Figure 2
From: Soluble expression, purification and characterization of the full length IS2 Transposase
Structure of plasmids used to create the IS 2OrfAB::GFP fusion construct. Modifications and alternations are indicated in red. (a) pGLO-ATG2, a derivative of the commercially available pGLO plasmid (Biotechnology Explorer GFP Chromatography kit, Bio-Rad Inc., Hercules, CA, USA) containing the GFPuv gene under the control of the PBAD promoter. An Eco RI-Nhe I cassetting site was created in the 5' multiple cloning site (MCS), to facilitate the cloning of the IS2orfAB fused frame gene. A unique Eco RI site was deleted from its position adjacent to the GFP stop codon and transferred to a position downstream of the PBAD promoter and 9 bp from an existing Nhe I site which encodes the first two amino acids of GFP. The mutagenizing primer for this last step also deleted the GFP start codon to create pGLO-ATG2. (b) pLL18, a pUC19 derivative with IS2 carrying the Kmr reporter gene [6]. IS2 in this construct contains the engineered orfAB gene described in Figure 1a (ii). (c) pLL2509A was created by removing the left inverted repeats and repositioning the existing Eco RI site to a location downstream of the PIRL promoter, effectively excluding this IS2 endogenous promoter from subsequent cloning of the cassetted orfAB gene. (d) pLL2521HK was created by the successive steps of adding (i) the 3'-located cassetting Nhe I site which included the removal of the orfAB stop codon and (ii) the 6XHIS-Tag, downstream of the Eco RI cassetting site. (e) pLL2522 was formed when the Nhe I-Eco RI cassetted orfAB (part d) was cloned into the corresponding 5' cloning site of pGLO-ATG2 (part a). bp: basepair; GFP: green fluorescent protein; IS: insertion sequences.