High-throughput split-protein profiling of T7 RNA polymerase with the high genetic switchability

The split T7 RNA polymerase (T7 RNAP) system is used in protein-fragment complementation assay (PCA) and as an AND-logic gate in synthetic biology studies. The self-complementary split sites are derived from the finding of simultaneous cleavage during T7 RNAP purification and the examination of the flexible area in its crystal structures. However, assisted complementary split sites are preferred for the successful PCA design, and all of the efforts performed so far are based on the expensive lengthy trial and error approaches. Here, we aimed to investigate every residue of T7 RNAP for discovering the high-contrast assisted complementary split sites by utilizing our previously developed high-throughput split-protein profiling (HiTS) method. Despite the success of the HiTS technique to decipher the assisted-complementary split positions in fosfomycin and erythromycin antibiotic resistance proteins, it could not find out any potential position in T7 RNAP. However, four self-complementing fragmentation sites residing at 69, 187, 237, and 598 residues were found with higher activity than the wild-type full-length T7 RNAP. Importantly, we found the fragments splitting at 598 was located in the beta-strand and showed stronger activity, whilst, none of the former studies discovered split sites in the structured area. We anticipate the ineffectiveness of the HiTS method is due to the biased insertion of the transposon (heterodimer interaction pair) by MuA transposase used in it, which leads to the low coverage at different positions of the target T7 RNAP gene sequence. We expect it can be overcome by integrating an unbiased approach with HiTS to insert the heterodimer interaction pair at every residue of T7 RNAP; however, it is still left to investigate.