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Authors: Jernej Turnsek
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Details of the conjugation episome used to generate the TpSil3p-EGFP expressing Thalassiosira pseudonana strain as reported in Faktorová et al (2019). The file contains:1/ a vector scheme summarizing all of its elements2/ expression cassette elements and their nucleotide sequences3/ fusion protein amino acid sequence4/ full vector DNA sequence with expression cassette elements annotated
Proper citation: Jernej Turnsek 2019. Faktorová_et_al_2019_pTpPuc3_TpSil3p-eGFP. protocols.io dx.doi.org/10.17504/protocols.io.8bdhsi6 Copy
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. Transfection of Diplonemids using AMAXA Nucleofactor apparatus. protocols.io dx.doi.org/10.17504/protocols.io.hf2b3qe Copy
Authors: Dr. Steven Wilhelm, Samantha Coy
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), Samantha R Coy's Protocols
Summary: Adapted from: Van Etten, J. (n.d.). Titering of Chlorella Viruses. Retrieved from http://ncv.unl.edu/vanettenlab/
Contact Dr. Steven Wilhelm ([email protected]) or Samantha Coy ([email protected]) for additional information regarding this protocol.
Proper citation: Dr. Steven Wilhelm, Samantha Coy 2017. Chlorella Virus Plaque Assay. protocols.io dx.doi.org/10.17504/protocols.io.hgqb3vw Copy
Authors: Dr. 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)
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.Modified from Wilhelm SW and CG Trick. 1995. Physiological profiles of Synechococcus (Cyanophyceae) in iron-limiting continuous cultures. Journal of Phycology, 31:79-85.
Proper citation: Dr. Steven Wilhelm 2017. A+ Media for Marine Phytoplankton. protocols.io dx.doi.org/10.17504/protocols.io.ibncame Copy
Authors: Nicholas Irwin, Elisabeth Hehenberger, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Here we describe a protocol for transforming Oxyrrhis marina with FITC-labelled dextran (a DNA analog) by electroporation. This protocol is reproducible, and results in a transformation efficiency of around 50%.We have confirmed the viability of transformed cells by allowing them to recover in the dark (and thus maintain fluorescence intensity) and then by observing fluorescent cells swimming normally, days after transformation (see the video here). As of yet we have been unable to demonstrate the actual uptake of plasmid DNA. We have tried using rhodamine labelled plasmids to demonstrate this but the results were inconclusive due to issues with cellular autofluorescence. We have also tried this method with a variety of expression plasmids encoding different promoters. For example, we tried the MOE promoter from Perkinsus marinus (a closely related species) and the generic eukaryotic CMV promoter. However no GFP fluorescence was detected in these samples. We have also tried transforming in vitro synthesized GFP encoding mRNA using this protocol. As of yet, no GFP production has been detected, but this may reflect non-cannonical translational systems in dinoflagellates as the same mRNA could be translated in starfish embryos.Although the development of expression systems in dinoflagellates remains a challenge, our results suggest that O. marina may be amenable to genetic transformation.
Proper citation: Nicholas Irwin, Elisabeth Hehenberger, Patrick Keeling 2016. Electroporation transformation of FITC-dextran into Oxyrrhis marina - an early branching dinoflagellate.. protocols.io dx.doi.org/10.17504/protocols.io.ginbude Copy
Authors: Aleksandra Kozyczkowska
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Proper citation: Aleksandra Kozyczkowska 2018. Transient tranfection of unicellular relative of animals, Creolimax fragrantissima, using Lonza Nucleofector. protocols.io dx.doi.org/10.17504/protocols.io.r65d9g6 Copy
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: Trace metals solution to be used in ESAW Media for Marine Phytoplankton
Proper citation: Ashley Humphrey 2016. Trace Metal Solution for ESAW Media. protocols.io dx.doi.org/10.17504/protocols.io.gdabs2e Copy
Authors: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Caecitellus sp. is a small phagotroph belonging to the biocosoecid order of the stramenopiles. Caecitellus is found across global oceans but it's role in marine ecosystems, as with many heterotrophic flagellates, is unclear. We isolated a strain of Caecitellus (small subunit rRNA gene was 99.53% identical to Caecitellus parvulus and Caecitellus paraparvulus) from a contaminated culture of Oxyrrhis marina and assessed its amenability to transformation. Fluorescein isothiocyanate conjugated to dextran was successfully introduced into Caecitellus sp. using a high voltage exponential decay pulse (1000 V, 10 µF, ∞ Ω) from a BioRad GenePulser Xcell (cuvette width 0.2 cm). Cell viability was confirmed by observing motility and fluorescence 24 h after electroporation (Figure 1). Lower voltage pulses did not result in FITC uptake. Estimates of transformation effifiency were difficult to obtain due to the small size of the cells.Unfortunately, the isolated strain of Caecitellus perished in an incubator malfunction. However, these results may be transferable to Caecitellus parvulus, a model for heterotrophic flagellates.
