Pwa pas e bad k gafm

The plasmids pBAD-(His₆)miRFP670 (Addgene no. 79984),^{29 (link)} pBAD-(His₆)miRFP709 (Addgene no. 79986),^{29 (link)} and pWA PAS-E BAD K-GAFm (Addgene no. 39866)^{25 (link)} were gifts from Vladislav Verkhusha. All plasmids were verified by DNA sequencing.

The plasmid for the coexpression of heme oxygenase to produce biliverdin IXα in cells was cloned using pWA PAS-E BAD K-GAFm (Addgene no. 39866), and the plasmid pCOLADuet1-hmuO1, which contains the Bradyrhizobium ORS278 gene for heme oxygenase 1 (hmuO1), and was obtained from Genscript. Two fragments from each plasmid were PCR-amplified and assembled by Gibson Assembly (NEB). From pCOLADuet1-hmuO1, the hmuO1 fragment and a fragment containing the ColA ori, Kan resistance, and flanking regions were amplified. From pWA PAS-E BAD K-GAFm, a fragment containing the rhamnose promoter elements and upstream flanking region as well as a fragment containing the rrnB terminator were each amplified. Primers for each amplification are listed as follows: hmuO1 sense: 5′-GGAGATATAACCATGGGCAGCAGCATGTTGGAC-3′; hmuO1 antisense: 5′-CAAAACAGCGAATTCTCAGCGGCCG-3′; pCOLA ori-Kan sense: 5′-TACAAACTCTTTAACCTAGGCTGCTGCCAC-3′; pCOLA ori-Kan antisense: 5′-TGGCCACGGCGCAACGCAATTAATGTAAGTTAGC-3′; rhamnose sense: 5′-GTTGCGCCGTGGCCAGGACCCA-3′; rhamnose antisense: 5′-GCCCATGGTTATATCTCCTTCTTAAGAAAATTATTTCTAGAG GGAAACCG-3′; rrnB sense: 5′-TGAGAATTCGCTGTTTTGGCGGATGAGAGAAG-3′; rrnB antisense: 5′-GCCTAGGTTAAAGAGTTTGTAGAAACGCAAAAAGGC-3′.