Jun 24, 2019
Version 2
Seminavis robusta protocol collection V.2
- Lev Tsypin1,
- Aaron Turkewitz2
- 1California Institute of Technology;
- 2University of Chicago
- Protist Research to Optimize Tools in Genetics (PROT-G)
Collection Citation: Lev Tsypin, Aaron Turkewitz 2019. Seminavis robusta protocol collection. protocols.io https://dx.doi.org/10.17504/protocols.io.4p8gvrw
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
We attempted to genetically transform S. robusta, but did not succeed. This collection provides protocols that we used to arrive at the transformations, and everything included should work. Under "Guidelines", you can find the plasmids that we attempted to use for the transformations.
Created: June 24, 2019
Last Modified: June 24, 2019
Collection Integer ID: 25056
Keywords: Diatoms, protist, transgenics, cultivation, marine, transformation, Seminavis robusta
Abstract
This is a collection of media, reagents, and protocols for the cultivation and manipulation of the diatom Seminavis robusta. Our hope is that these documents will help future researchers develop genetic tools for studying this organism.
Guidelines
In our (unsuccessful) attempts to transform S. robusta to confer resistance to puromycin or glyphosate, we used the attached plasmid sequences: pBS_ActAgroCP4GlyphR (an Agrobacterium tumefaciens derived glyphosate resistance cassette flanked by S. robusta sequences upstream and downstream of its Actin 1 homolog), pBS_ActEF_AgroCP4GlyphR (an Agrobacterium tumefaciens derived glyphosate resistance cassette flanked by S. robusta Actin 1 5' region upstream and Elongation Factor 1 3' downstream), and pBS_Act_PurR (a puromycin acetyltransferase glanked by S. robusta sequences upstream and downstream of Actin 1). All the plasmids used a pBluescript II SK (+) backbone and the resistance gene cassettes were codon optimized for the S. robusta genome.
pBS_Act_AgroCP4GlyphR.gb pBS_Act_PurR.gb pBS_ActEF_AgroCP4GlyphR.gb
pBS_Act_AgroCP4GlyphR.gb pBS_Act_PurR.gb pBS_ActEF_AgroCP4GlyphR.gb 