Many of the gene regulatory networks used within the field of synthetic biology have extensively employed the AraC and LacI inducible transcription factors. However, there is no Escherichia coli strain that provides a proper background to use both transcription factors simultaneously. We have engineered an improved E. coli strain by knocking out the endogenous lacI from a strain optimal for AraC containing networks, and thoroughly characterized the strain both at molecular and functional levels. We further show that it enables the gradual and independent induction of both AraC and LacI in a simultaneous manner. This construct will be of direct use for various synthetic biology applications.