Figure 3

Activities of TetX detected using the pYR _tetOR system and fluorescence-based assays. A-D) Induction ratios in the presence of the active enzymes (open circles) or mutated inactive enzymes (solid circles). Best fit lines using the enzyme kinetics model (see text and Methods for details) are shown. A, C and D were performed in the presence of 0.0002% arabinose, which was the minimum arabinose concentration where consistent full repression could be obtained under these conditions. B was perfomed in 0.02% arabinose. E) A Western blot (anti-6xHis) showing the expression level of TetX at the point where drugs were added. Bands indicated by arrows correspond to the enzymes and bands indicated by the triangle correspond to purified TetR-6xHis protein. The enzyme bands show the total amount of enzyme from ~1 μL cell pellet, and the TetR bands show the amount of 6xHis tagged protein from 1 μL solution containing the indicated concentration protein.