symmetrical palindromic sequences hixL and hixR flank the invertible DNA segment and serve as the recognition sites for cleavage and strand exchange. Our system uses hixC, a composite symmetrical hix site that shows higher binding affinity for Hin and a 16-fold slower inversion rate than wild type sites hixL and hixR [13, 14].
HinLVA flips and sorts hixC-flanked DNA segments in vivo
The sqPCR results suggest that flipping has not yet reached equilibrium after 11 hours of HinLVA activity in the absence of RE. Plasmid supercoiling might be a limiting factor. Hin-mediated inversion requires a negatively supercoiled plasmid DNA substrate [15, 16]. The loss of four negative supercoils after each inversion event  might require cells to undergo cell division to reset optimal supercoiling before a second inversion event can occur. Based on the 4 hour lag time and 36 minute maximum doubling rate of the cotransformed cells, we estimate that no more than 12 doublings occurred before sqPCR analysis. Twelve cell divisions appear to be insufficient to allow the distribution of rearrangements to reach equilibrium.
Two-pancake permutations and their phenotypes
BPP Plasmid construct a
mRFP expression on amp, IPTG c
Growth on tet, amp, IPTG d
RR-hixC-tetA(C) rev -RBS rev -hixC-pLac rev -hixC
RR-hixC-pLac-hixC-tetA(C) rev -RBS rev -hixC
RR-hixC-RBS-tetA(C)-hixC-pLac rev -hixC
RR-hixC-pLac rev -hixC-RBS-tetA(C)-hixC
RR-hixC-tetA(C) rev -RBS rev -hixC-pLac-hixC
RR-hixC-pLac rev -hixC-tetA(C) rev -RBS rev -hixC
Modeling and detection of phenotypic output
As an initial step towards carrying out flipping in vivo, we manually constructed all eight pancake permutations (excluding the RE) and transformed them into cells to confirm their phenotypes. We observed several unexpected outcomes. In cells that contain a strong pLac repressor (lacIQ), BPP plasmids (1, 2) and (-2, -1) showed significant tetracycline resistance without activation of pLac by IPTG. We also observed that HinLVA-mediated inversion does not require induction of the pLac promoter on the HinLVA plasmid, indicating general leakiness of pLac promoter activity probably due to more lacIQ binding sites than available repressor protein . The addition of IPTG appears to slow the growth of (1, 2) transformants; this might be result of toxic TetA(C) over expression . We expected to detect mRFP expression from all four plasmids that contain reversed pLac. However, reversed pLac fails to induce mRFP expression when it is positioned after tetA(C) (i.e., RBS-mRFP-hixC-RBS-tetA(C)-hixC-pLac rev -hixC). Increased distance from mRFP or the DNA structure of tetA(C) [20, 21] might block transcription of mRFP.
We found it surprising that four constructs in which pLac is not in the proper position and/or orientation to drive expression of tetA(C) were able to confer tetracycline resistance; in the presence of IPTG, three of these showed more robust growth than cells carrying (1, 2). When the pLac promoter was removed from the construct, cells were still tetracycline resistant (data not shown), thus pLac is not required for expression in the pBR322-derived cloning vector we had been using (pSB4A3). Read-through transcription by RNA polymerase binding to the antibiotic resistance marker promoter or degenerate promoter sequences within the vector backbone  could result in tetA(C) expression in the tetracycline resistant scrambled permutations (1, -2), (-1, 2), (-2, 1), and (2, 1). We constructed an "insulated" vector (pSB1A7) containing forward and reverse double transcription terminator sequences to shield RBS-tetA(C) from read-through transcription. In pSB1A7, there was no expression of RBS-tetA(C) (forward or reverse) when pLac was removed from the construct. Arrangements (1, 2) and (-2, -1) produced tetracycline resistance, as expected. Surprisingly, we also observed tetracycline resistance in the insulated vector when pLac was reversed relative to tetA(C) in arrangements (-1, 2) and (-2, 1), suggesting reverse promoter activity from pLac. Unlike forward transcription initiated from pLac, backwards t