University of Regina

2 ARTICLES PUBLISHED IN JoVE

Biology

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Alla Gagarinova^*1, Mohan Babu^*2,3, Jack Greenblatt^1,2, Andrew Emili^1,2

¹Department of Molecular Genetics, University of Toronto, ²Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, ³Department of Biochemistry, Research and Innovation Centre, University of Regina

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Biology

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Mohan Babu^1,2, Olga Kagan¹, Hongbo Guo¹, Jack Greenblatt^1,3, Andrew Emili^1,3

¹Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, ²Deparment of Biochemistry, Research and Innovation Centre, University of Regina, ³Department of Medical Genetics and Microbiology, University of Toronto

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.