Figure 5.

HinLVA flips adjacent hixC-flanked segments simultaneously. (A) A map of the BPP plasmid including the RBS-mRFP_rev reporter followed by hixC-tetA(C)_rev-hixC-pLac-hixC (-2, 1) is shown at the top. In this arrangement, pLac cannot drive mRFP expression. Simultaneous inversion of both segments converts (-2, 1) into (-1, 2); the pLac promoter is directed towards mRFP so that mRFP expression is turned on. (B, inset C) White light photograph of colonies ~18-hours after cotransformation with BPP plasmid (-2, 1) and HinLVA. (D, inset E) mRFP protein production visualized under ultraviolet light (solid white arrow) indicates a simultaneous flip of two adjacent pancakes that placed pLac in the reverse orientation adjacent to RBS-mRFP_rev. Some colonies do not glow red (open arrow), indicating a lack of double segment flipping or subsequent conversion into other arrangements that lack mRFP expression (e.g., (1, 2)).