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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.s6cehaw 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 Bathycoccus.

Proper citation: Francois-Yves bouget 2017. Selection of stable transformants in Ostreococcus tauri and Bathycoccus prasinos. protocols.io https://dx.doi.org/10.17504/protocols.io.hc4b2yw Copy   

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Authors: Valeria Sabatino, Monia Teresa Russo, Shrikant Patil, Giuliana d’Ippolito, Angelo Fontana, Maria Immacolata Ferrante
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol allows transformation of two species of Pseudo-nitzschia through biolistic method.With this method it is possible to insert one (transformation) or two (co-transformation) plasmids in the cells. 

Proper citation: Valeria Sabatino, Monia Teresa Russo, Shrikant Patil, Giuliana d’Ippolito, Angelo Fontana, Maria Immacolata Ferrante 2017. Biolistic Transformation of Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis. protocols.io https://dx.doi.org/10.17504/protocols.io.hp5b5q6 Copy   

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Authors: Yutaka Hanawa, Yoshihisa Hirakawa, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Yutaka Hanawa, Yoshihisa Hirakawa, Patrick Keeling 2018. Electroporation of Oxyrrhis marina. protocols.io https://dx.doi.org/10.17504/protocols.io.vcne2ve Copy   

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Authors: Francois-Yves Bouget, Valérie Vergé et Jean-Claude Lozano
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes the quantification of luciferase activity in Ostreococcus and Bathycoccus cells that have been transformed with a firefly luciferase  coding sequence upstream of the OTTH595 high affinity transporter promoter. Using this methods we were able to show that O. tauri promoters work in Ostreococcus RCC809 and Bathycoccus.  

Proper citation: Francois-Yves Bouget, Valérie Vergé et Jean-Claude Lozano 2017. Transient luciferase expression in Ostreococcus (OTTH595, RCC809) and Bathycoccus. protocols.io https://dx.doi.org/10.17504/protocols.io.hcib2ue Copy   

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

Proper citation: Angela Piersanti 2017. Euplotes crassus transfection using Lipofectamine 2000 with repetitive exposition (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.hhtb36n 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 Lipofectamine 2000 as vehicle (provisional). protocols.io https://dx.doi.org/10.17504/protocols.io.hjeb4je Copy   

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