Fig. 2
From: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
Directed integration of MEL1His gene in the K. lactis LAC4 chromosomal locus. a The linearized expression cassette (MEL1His-1 or MEL1His-2) is guided by the 3´and 5´ends of PLAC4-PBI (black boxes) to the LAC4 promoter region that is reconstructed after the integration. P6 primer anneals upstream PLAC4 of the original strain and P7 primer anneals upstream PLAC4-PBI inside the expression cassette. Primer pairs P6-P5 and P7-P5 were used to analyze if the integrations were single or multiple copy, respectively. b During Southern blot analyses, the genomic DNA was digested with SpeI and ApaI, and bands separated by electrophoresis before Southern transference. The probe (white box) when hybridizes with single copy bands reveals a fragment of 12.4 Kb that increases in 7.4 Kb per additional cassette integrated in multicopy strains. c Immunological detection and colour reaction allowed to determine the number of copies of the integrated cassette in KGM strains. MW, molecular weight marker (Kb)