ranscription 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 genetic elements within a transgene at a single insertion site eliminates the problem of genomic position effects associated with independently introducing variants of transgenes at different loci. Furthermore, adjacent genetic elements could be rearranged at a single locus (e.g., switching the positions of a promoter and transcriptional insulator to test how well the insulator blocks transcription). The ability of Hin recombinase to invert large and small DNA fragments and adjacent flippable elements demonstrates the potential flexibility of the Hin/hix system.
The capability of HinLVA to flip adjacent DNA segments indicates that this system could be scaled up to accommodate more complex pancake stacks. As an application in comparative genomics, flippable DNA segment arrays could serve as a model to improve our understanding of syntenic genome rearrangements that have occurred during evolution. Chromosomal regions exist as syntenic modules arranged in different orders and orientations in the genomes of related species. Each syntenic module can be considered a burnt pancake that has a particular order and orientation. Phylogenetic relationships between species can be inferred by using BPP mathematical modeling to compute the minimum number of rearrangements that link two syntenic genomes (see  for review). Hin-mediated rearrangements of an array containing different sized DNA fragments would help refine the mathematical model by accounting for the impact of differences in sequence composition and lengths of syntenic modules. Using Hin/hix to sort DNA fragment permutations in vivo expands the horizons for the emerging field of applied DNA-based computing.
Construction of parts and plasmids
All genetic elements reported here are standardized parts (prefixed "BBa_" and "pSB") that are flanked by universal "BioBrick" cloning sites  and are documented and distributed by the MIT Registry of Standard Biological Parts .E. coli strain JM109 was used for cloning and as the chassis for our system. The recombinational enhan