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The switch as constructed was able to transition into three out of the five end
states (excluding initial state 0). Thus, we could detect the following sequences:
Hin alone, FimB alone, or Hin followed by FimB. It was unable to transition to the
other two states, FimB followed by Hin and (Hin:FimB:Hin, FimB:Hin:FimB),
probably due to poor transition rates of the fim inversion. The mechanism for the
poor transition rate was not determined.
In order to construct a fully functioning heritable switch as envisioned in this
paper, robust inversion systems are a necessity. The hin system, using hixC, is
quite robust to introduction of exogenous sequences, including strong hairpins
and other recombination sites. The fim system, however, seems to be less robust,
perhaps because it is more sensitive to the positioning of the IHF and LRP bind-
ing sites. Or it is possible that there are yet to be discovered key mechanisms not
considered in the design. A greater understanding of the fim inversion system is
necessary for the development a robust system. In this study there was not an op-
portunity to construct a variety of fim inversion systems with many different ar-
rangements of IHF, LRP, mirrored pairs, terminators, in different permutations to
concretely discover the exact cause of the inversion repression. With the existing
synthesis technology, a study that would rigorously test the necessary elements for
fim inversion would have been very costly and time consuming. But perhaps with
advanced DNA synthesis and assembly technologies, such tests may be feasible
in the near future. Because of the apparent power and theoretical efficacy of these
devices for encoding states it seems a useful program on which to embark.
Despite its limitations, this work creates a proof of principle mechanism for
production of finite-state DNA read/write systems and has uncovered key chal-
lenges that might not have been clear without the construction. With the large
variety of enzymes that edit DNA, successful harnessing of DNA recombination
systems could lead to powerful applications in biological control and sensing.
Materials and Methods
construction
All standard molecular techniques were performed using established protocols
[33]. The double inversion switch was synthesized de novo in multiple steps.
First, a simplified version of the switch, which had the fim and hin mirrored pairs
and the middle terminator replaced by restriction sites, was synthesized using
the protocol of Rouillard and colleagues [34]. The product was cloned into the
pPROBE vector [35], which harbors gfp encoding the green fluorescent protein
along with flanking terminators, resulting in pTSH49. Once the sequence was
verified, the mirrored pairs and the terminator were cloned in via double strand
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