Figure 2

Schematic diagram of the utilized selectable marker and reporter gene plasmids. (A) Plasmid pPmr3 carries the selectable S. rimosus aphVIII gene driven by an artificial tandem promoter from the hsp70A and rbcS3 genes of V. carteri and a 3′-flanking sequence derived from the rbcS3 gene of V. carteri [8]. (B) Plasmid pNitAph contains the chimeric aphVIII selectable marker gene derived from plasmid pPmr3; however, the aphVIII gene is only driven by the V. carteri rbcS3 promoter (no tandem with hsp70A); it also contains the 3′-flanking sequence of the V. carteri rbcS3 gene. In addition, pNitAph contains the V. carteri nitA promoter region and also the 3′ flanking sequence of the nitA gene. An artificial multiple cloning site was introduced between the 5′ and 3′ control elements of nitA to allow for integration of a protein coding sequence of interest. (C) Plasmid pNitLucX contains the same nitA cassette as pNitAph (but no selectable marker); in addition, the G. princeps g-luc reporter gene was cloned into the multiple cloning site between the 5′ and 3′ control elements of nitA. (D) Plasmid pNitLucA is a derivative of pNitAph; the G. princeps g-luc reporter gene was inserted into the multiple cloning site. Amplified fragments of rbcS3/aphVIII (gPCR1) and g-luc (gPCR2) are indicated. V.c., Volvox carteri; S.r., Streptomyces rimosus; G.p., Gaussia princeps; g-luc, luciferase gene.