Proper citation: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling 2019. Electroporation of Caecitellus sp. with FITC-dextran. protocols.io dx.doi.org/10.17504/protocols.io.35kgq4w Copy
Authors: Glen Wheeler, Rowena Stern, Cecilia Balesteri
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Glen Wheeler, Rowena Stern, Cecilia Balesteri 2017. Method for electroporation of Isochrysis galbana CCMP1323. protocols.io dx.doi.org/10.17504/protocols.io.hmab42e Copy
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 Corallochytrium limacisporum with electroporation using Neon SystemVersion 1. protocols.io dx.doi.org/10.17504/protocols.io.hmwb47e Copy
Authors: Lu Wang, Brittany Sprecher, Huan Zhang, and Senjie Lin
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Lu Wang, Brittany Sprecher, Huan Zhang, and Senjie Lin 2019. Co-Incubation protocol for transforming heterotrophic dinoflagellates (e.g. Oxyrrhis marina). protocols.io dx.doi.org/10.17504/protocols.io.7pphmmn Copy
Authors: RACHELE CESARONI, Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: RACHELE CESARONI, Rachele Cesaroni 2019. Euplotes crassus transfection using Lipofectamine 2000 as vehicle (provisional). protocols.io dx.doi.org/10.17504/protocols.io.2aigace Copy
Authors: Imen Lassadi
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Attempts to transfect Hematodinium was performed using a plasmid that will express eGFP under the control of DVNP regulatory sequences. See attachment for plasmid information.
Proper citation: Imen Lassadi 2019. Plasmid used for transfection trails of Hematodinium . protocols.io dx.doi.org/10.17504/protocols.io.4nigvce Copy
Authors: Jeffrey Lewis, Amanda Scholes
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Lewis Lab
Summary: A generalizable protocol for measuring relative changes in gene expression via qPCR (cDNA synthesis, qPCR primer optimization, and qPCR analysis), with specific optimization for the budding yeast Saccharomyces cerevisiae.
Proper citation: Jeffrey Lewis, Amanda Scholes 2020. Quantitative (q)PCR and Differential Expression Analysis. protocols.io dx.doi.org/10.17504/protocols.io.bbgpijvn Copy
Authors: Jacob Harris
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab
Summary: The FlowCam can be used to take quick cell density measurements of liquid samples.
Proper citation: Jacob Harris 2016. FlowCam Standard Operating Procedure. protocols.io dx.doi.org/10.17504/protocols.io.f2tbqen Copy
Authors: Vincent Bielinski, Chris Dupont
Group: Protist Research to Optimize Tools in Genetics (PROT-G), JCVI West Protocols
Summary: This protocol is the adaptation of previously published methods for use in isolating intact high molecular weight DNA from stramenopiles for long-read sequencing. The advantages here are minimal amount of centrifugation steps on isolated DNA, as well as the long-term storage of the DNA while in plugs to allow experimental flexibility.
Proper citation: Vincent Bielinski, Chris Dupont 2018. Modified HMW DNA Isolation from Stramenopiles with Agar Plugs. protocols.io dx.doi.org/10.17504/protocols.io.wenfbde Copy
Authors: Joshua Bugge, Deborah Robertson
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Robertson Lab
Summary: An electroporation-mediated genetic transformation or the marine diatom Thalassiosira pseudonana was developed. Using a sorbitol-based buffer, T. pseudonana cells were successfully transformed with the Tpfcp/nat plasmid at an efficiency of 2820 per 108 cells. This represents a six-fold improvement compared with previously published methods.
Proper citation: Joshua Bugge, Deborah Robertson 2019. Electroporation of Thalassiosira pseudonana. protocols.io dx.doi.org/10.17504/protocols.io.yvqfw5w Copy
Authors: Elin Einarsson
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: We have created plasmids for transfection of the parasite Perkinsus marinus using Golden Gate cloning. We have explored the possibility to either FACS sort (GFP/mCherry fused to MOE membrane protein) or using drug selection (bleomycin, blasticidin S or puromycin) to enrich transfected cells.
Proper citation: Elin Einarsson 2019. Golden Gate plasmids used for transfection of Perkinsus marinus. protocols.io dx.doi.org/10.17504/protocols.io.37egrje Copy
Authors: Larry Klobutcher
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Larry Klobutcher 2017. Growth of Dunaliella salina on artificial seawater.. protocols.io dx.doi.org/10.17504/protocols.io.gwpbxdn Copy
Authors: Inbal nussbaum, Daniella Schatz
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: We used the NEPA electroportor to transform Emiliania huxleyi cells. We were able to establish that this method can be used to introduce proteins into the cells.
Proper citation: Inbal nussbaum, Daniella Schatz 2018. NEPA electroporation of Emiliania huxleyi cells. protocols.io dx.doi.org/10.17504/protocols.io.p4zdqx6 Copy
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