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. 2015 Jul 6;112(29):8953–8958. doi: 10.1073/pnas.1507606112

Fig. 1.

Fig. 1.

Construction of bidirectional promoter cassettes and multiplexed promoter exchange strategy. (A) Overview of promoter exchange strategy. (B) The bidirectional promoter cassettes were generated by PCR amplification of six yeast selectable makers with primers containing promoter/insulator/RBS combinations (SI Appendix). The generated promoter cassettes were cloned into TOPO vector 2.1 for their future maintenance. These bidirectional promoter cassettes were tested on the Reb gene cluster, and those that produced rebeccamycin were used for promoter engineering (SI Appendix). (C) The method to generate the 500-bp homology arms is outlined. A promoter cassette is inserted into a gene cluster using 40-bp homology arms and the region around this insertion is PCR-amplified to generate 500-bp homology arms. (D) The number of colonies generated from the insertion of one, two, or three promoter cassettes with 40-bp or 500-bp homology arms were compared using the Reb gene cluster (cluster A) and an unrelated eDNA-derived type II polyketide synthase gene cluster (cluster B). A representative collection of yeast colonies (>10) from each experiment was examined by PCR to determine the frequency with which all promoter cassettes were correctly inserted. The solid portion of the bars represents the fraction of colonies with correctly inserted promoter cassettes.