Implantation of unmarked regulatory and metabolic modules in Gram-negative bacteria with specialised mini-transposon delivery vectors

► A new set of mini-Tn5-based delivery vectors for chromosomal insertions is presented. ► These tools allow cloning and markerless insertion of expression cargoes. ► Antibiotic-sensitive hosts can be obtained upon the action of the Flp recombinase. ► Regulated expression of gene(s) using the LacIQ/Ptrc and XylS/Pm can be achieved. ► Application of these systems in metabolic engineering endeavours is demonstrated.