The ability of genetically modified probiotic (beneficial) bacteria to deliver therapeutic compounds is a promising new experimental method for delivering fragile and complex bio-molecules without injection. The long-term goal of this study is to improve the genetic stability of a probiotic organism for use as a living drug delivery vehicle. Escherichia coli Nissle 1917, a non-pathogenic E. coli, currently used in probiotic treatment of intestinal disorders, is an attractive candidate drug delivery system. Although recombinant DNA methods were originally pioneered in E. coli and this organism remains the most common host for bacterial production of recombinant products, some problems still occur. Degradation of strain performance is not unusual in industrial fermentations and is often the result of genetic instability due to mobile elements originating from the bacterial chromosome. This proposal outlines a program for removing mobile elements from the E. coli Nissle 1917 chromosome using well characterized recombination methods. Sequences to be deleted are chosen on the basis of their similarity to regions in pathogenic E. coli, the presence of many mobile elements and identifiable prophage sequences and similarity to genes that will be used in therapeutic applications of this strain. The primary goal of the proposed research program is to produce variants of E. coli Nissle 1917 that lack all pathogenic and mobile genetic elements. Individual deletion strains will be tested for viability and their ability to persist in the mouse gut. Deletion strains that exhibit no loss of viability or persistence will be noted and the deletions cumulatively combined. Deletion strains with diminished viability or persistence will serve as hosts for gene addition studies, to determine which genes are required to restore viability and/or persistence. ? ? ?