Methodology for markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation

The probiotic strain Escherichia coli Nissle 1917 (EcN) is a common bacterial chassis in synthetic biology developments for therapeutic applications given its long track record of safe administration in humans. Chromosomal integration of the genes of interest (GOIs) in the engineered bacterium offers significant advantages in genetic stability and to control gene dose, but common methodologies relying on transformation of EcN are inefficient. In this work, we implement in EcN the use of bacterial conjugation in combination with markerless genome engineering to efficiently insert multiple GOIs at different loci of EcN chromosome, leaving no antibiotic resistance genes, vector sequences, or scars in the modified bacterium. This straightforward approach accelerates the generation of multiple modifications in EcN chromosome for the generation of living bacterial therapeutics.