Supplementary Figure 3: Bioactivity and sequence verification of streptothricin and streptomycin inactivated strains. | Nature Biotechnology

Supplementary Figure 3: Bioactivity and sequence verification of streptothricin and streptomycin inactivated strains.

From: Hidden antibiotics in actinomycetes can be identified by inactivation of gene clusters for common antibiotics

Supplementary Figure 3

Streptothricin (A) and streptomycin (B) CRISPR-mediated gene inactivations were verified by agar plug bioassays with susceptible and resistant E. coli. Representative data is shown for one clone of each species/gene pair that was successfully knocked out. Two independent fermentations and bioassays for each strain gave similar results. (C) Sanger sequencing of CRISPR-engineered strains. Sequence alignments highlight the sgRNA target site (blue) and PAM (green). HR results in the introduction of a stop codon (red) and in some cases a HindIII site (gray). NHEJ resulting in indels (yellow) was also detected. (D) Attempts to PCR amplify the targeted gene failed to yield an amplicon in some cases and was interpreted as an undefined deletion in the genome. Representative results are shown for several clones (Δ1, Δ2, etc.) of three streptothricin producers targeted for orf15. 16S PCR and Sanger sequencing was used to verify template DNA integrity and the strain’s identity. Deletions shown in this figure were generated using the pCRISPR-Cas9 system except for WAC6128 Δorf17 generated using pCRISPomyces.

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