We have constructed the attenuated hyper-invasive Salmonella enterica serovar Typhimurium (hereinafter S. Typhimurium), that are attenuated, yet capable of synthesizing selected protein and harboring an improved DNA vaccine vector encoding a selected protein. The programmed self-destructing features designed into these S. Typhimurium strains allow release of the cell contents by cell lysis after bacteria accumulated in host tissues. This proposal was promoted by the idea that if the selected proteins are able to trigger tumor cell apoptosis, then the engineered self-destructing Salmonella could serve as a programmed 'bio-time-bomb' to destroy tumor tissues by release of tumor killing materials after colonization in tumor tissues. Colorectal cancer is the second leading cause of cancer-related deaths in the United States (after lung cancer). A major challenge in treating cancer is the difficulty of bringing therapy to poorly perfused areas of solid tumors, which are often most resistant to chemo- and radiotherapy. Motile facultative S. Typhimurium strains, which are specifically attracted to compounds produced by quiescent cancer cells and could overcome the traditional therapeutic barrier. It is known that attenuated S. Typhimurium preferably target and penetrate into tumor tissue allowing to accumulate about 2,000-fold more in tumors than in other organs, where they could overcome diffusion limitations and attack quiescent cancer cells that are impervious to standard chemo- and radiotherapy. Therefore we wish to explore the potential to use the engineered self-destructing Salmonella as a colorectal cancer cure. Our objective is to design, construct and evaluate a novel inexpensive rapidly modifiable recombinant attenuated S. Typhimurium (RAS) delivery system that will be a 'time bomb' with multiple functional biotherapy agents as an optimal colorectal cancer cure to overcome chemo- and radiotherapeutic resistance. The (RAS) delivery system with newly developed features will (i) be hyper- invasive, (ii) selectively synthesize bacterial serine and aspartate chemoreceptors to facilitate maximal colonization of tumor tissues, (iii) displays regulated delayed synthesis of S. Typhimurium T3SS effector SopE2 that stimulates innate immune responses, and (iv) exhibits regulated delayed lysis to efficiently deliver the bacterial virulence factor TlpA, that directed by Salmonella promoter preferentially activated inside tumors, to induce apoptosis in tumor cells, and an improved DNA vaccine vector encoding a tumor-specifically synthesized death ligand Fas to trigger tumor cell death and also to attract immune cells to attack tumor cells. This RAS delivery system will have features to render it completely safe for humans administered the engineered Salmonella and to be unable to persist in immunized individuals or survive if excreted. Furthermore, we believe that the designing, constructing and evaluating an engineered Salmonella-based cancer cure would represent a highly effective means to reduce systemic toxicity of cancer treatment and perhaps lead to eradication of tumors from the host. ) <P> Public Health Relevance: The engineered self-destructing Salmonella could serve as a programmed 'bio-time-bomb' to destroy tumor tissues by release of tumor killing materials after colonization in tumor tissues. The designing, constructing and evaluating an engineered Salmonella-based colorectal cancer cure would represent a highly effective means to overcome chemo- and radiotherapeutic resistance, to reduce systemic toxicity of cancer treatment and perhaps lead to eradication of tumors from the host.