Significance Genome fluidity is directly associated with the emergence and evolution of many bacterial pathogens. Horizontally acquired genetic elements, which harbor fitness traits, are open source for the bacterial evolution. The genome of Vibrio cholerae is equipped with multiple mobile genetic elements (MGEs), which are crucial for disease development and survival. The present study engineered the genome of a well characterized clinical V. cholerae strain N16961 and reported ( i ) stability of MGEs in cholera pathogen, ( ii ) interactions between the core genome and MGEs, and ( iii ) role of MGEs in the modulation of cellular proteome. The engineered strain could be a potential candidate for understanding evolution of cholera pathogen and development of new therapeutic interventions.