Phusion or q5 dna polymerase

The bacterial strains used and generated during this study are listed in Table 1. Escherichia coli and Streptomyces strains were cultured, maintained, and manipulated genetically following the methods of Sambrook et al. (15 ) and Kieser et al. (16 ), respectively. The plasmids and oligonucleotides used or constructed during this work are listed in Table 2 and Table 3, respectively. Mobilization of phage P1-derived artificial chromosome (PAC) clones into Streptomyces strains was performed as described previously (17 (link)). Molecular biology enzymes, reagents, and kits were used according to the manufacturer's instructions. High-fidelity PCR amplification was performed with Phusion or Q5 DNA polymerase following the manufacturer's instructions (NEB, Ipswich, MA) with a nucleotide proportion of 15A:15T:35G:35C to improve the amplification efficiency of high-moles-percent G+C Streptomyces DNA. 3-Amino-5-hydroxybenzoic acid (AHBA) was purchased from Sigma (catalog no. PH011754) and dissolved in dimethyl sulfoxide.

To construct riboswitch and CDS plasmids for this study, we started with the pFTV1 vector backbone, which contains mRFP1 modified to contain an N-terminal SacI restriction site^{41 (link)}. We used the Riboswitch Calculator to design candidate riboswitch sequences and the Operon Calculator to codon-optimize the MS2 coat protein CDS and design an optimal RBS sequence (Source Data)^{39 (link),64 (link)}. We designed and ordered gBlocks, containing primer binding sites and additional restriction sites, and PCR primers for both the riboswitches and MS2 coat protein CDS (Integrated DNA Technologies). We PCR amplified the gBlocks using Phusion or Q5 DNA polymerase (New England Biolabs). For the riboswitches, we digested the riboswitch amplicons and pFTV1 vector backbone with XbaI and SacI-HF (New England Biolabs). For the MS2 coat protein CDS, we digested the CDS amplicon and pFTV1 vector backbone with XbaI and NotI-HF (New England Biolabs). For both the riboswitches and CDS, we ligated the digested inserts with digested backbone using T4 DNA ligase (New England Biolabs), and heat-shock transformed the ligation product into chemically competent DH10B. We then performed Sanger sequencing to verify that the insert had been cloned correctly.