+NeoR plasmid contains V5-tagged aminoglycoside 3'-phosphotransferase gene (APT; conferring resistance to neomycin) flanked by partial regulatory sequences derived from the hexokinase gene of the kinetoplastid Blastocrithidia (strain p57).Plasmid GenBank accession number = MN047315

Proper citation: Binnypreet Kaur, Drahomira Faktorova, Julius Lukes Group 2019. Transformation of Diplonema papillatum using V5+NeoR construct. protocols.io https://dx.doi.org/10.17504/protocols.io.4digs4e Copy   

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Authors: Joshua Rest, Jackie Collier
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: To develop broadly useful methods for the genetic manipulation of Labyrinthulomycetes, it is essential to understand the similarities and differences in regulation of gene expression among them. Toward this end we have used FIMO from the MEME suite (http://meme-suite.org/doc/fimo.html) to identify motifs similar to yeast transcription factor binding sites in each of the three available genome sequences: Aplanochytrium kerguelense PBS07, Schizochytrium aggregatum ATCC 28209, and Aurantiochytrium limacinum ATCC MYA-1381. We then make logograms to illustrate yeast-like GAL4 binding sites in each of the three genomes. 

Proper citation: Joshua Rest, Jackie Collier 2017. Code: Discovery of regulatory motifs in Labyrinthulomycete genomes. protocols.io https://dx.doi.org/10.17504/protocols.io.hg8b3zw Copy   

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

Proper citation: Brittany Sprecher, Lu Wang, Huan Zhang, and Senjie Lin 2017. Co-Incubation protocol for transforming heterotrophic dinoflagellates (e.g. Oxyrrhis marina). protocols.io https://dx.doi.org/10.17504/protocols.io.hmzb476 Copy   

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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 recommend 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 2018. Electroporation of natural communities in sea water. protocols.io https://dx.doi.org/10.17504/protocols.io.h3nb8me Copy   

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Authors: Jackie Collier
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: This method adapted from instructions provided by Daiske Honda, Konan University. 

Proper citation: Jackie Collier 2018. Cryopreservation of labyrinthulomycetes in trehalose. protocols.io https://dx.doi.org/10.17504/protocols.io.pgtdjwn Copy   

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