The type III secretion system (T3SS) of gram-negative bacteria involves dedicated protein translocation machinery that directly injects proteins into target cells. Pathogenic bacteria already benefit from this unique system. The successful functional cloning of this useful tool into non-pathogenic bacteria would help establish novel clinical and basic biotechnology strategies in areas such as vaccine administration, the development of screening systems for anti-T3SS drugs and the target-specific delivery of bioactive compounds. In this study, we successfully cloned the Vibrio parahaemolyticus T3SS1 genetic locus into a non-pathogenic Escherichia coil K-12 strain. Assays performed here revealed that the T3SS1 cloned into the E. coil K-12 strain has the ability to translocate V. parahaemolyticus T3SS1 secreted proteins. Importantly, we also observed this system to allow the E. colt K-12 strain to inject foreign protein, as well as the V. parahaemolyticus T3SS effector, into cultured cells. These results demonstrate a prospective useful tool with experimental and therapeutic applications. (C) 2012 Elsevier Inc. All rights reserved.