SY
Shuichi Yanagisawa
  • Faculty, Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
Research fields
  • Molecular Biology, Plant Science
CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii
Authors:  Shaojun Long, Kevin M. Brown and L. David Sibley, date: 03/20/2018, view: 10906, Q&A: 0
Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this method provides a robust approach to defining protein networks. This approach detects interaction within intact cells, it is applicable to both soluble and insoluble components, including large proteins complexes that interact with the cytoskeleton and unique microtubule organizing center that comprises the apical complex in apicomplexan parasites.
Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii
Authors:  Kevin M. Brown, Shaojun Long and L. David Sibley, date: 02/20/2018, view: 16030, Q&A: 0
Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii’s 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T. gondii. Here we provide a step-by-step protocol for examining protein function in T. gondii using the AID system in a tissue culture setting.
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