Modification of the nisin promoter PnisA for combined OTS and nisin expression in L. lactis. (A) Modified PnisA (black arrow) of vector pNZ-RBSpylTS and its derivatives with deleted RBS upstream of pylT (encoding tRNAPyl) to prevent initiation of translation. RBS locations are indicated by orange triangles, the original RBS location is crossed out. In blue arrows, the components of the OTS, pylT and pylS are depicted. The green arrow labeled with nisA∗ represents the nisin precursor gene, either encoding the WT (nisA) or variants with in-frame stop codons [nisA(amber)]. The size of the depicted genes is not drawn to scale. (B) Heterologous expression of archaeal PylRS controlled by modified PnisA. Cell extracts from L. lactis pNZ-RBS, pNZ-RBSpylTS, and pNZ-RBSpylTSnisA were applied to a Streptactin column and the two elution fractions were analyzed by immunoblotting with a Streptactin–HRP conjugate. Only cells expressing PylRS led to detection by the antibody (upper picture). Strep-tagged Page Ruler Standard (Thermo Fisher Scientific) served as positive control, apparent molecular weight and calculated value for PylRS are indicated. The corresponding 10% SDS-PAGE stained with Coomassie brilliant blue revealed that all analyzed fractions contained protein (picture below).