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Figure 1 | Microbial Cell Factories

Figure 1

From: A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis

Figure 1

Host-vector system for insertion of "your favorite gene (YFG)" downstream of the LAC4 promoter. The K. lactis LAC4 gene encoding β-galactosidase is divergently transcribed with the lactose permease gene (LAC12) and both genes are essential for growth on lactose. Disruption of LAC4 is required to apply selection. In the lac4::ScURA3 strain (VAK367-D4) the sequences between +358 and +1181 of the LAC4 ORF were replaced by the ScURA3 gene by homologous recombination. The expression cassette contains YFG fused to the LAC4 promoter, flanked at the 3'-end by the TEF1 terminator, the KlGAL80 promoter and the 5'-end of LAC4. It can be excised from the vector backbone by cleavage with e.g. Hpa I. The resulting fragment can replace the ScURA3 gene and restore the LAC4 ORF by homologous recombination giving the P GAL80 -LAC4 fusion gene. Desired recombinants are selected for growth on lactose plates. Bottom: Genome structure of BVDV with E2 gene, which was inserted as an example YFG in the pilot experiment giving strain VAK726.

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Microbial Cell Factories

ISSN: 1475-2859

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