This work used the following E. coli strains: NEB 5-alpha (NEB, C2987; not authenticated), BL21-AI (Thermo Fisher, C607003; not authenticated), bMS.346 and bSLS.114. bMS.346 (used previously20 (link)) was generated from E. coli MG1655 by inactivating the exoI and recJ genes with early stop codons. bSLS.114 (used previously29 (link)) was generated from BL21-AI by deleting the retron Eco1 locus by lambda Red recombinase-mediated insertion of an FRT-flanked chloramphenicol resistance cassette, which was subsequently excised using FLP recombinase42 (link). bCF.5 was generated from bSLS.114, also using the lambda Red system. A 12.1kb region was deleted that contains a partial lambda*B prophage that is native to BL21-AI cells within the attB site, where temperate lambda integrates into the bacterial genome43 (link).
Phage retron recombineering cultures were grown in LB, shaking at 37 °C with appropriate inducers and antibiotics. Inducers and antibiotics were used at the following working concentrations: 2 mg/ml L-arabinose (GoldBio, A-300), 1 mM IPTG (GoldBio, I2481C), 1mM m-toluic acid (Sigma-Aldrich, 202-723-9), 35 μg/ml kanamycin (GoldBio, K-120), 100 μg/ml carbenicillin (GoldBio, C-103) and 25 μg/ml chloramphenicol (GoldBio, C-105; used at 10 μg/ml for selection during bacterial recombineering for strain generation).
Phage retron recombineering cultures were grown in LB, shaking at 37 °C with appropriate inducers and antibiotics. Inducers and antibiotics were used at the following working concentrations: 2 mg/ml L-arabinose (GoldBio, A-300), 1 mM IPTG (GoldBio, I2481C), 1mM m-toluic acid (Sigma-Aldrich, 202-723-9), 35 μg/ml kanamycin (GoldBio, K-120), 100 μg/ml carbenicillin (GoldBio, C-103) and 25 μg/ml chloramphenicol (GoldBio, C-105; used at 10 μg/ml for selection during bacterial recombineering for strain generation).