Fig. 1

Construction of pDOC-GG and its use to produce Gene Doctoring donor plasmids and perform chromosomal modifications. The first three stages describe the production of pDOC-GG. 1) Three DNA fragments (brown arrows) were designed, based on pDOC-K, to form the backbone of pDOC-GG. Significant features of pDOC-K are displayed. White boxes represent multiple cloning sites (MCS) 1 and 2; dark green arrows represent PCR primers sacB_GA_F and sacB_GA_R used to obtain fragment pDOC_sacB. Other fragments were produced by chemical synthesis. 2) Full features of the three fragments designed from pDOC-K and two further fragments to introduce new genetic elements. Light green arrows represent promoters. 3) Significant features of pDOC-GG, which was produced by Gibson assembly of the five fragments. The last three stages describe the construction of mutagenesis cassettes in pDOC-GG, and use of the resulting donor plasmid for Gene Doctoring. 4) BsaI overhangs (coloured boxes) for homologous regions (HR) 1 and 2 should be complementary to the CTAC and ACGA overhangs generated on pDOC-GG. Multiple intervening parts (Pt. A – X) are assembled sequentially by design of appropriate complementary BsaI overhangs. 5) and 6) The donor plasmid is used for Gene Doctoring as described by Lee et al. [1]