Figure 1

Cloning and purification of GST-GFP. A: Schematic of vector construction. (1) A spacer was cloned into the pET 15b vector/plasmid in proximity to the hexa-histadine tag and thrombin site, pJB-HTS (2) This fragment was cloned from the pET 15b vector (3) into the pGex-6p-1 vector to add a GST upstream, pJB-GST-HTS. (4) Red shifted GFP was isolated by primer extension with a spacer and a second hexa-histadine tag and (6) cloned into pJB-GST-HTS yielding pJB-GST-HTS-HS-GFP. B: Gel electrophoresis screening of DNA prepared from colonies following step 1—pJB-HTS—were digested with BglII/HindIII (top) the loss of a 500 bp band (lane 1) and appearance of a 318 bp digestion fragment indicates positive colony for spacer insertion. Arrows indicates 500 bp and 250 bp bands on marker. DNA was digested with NcoI (bottom) to screen for positive insertions. One was chosen for sequencing and further cloning (asterisk). C: Basal GFP expression of a pJB-GST-HTS-HS-GFP containing colony is observed microscopically (brightfield overlaid with epifluorescence) with the scale bar indicating 100 μm. After induction, bacteria express significant green color under UV illumination (bottom) indicating high levels of GFP expression. D: GST-GFP electrophoresed as predicted for the known molecular weight (2) before and (3) after cleavage with thrombin as compared to the (1) molecular weight ladder with arrows indicating 55 kDa, 35 kDa, 25 kDa, respectively.