Experiments with plasmid-borne reporters were performed in Escherichia coli strain DH5α [F−, λ−, φ80lacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, thi-1, gyrA96, relA1], whereas experiments with chromosomally encoded reporters were performed in E. coli strain W3110 [F−, λ−, IN(rrnD-rrnE), rph-1]. With the exception of assays for promoter function on the chromosome, all experiments utilized plasmid pSB3C5 and the construct of interest was cloned via standard procedures between the BioBrick cloning sites (35 (link)). Each construct contained the Bba_B0032 ribosome binding site and the Bba_B0015 transcriptional terminator. The sequences for the fluorescent reporter proteins, GFP (Bba_E0040), dsRed (Bba_E1010), Gemini (Bba_E0051), the ribosome binding site (Bba_B0032) and the terminator (Bba_B0015) can be found at the Registry of Standard Biological Parts (www.partsregistry.org ).
Plasmid constructs contained the following elements: promoter-TACTAGAG-B0032-TACTAG-ORF(dsRed, GFP, Gemini)-TACTAGAG-B0015, where the ORF was exchanged using standard PCR-based techniques. For chromosomal insertions, test constructs were fused to a kanamycin resistance marker (Bba_P1003, Registry of Standard Biological Parts,www.partsregistry.org ) using SOEing PCR (36 (link)). PCR products were recombined onto the chromosome using the λ-red recombination system, encoded on the plasmid vector pSIM5, as described earlier (37 (link)). After verification of successful cassette insertion by sequencing, pSIM5 was cured from the strain. For the tonB locus, the SOEing primers used are as follows (with homology to the locus listed in bold).
Two strains were used to control for cellular auto-fluorescence. DH5α transformed with pSB3C5 was used as a negative control for experiments using plasmid-based constructs. For experiments testing promoter function from the tonB locus, the kanamycin resistance marker with no reporter construct was recombined downstream of the tonB locus using primers tonB-BioBrickPrefix-fwd, P1003-tonB-rev.
Plasmid constructs contained the following elements: promoter-TACTAGAG-B0032-TACTAG-ORF(dsRed, GFP, Gemini)-TACTAGAG-B0015, where the ORF was exchanged using standard PCR-based techniques. For chromosomal insertions, test constructs were fused to a kanamycin resistance marker (Bba_P1003, Registry of Standard Biological Parts,
tonB-BioBrickPrefix-fwd
BioBrickSuffix-rev
cgaacttttgctgagttgaaggatcagCTGCAGCGGCCGCTACTAGTA
BioBrickSuffix-fwd
TACTAGTAGCGGCCGCTGCAGctgatccttcaactcagcaaaagttcg
P1003-tonB-rev
Two strains were used to control for cellular auto-fluorescence. DH5α transformed with pSB3C5 was used as a negative control for experiments using plasmid-based constructs. For experiments testing promoter function from the tonB locus, the kanamycin resistance marker with no reporter construct was recombined downstream of the tonB locus using primers tonB-BioBrickPrefix-fwd, P1003-tonB-rev.
