Complete set of ORF clones of Escherichia coli ASKA library (A Complete Set of E. coli K-12 ORF Archive): Unique Resources for Biological Research

Based on the genomic sequence data of Escherichia coli K-12strain, we have constructed a complete set of cloned individualgenes encoding Histidine-tagged proteins with or without GFPfused for functional genomic analysis. Each clone encodes aprotein of predicted ORF attached by Histidines and seven spaceramino acids at the N-terminal end, and five spacer amino acidsand GFP at the C-terminal end. SfiI restriction sites are generatedat both the N- and C-terminal boundaries of ORF upon cloning,which enables easy transfer of ORF to other vector systems bycutting with SfiI. Expression of cloned ORF is under the controlof an IPTG-inducible promoter, which is strictly repressed bylacI^q repressor gene product. The set of cloned ORFs describedhere should provide unique resources for systematic functionalgenomic approaches including (i) construction of DNA microarray,(ii) production and purification of proteins, (iii) analysisof protein localization by monitoring GFP fluorescence and (iv)analysis of protein–protein interaction.