To replace many of the current methods that have several pitfalls such as inefficient, mutagenic and clinically inapplicable. To illustrate the utility of this system, we have generated a strain of P. aeruginosa with Cycloheximide diminished cytotoxicity to deliver Cre, a well characterized protein which functions only in the nucleus to interact with DNA and exerts its recombinase activity. The availability of numerous Cre-dependent reporters has conveniently allowed us to observe the effects of Cre delivery to both differentiated and pluripotent cells. The ability of bacterially delivered ExoS54-Cre to induce high levels of b-galactosidase activity in TE26 cells indicates that the process of type III secretion and the presence of the ExoS54 signal sequence do not interfere with the ability of this protein to properly localize to the nucleus of host cells or exert its biological function. Our experimental data support the notion that Cre-mediated DNA recombination can only occur when cells are in S phase or when the target loxP sites are freely available, as evident from the observation that the LacZ positive recombinant TE26 cells increase in proportion to the S phase cells during the Cre delivery by the bacteria. For TE26 cells, one full cell cycle takes around 34 hours to complete under our culture condition and a transition time from G2 to S phase takes about 20 hours, thus to achieve close to 100% efficiency of recombination, the injected Cre protein needs to be stable for a time period when all of the cells go through the S phase, which is at least 20 hours for TE26 cell. Apparently, the intracellular stability of the injected ExoS54-Cre is much shorter than the required 20 hours, presumably due to proteasome mediated breakdown. It is possible to achieve 100% Cre-mediated recombination by combining several approaches, including an increase of S-phase cell fraction by synchronization, an increase of the half life of the translocated Cre by utilizing a proteosome inhibitor, and successive infections for Cre to encounter S phase for every cell in the population. While embryonic stem cells are notoriously sensitive, the presence of EYFP positive colonies after bacterial delivery of ExoS54-Cre indicates that these cells are susceptible to P. aeruginosa infection, but neither infection nor protein delivery significantly affects the cellular morphology of R26R-EYFP mESCs. This finding is in accordance with the results of a recent study which demonstrates that mESC infection with other T3SS expressing bacteria, such as Shigella flexneri, does not alter pluripotency, as evaluated by Oct4 expression levels. Given the inherent difficulty of manipulating embryonic stem cells, we have achieved relatively high delivery efficiency in a short time with a single infection. The infection efficiency may further be increased with multiple rounds of protein delivery. We are currently investigating the proficiency of repeated protein delivery to modify gene expression.