Establishing a functional PylRS-based OTS in L. lactis by utilization of the NICE system (Kuipers et al., 1998) required a modification of the nisin inducible promoter PnisA. The ribosomal-binding site (RBS) located within the WT PnisA was disrupted by mutating the sequence AAGGAG to AATTCG (van Gijtenbeek et al., 2016) to hinder unwanted translation of pylT encoding the orthogonal tRNA. As depicted in Figure 3A, an additional RBS was added upstream of pylS as well as of nisA(amber). Despite these genetic rearrangements, the new variant of PnisA remained functional, because expression of N-terminally Strep-tagged PylRS was detectable by Streptactin immunoblotting. After induction of PylRS expression by nisin, cell extracts of L. lactis NZ9000 pNZ-RBSpylTS and pNZ-RBSpylTSnisA as well as the empty vector pNZ-RBS as negative control were applied to a Streptactin column. After purification, immunoblotting revealed a band of matching molecular weight by a Streptactin HRP conjugate in elution fractions resulting from PylRS expressing cells in contrast to cells bearing the control vector (Figure 3B).