Standardization of the regulatory region and operator parts. (A) The regulatory region of a plasmid was divided into the upstream multi-cloning region and the downstream standard region for standardization of the assembly. The upstream multi-cloning region contains the EcoR I, Xba I, Sal I and BamH I restriction sites. The downstream standard region contains the Bgl II and Mlu I restriction sites. The operator part with a -35/-10 sequence is inserted into the upstream multi-cloning region, while that without a -35/-10 sequence is inserted into the downstream standard region. The sets of restriction sites allow insertion of the operator parts between the pre-connected parts. (B) The operator sequence is flanked by the sticky ends of BamH I and Mlu I in the operator part for the downstream standard region. The operator parts are inserted to the reporter plasmid digested with Bgl II and Mlu I. The BamH I sticky end of the operator part comes together with the Bgl II sticky ends of the plasmid. (C) The LacI operator part was designed as a downstream part. The BamH I sticky end (Bold & Italic) is added to the left side of the LacI operator sequence (small letters). The Bgl II restriction site (Italic), spacer (Bold), and Mlu I sticky end (small letters & Italic) are added on the right side of the LacI operator. (D) Although the original Bgl II site on the reporter plasmids disappears by the insertion of the operator part, the Bgl II site contained on the inserted part works as a new Bgl II site of the plasmid. This recovery of Bgl II site allows repeating insertions of the next operator parts.
Ayukawa et al. BMC Genomics 2010 11(Suppl 4):S16 doi:10.1186/1471-2164-11-S4-S16