Fig. 2

Insertion of six base pairs by the modified Gibson approach into two distinct sites within dLight. a Depiction of the dLight-GFP (green fluorescent protein) synthetic gene (colored segments, A–C). Fragment A includes sequences spanning from the promoter of the plasmid and intracellular linker I (ILI; purple); fragment B spans from ILI to ILII (orange); fragment C, ILII to the end of the GFP (green). Backbone of plasmid is light grey. The mutations (cyan or yellow) are situated between the purple and orange fragments, and orange and grey fragments, respectively. Sizes (# of bps) of the fragments are noted within the fragments. Intrinsic and unique digestion sites are also noted (BamHI and BsrGI). Primers used for amplification of each fragment are noted on the right (with corresponding colors). b Image of PCR products from step I on 1% agarose gel. DNA ladder sizes (in Kbp) are noted on the right of third ladder. For clarity, we have cropped the images of the gel to show the relevant bands. Full length agarose gels for all figures are provided in Additional file 3: Fig. S5. c The assembled Gibson products after amplification by hSyn promoter_F and the ILIII_R primers, and FP_R primers. d Image of the agar plates with colonies obtained after ligation of assembled Gibson products to the plasmid (at 1:2 ratio, V:V; top—ILI, bottom—ILII), and controls (left images; backbone plasmid without inserts). e Top—Amino acid sequences of WT, and expected insertion within ILI and ILII, middle and bottom sequences, respectively. Bottom—The resulting sequences and chromatograms from DNA isolated from colonies (dashed boxes shows the correct modifications)