Plasmid construction

Plasmid pRRG13 (fig. S1A, upper right side) was generated by inserting the tetA promoter region (pA) in front of the gfp gene in plasmid pUA66. Promoter pA was polymerase chain reaction (PCR) amplified from the Tn10 region of the natural plasmid R100 using oligonucleotides pTetA1XhoI and pTetA2BamHI. The resulting band was inserted in pUA66 using Xho I and Bam HI restriction endonucleases. The TetR_mkate2 fusion protein was generated by binding the N terminus of protein mKate2 to the C-terminal end of TetR through a flexible linker made of Ser-Gly-Gly-Gly-Gly peptide. For this purpose, we constructed plasmid pRRG54. Plasmid pRRG54 was built by Gibson assembly out of three DNA fragments. The first one was the PCR product of amplifying mkate2 with primers isomkatefusdir and isomkate_rev from plasmid pRAF33. The second fragment was PCR amplified using primers isotetRfusrev and isotetRfus_dir using plasmid R100 as template, and contained tetR. The third fragment was generated by linearizing expression vector pBAD33 using Xba I digestion. Plasmid pRRG62 (fig. S1A, upper left side) was constructed by substituting the replication origin of pRRG54 (p15A) with the replication origin of plasmid pSEVA121 (RK2). This way, we constructed an expression vector for TetR_mkate2 with a lower plasmid copy number, which allowed us to control TetR expression more tightly. To generate plasmid pRRG62, TetR_mkate2 was PCR amplified from pRRG54 using primers pbadseva1 and pbadseva2. The resulting band was digested with Pac I and Spe I restriction endonucleases and inserted in pSEVA121 (37). Plasmid pRRG63 (fig. S1A) contains TetR_mkate2 and its target promoter pA::GFP transcriptional fusion. To generate this plasmid, pA::GFP was PCR amplified from plasmid pRRG13 using primers pua66terb1007 and ptetAgfpkm_revPacI. This PCR fragment was inserted into plasmid pRRG62 using Pac I and Hind III restriction endonucleases. This way, the bidirectional transcriptional terminator BBa_B1007 (http://parts.igem.org) was inserted, isolating TetR_mkate2 and pA::GFP cistrons. In plasmid pRRG74, TetR_mkate2 is expressed from promoter pR, while the pA::GFP fragment is located in the region occupied by pA::TetA in Tn10. Plasmid pRRG74 was generated by Gibson assembly, fusing three DNA fragments. The first one included mkate2, amplified using oligonucleotides pRRG74_mkatedir and pRRG74_mkaterev from plasmid pRAF22. The second one, containing the gfp gene, was amplified from pUA66 using primers pRRG74_GFPdir and pRRG74_GFPrev. Last, the third one contained the Tn10 region that includes tetR, pR, and pA. It was amplified from plasmid R100 using primers pRRG74_ptetArev and pRRG74_TetRrev.