Cloning of the wild-type and cysteine mutants of mazE and mazF genes

For cloning of the wild-type (WT) genes, the codon optimized genes of each TA system were PCR amplified from the pET Duet-1 vector. PCR amplified products were gel purified and in vitro recombined using Gibson assembly with either pET-15b vector for protein purification, or pPNLS vector for YSD (Gibson et al., 2009). Recombined products were transformed into E. coli and plasmid identities were confirmed by Sanger sequencing. The base pair and amino acid sequences of the proteins used in the study along with their molar extinction coefficients are provided as supplementary text.

The WT mazF6 gene cloned in pET-15b vector was used as a template to introduce the cysteine mutants by PCR as described earlier (Chattopadhyay et al., 2022a) and the amplified products were then PCR purified and in vitro recombined using Gibson assembly with either pET-15b or pPNLS. Selected individual cysteine mutants of MazE3, MazE9, MazF9 cloned in pETCON vectors for YSD were synthesised by GenScript (United States).