Fig. 2

Screening of pyrE knockout, pyrE repair and integration at the pyrE locus. PyrE orotate phosphoribosyltransferase; BCV53_03770: transcriptional regulator, PheB G. stearothermophilus 2,3-dioxygenase, LHA left homology arm (299 bp), RHA right homology arm (1200 bp), MW 2-log DNA marker (NEB), molecular weight marker, WT G. thermoglucosidasius NCIMB 11955 wild type, ΔpyrE G. thermoglucosidasius pyrE knockout mutant. The chromosomal region at the pyrE locus is illustrated for WT and ΔpyrE. All screening was conducted using primers pyrE_C1_F (1) and pyrE_C2_R (2), giving the expected PCR product of 2101 bp for WT, 1876 bp for ΔpyrE and 3490 bp for the integration of pheB. Lanes 2–7 (a) and 1–12 (b) represent the screened DNA samples from randomly selected uracil auxotrophic colonies for WT (a) and TM89 (b). Lanes 2–4 and 6–8 (c) represent the screened DNA samples from randomly selected uracil prototrophic colonies derived by repairing of pyrE with pMTL-LS2 in both WTΔpyrE and TM89ΔpyrE. Lanes 1–12 (d) represent the screened DNA samples from randomly selected uracil prototrophic colonies derived from the integration of pheB at the pyrE locus. Of the 11 screened colonies, 5 had the expected PCR band size. Correct integrants are plated on TSA and sprayed with catechol for phenotypic validation (d)