tial to be scaled up to accommodate larger and more challenging problems. Hin/hix may provide a flexible new tool for manipulating transgenic DNA in vivo.
The biological equivalent of a burnt pancake is a functional module of DNA such as a promoter or coding region (Fig. 1a). Similar to burnt pancakes in the BPP, DNA modules have directionality (5' to 3'), require a specific order of the units (e.g., promoter followed by coding region) and can be flipped (cut, inverted, and spliced in vivo by cellular machinery). We designed a modular system in which pancake stacks are assembled from flippable DNA segments. Flipping of the DNA segment "pancakes" is mediated by a Salmonella typhimurium-derived DNA recombination system. In Salmonella, Hin DNA recombinase catalyzes an inversion reaction that regulates the expression of alternative flagellin genes by switching the orientation of a promoter located on a 1 kb invertible DNA segment [9, 10]. Two palindromic 26 bp hix sequences flank the invertible DNA segment and serve as the recognition sites for cleavage and strand exchange. A ~70 bp cis-acting recombinational enhancer (RE) increases efficiency of protein-DNA complex formation . We have reconstituted the genetic elements required for DNA inversion as a collection of modular genetic elements for use in E. coli. Our system is a proof-of-concept genetic computing device that manipulates plasmid DNA processors within living cells.
Results and discussion
Design and construction of a Hin/hix-based DNA recombination system
DNA inversion occurs very rapidly in vitro. Protein-DNA complex assembly, strand cleavage, inversion, and ligation occur in less than 1 minute . Therefore, we engineered Hin/hix inversion to be more tractable to regulation and kinetic studies by decreasing inversion efficiency. Hin was cloned from S. typhimurium by PCR. An ssrA LVA protein degradation tag  was added to the C-terminal DNA binding domain to prevent over accumulation of Hin and to achieve tighter control of DNA inversion. In Salmonella, the asymmetrical 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 p