Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory - Genetic Engineering: Recent Developments in Applications - page 179

inversion in both directions without impediment [13], as the hin mirrored pair
behaved unpredictably due to its native mirrored-pair like structure (data not
shown). HixC, however, does have a side effect of resulting in a higher rate of self-
excision of the DNA when in a directly repeated orientation.
Figure 3. Culture PCR results for the response to the different input sequences . {FimB}, {HinB}, {FimB,
HinB} and {HinB, FimB}. Each lane probes for the existence of a state or states, as shown in the legend.
Because of the possible arrangements of DNA, lanes E and F could result in either short or long PCR products,
indicating states {1, 3}, and {2, 4} respectively. See Figure 2 for relative location of the primers. No State 5 was
observed when Hin and FimB were simultaneously expressed.
The fim inversions performed as expected, except with a much lower inver-
sion efficiency than hin. By culture PCR, we determined that some fraction of the
cells were inverting, resulting in strong PCR product bands. However, of the 50
isolates sequenced, none of them had the inverted fim orientation, even though
sequencing of the PCR product showed clean fim inversion. It would be possible
to screen more perhaps by doing a serial dilution and a binary tree search. It ap-
pears that the intercalated switch construct has low fim inversion efficiency, with
an unknown rate.