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In all inversions, inducing the culture at low OD rather than stationary phase
resulted in a greater number of inversions. For hin inversion, this is consistent
with the fact that Fis production is eliminated during stationary phase [32]. How-
ever, it does not explain the increase in fim inversion. Perhaps IHF or LRP is
regulated in a similar fashion as Fis, or there is an unknown protein involved in
fim inversion.
The rarity of fim inversion had consequences for the second generation (states
3 and 4) inversions. State 4, the hin then fim inversion, although rare, was ob-
served by PCR product sequencing, as a large fraction of hin inversion occurred,
and a small fraction of fim inversions then occurred. However, state 3, the fim
then hin inversion, was never observed. This is perhaps the result of only having a
small fraction of fim inversion occurring, and among the smaller pool of inverted
fims, the occurrence of hin inversion was very small. It may also be that the hin
inversion efficiency was lower than normal due to the DNA rearrangement via the
fim inversion, further suppressing the occurrence of state 3 plasmids.
Many of the problems encountered in creating a functional double inversion
switch stemmed from two principal causes. First, the necessity for the mirrored
pair introduced a very long hairpin structure that is both difficult to construct and
maintain within E. coli. Perhaps strains more tolerant of such structures would
be useful. Also, such hairpins have made sequencing very challenging: constructs
containing the mirrors had to be sequenced piecemeal, as the sequencing reaction
would stop at the very edges of the hairpin. Additionally, the hairpins acted as ter-
minators of unknown strength for the internal constitutive promoters, although
this had a positive effect on our switch design.
The second problem is our lack of understanding about the exact structure of
protein-DNA complex that forms when FimB binds to the inversion sites. The as-
sumption of non-interference between recombinases was clearly violated. Perhaps
a better understanding of the structure of FimB, including its reaction with the
enhancer and DNA bending proteins, would reveal insight into why the switch
did not perform as expected. As a whole, however, the switch did demonstrate
history dependent configurations with states 0, 1, 2, and 4 visited as expected
although not with the frequency desired.
conclusions
A heritable inversion switch was designed and constructed using both the fim and
the hin inversion recombination systems. The design allowed for encoding of state
information to the DNA, which would be inherited generation after generation.
The design also displayed finite state machine-like behavior, as the reporter would
transition to and from different states, recording path traversal as it went along.
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