Using transposon-sequencing to probe whole cell protein-protein interactions

Aim

This project aims to identify all protein-interaction partners of a single target protein as proof of principle for a novel protein-protein interaction study. The project design makes use of existing protein-interaction assays in conjunction with genomic mutagenesis screens, to identify the complete protein interactome for a given protein.

Brief project outline

This project involves construction of genomic mutants in a defined E. coli background that encodes a tagged bait protein of interest. Screening the library under specific selection conditions and sequencing output mutants will identify protein-protein interactions on an unprecedented scale.

Genomics-based innovative aspect of proposal

Our method for rapid identification of disrupted genomic loci coupled to well-established protein-interaction screens to identify protein interaction partners on a genomic scale is an innovative use of amplicon sequencing. If successful, this would be the only whole genome screen of its kind and a major new capability for studying protein-protein interaction.  The types of dataset generated also have massive implications for mapping synthetic protein networks. 

Broad applicability of the technique

Extension of the transposon-sequencing method to identify protein interaction partners would benefit the field of microbiology.  The method is applicable to any bacterial genome.  As an indication of the broad applicability of this method, the original description of the bacterial two hybrid system has been cited 1,204 times since 1998.  The methods have been adopted internationally and many labs already have the existing infrastructure and expertise to readily apply our proposed new method.  The infrastructure to complete this project is already available at UQ, and directly correlates with existing research interests here. If successful, this approach can be applied to yeast giving unprecedented insight into eukaryotic biology. The data will be made publicly available following publication of the data and will be deposited with a nucleic acid repository.