We developed a recombinant PCR method for in vitro creation of linear constructs for the replacement of every protein-coding gene in the S. sanguinis SK36 genome (Figure S1A). Based on the complete S. sanguinis SK36 genome sequence24 (link), three sets of primers (F1/R1, F2/R2 and F3/R3) were designed to amplify the S. sanguinis sequence upstream from each targeted gene, the aphA-3 gene, encoding Kmr45 (link) and the S. sanguinis sequence downstream from each targeted gene, respectively.
For most of the mutagenized genes, the R1 and F3 primers were designed to delete the coding region from 6 bp after the start codon to 30 bp before the stop codon. Stop codons were inserted in all three frames to prevent fusion of the N-terminus of the targeted open reading frame with the Kmr protein. The last 30 bp were retained to preserve potential ribosomal binding sites used by adjacent downstream genes. The upstream retained region was extended from 6 bp to 100 bp when two neighboring genes were located head-to-head in opposite orientation to prevent deletion of potential promoters for flanking genes. Primers R1 and F3 contained 25-nt sequences that are complementary with the antibiotic selection cassette at their 5' ends. The P1, P2, and various T1 primers were designed for sequencing to confirm mutants. The sequence of every primer is documented in Table S1.
Three PCR amplicons were created using F1/R1, F2/R2 and F3/R3. All PCR reactions were performed at 94°C for 1 min, and 30 cycles of 94°C for 30 sec, 54°C for 30 sec and 68°C for 1.5 min. After DNA purification by PureLink 96 PCR purification kits (Invitrogen), the three PCR amplicons were combined in equal amounts in one tube and amplified again using the F1 and R3 primers to obtain the final linear recombinant PCR amplicon. Conditions were 94°C for 2 min, 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 68°C for 3.5 min, and finally 68°C for 4 min. High-fidelity Platinum® Taq DNA polymerase (Invitrogen) was used in all reactions.
For most of the mutagenized genes, the R1 and F3 primers were designed to delete the coding region from 6 bp after the start codon to 30 bp before the stop codon. Stop codons were inserted in all three frames to prevent fusion of the N-terminus of the targeted open reading frame with the Kmr protein. The last 30 bp were retained to preserve potential ribosomal binding sites used by adjacent downstream genes. The upstream retained region was extended from 6 bp to 100 bp when two neighboring genes were located head-to-head in opposite orientation to prevent deletion of potential promoters for flanking genes. Primers R1 and F3 contained 25-nt sequences that are complementary with the antibiotic selection cassette at their 5' ends. The P1, P2, and various T1 primers were designed for sequencing to confirm mutants. The sequence of every primer is documented in Table S1.
Three PCR amplicons were created using F1/R1, F2/R2 and F3/R3. All PCR reactions were performed at 94°C for 1 min, and 30 cycles of 94°C for 30 sec, 54°C for 30 sec and 68°C for 1.5 min. After DNA purification by PureLink 96 PCR purification kits (Invitrogen), the three PCR amplicons were combined in equal amounts in one tube and amplified again using the F1 and R3 primers to obtain the final linear recombinant PCR amplicon. Conditions were 94°C for 2 min, 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 68°C for 3.5 min, and finally 68°C for 4 min. High-fidelity Platinum® Taq DNA polymerase (Invitrogen) was used in all reactions.
