Without a doubt, in the past 20 or so years, we have achieved the power of biology in diferent ways. In the present, we have many tools for developing novel technologies and applications for organism modiications that ultimately let us know many aspects of organisms' biology and, therefore, apply that knowledge for technological purposes. Of all the model organisms and tools for genetic modiication available, Escherichia coli stands out as a model organism and what we would like to call "molecular biologist tool box." In the present chapter, we aim to review our current knowledge regarding genetic modiications and tools for modifying E. coli to generate plasmid vectors, single and multiple gene knockouts, whole genome editing, biosensor generation and applications and synthetic gene circuits and genomes.