romoter 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 transcription did not respond to IPTG as determined by cell growth; IPTG induction of forward transcription from pLac led to overexpression of tetA(C) and subsequent cell death . Due to the backwards promoter activity of pLac, our manually built set of permutations are not distinguishable by phenotype, thus phenotype alone is insufficient to perform computation using pLac and RBS-tetA(C). The observations described above demonstrate that the construction of synthetic biological devices can reveal unexpected characteristics of well-studied DNA elements (e.g., pLac).
We have demonstrated that a modified Hin/hix DNA recombination system can be used in vivo to manipulate at least two adjacent hixC-flanked DNA segments; HinLVA and hixC are sufficient for DNA inversion activity. The RE is not required, although it may play some role in preventing aberrant flips that lead to plasmid knotting  and subsequent plasmid loss . Thus, the RE might be added to BPP plasmids to increase DNA recombination efficiency. Once phenotypic output is optimized for this system, the kinetics of flipping (i.e., number of flips per unit of time) could be determined by comparing Markov Chain model simulation output to in vivo pancake sorting. Comparing actual cell survival to the survival probabilities predicted by our model should also enable us to determine whether flipping is biased for different sized DNA fragments.
The Hin/hix DNA recombination system could be used for other biological engineering applications. We have developed a set of modular genetic elements (hixC, RE, and HinLVA) that expands the repertoire of molecular tools for enzyme-mediated DNA manipulation in vivo. As with Cre/loxP from P1 bacteriophage  and Flp/FRT from yeast , Hin/hix may open avenues for recombinase-mediated transgene engineering. For instance, hixC-flanked promoters and other regulatory elements could function as flippable genetic toggle switches to regulate gene expression just as Hin mediates the expression of flagellin genes in Salmonella. Manipulation of