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Identifying chemotaxis protein‐protein interactions in Epulopiscium sp. Type B using a yeast two hybrid system
Author(s) -
Levesque Brié,
Piefer Andrew J.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1039.3
Subject(s) - chemotaxis , bacillus subtilis , yeast , saccharomyces cerevisiae , biology , escherichia coli , two hybrid screening , gene , genetics , homology (biology) , protein–protein interaction , bacteria , microbiology and biotechnology , receptor
Epulopiscium sp. Type B (Epulo) is a large symbiotic aquatic bacterium that resides in the gut of surgeon fish. From genomic data, Epulo is predicted to have a chemotactic sensory and motility system similar to Escherichia coli and Bacillus subtilis . Our experiments are designed to test the protein‐protein interactions between methyl accepting chemotaxis proteins (MCPs) and the Che proteins, CheA (a sensory kinase) and CheW (a coupling protein), through the use of a yeast‐2‐hybrid (Y2H) genetic assay. We hypothesize based on genetic homology that Epulo chemotaxis proteins will behave in a similar fashion to their orthologs in B.subtilis and E.coli . The Y2H tests protein‐protein interactions through metabolic and drug selection in the yeast Saccharomyces cerevisiae . The genes in question; MCP3, MCP6 and CheW will be cloned into pGADT7 vector while CheA will be inserted into pGBKT7. Preliminary results confirmed an interaction between CheA and CheW; other interactions have yet to be detected; possibly because CheW is mildly toxic to the yeast strain Y187. Alternatively, a CheA+CheW+MCP ternary complex may be necessary for MCP binding. This research is funded by a Hartwick College Faculty Research Grant awarded to AJP.