Molecular cloning is the core of synthetic biology, as it comprises the assembly of DNA and its expression in target hosts. At present, however, cloning is most often a manual, time-consuming, and repetitive process that highly benefits from automation. The automation of a complete rational cloning procedure, , from DNA creation to expression in the target host, involves the integration of different operations and machines. Examples of such workflows are sparse, especially when the design is rational (, the DNA sequence design is fixed and not based on randomized libraries) and the target host is less genetically tractable (, not sensitive to heat-shock transformation). In this study, an automated workflow for the rational construction of plasmids and their subsequent conjugative transfer into the biotechnological platform organism is presented. The whole workflow is accompanied by a custom-made software tool. As an application example, a rationally designed library of transcription factor-biosensors based on the regulator Lrp was constructed and characterized. A sensor with an improved dynamic range was obtained, and insights from the screening provided evidence for a dual regulator function of Lrp.