Screening and validation of the aor double KO strain with restored pyrE. (A) PCR screening of ∆aor2 and aor1 KO strain; (B) PCR screening of uracil autotrophic aor double KO strain for restored pyrE allele; (C) Southern Blot analysis of aor1 KO strain. M = NEB 2-log DNA ladder; 1 – 6= aor2-seq-F and aor2-seq-R primer pair; 7 – 12= aor1–559 s-F and aor1–559 s-R primer pair; 13 – 18= ACE-pyrE-F and ACE-pyrE-R primer pair; 1, 7 and 13= Non-template controls; 6, 12, 18 and 23= C. autoethanogenum WT genomic DNA control; 2 – 5, 8 – 11, 14 – 17= clones of aor double KO strain with restored pyrE; 19 – 22= HindIII digested genomic DNA of aor1 KO strain. Arrows and the accompanying numbers denote the fragment sizes of DNA ladder in kilobases.