Phusion hot start flex master mix

Synthetic, codon-optimised genes for E. coli expression encoding KisK module 6 (A-PCP architecture, UniProt code A0A2P9IC10) and MbtH-like protein KisM (UniProt code A0A2P9IBI0) from kistamicin biosynthesis NRPS gene cluster Actinomadura parvosata subsp. kistnae (Nonomuraea sp. ATCC55076) were obtained from Eurofins Genomics MWG (ESI Table S5†). Both KisK constructs (the wild type A-PCP construct as well as double A-domain mutant) and MbtH protein encoding plasmids were generated using In-Fusion® HD Cloning kit (Clontech) as described in section above. Both KisK constructs were cloned into the pET-GB1-1d series plasmid and KisM-into a pCDF vector. DNA fragments were amplified by PCR from a synthetic gene using primers listed in ESI Table S5.† To improve amplification by PCR, the NEB Phusion® Hot Start Flex Master Mix was supplemented with 540 mM betaine, 1.34 mM DTT, 11 μg mL^–1 BSA and 1.34% DMSO.24 (link)

Tcp11 constructs with an alternative module architecture (module 4 C-A-PCP-E-C, module 5 A-PCP-E-C and module 6 A-PCP) were cloned into a pET-GB1-1d vector22 (link) using In-Fusion® HD Cloning kit (Clontech). PCR primers were designed that share 15 bases of homology with adjacent DNA fragments. Then these primers (ESI Table S3†) were used to PCR amplify both the insert/(-s) and plasmid DNA. The plasmid DNA, containing the gene of NRPS module of interest, was used as the template DNA for a PCR reaction. Fragments were amplified using Phusion® Hot Start Flex Master Mix (NEB) and the appropriate forward and reverse primers (ESI Table S3†). The PCR products were analysed on a 0.8% agarose gel in TAE buffer and the DNA subsequently gel-extracted and purified using the Wizard® SV gel and PCR clean-up kit (Promega). The extracted PCR products (insert/(-s) and vector) were combined in the In-Fusion® cloning reaction as per the manufacturer's instructions. In-Fusion® cloning reactions were incubated for 15 min at 50 °C, then placed on ice and 2.5 μL of the reaction mixture was used for transformation of NEB 10-beta competent E. coli cells.