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meaning that there are many copies of our device in the cell such that the state of
the device could possibly be different for each copy of the plasmid. Since Hin and
FimB are reversible enzymes, each cell will harbor a mixture of states of the device
as described below. The plasmids for the input and DNA response elements of the
device are shown in Figure 2A.
Figure 2. Plasmids used in the study. A) Plasmid maps for constructs containing the invertases and the memory
switch. B) Detail of structure of the double inversion switch annotated with the primers used to diagnose state
as described in the text.
recombinase optimization
The double inversion circuit requires two independently induced recombinases,
FimB and Hin, to be expressed. In addition, they have to be repressed indepen-
dently; that is, ideally, one can be induced while the other is repressed. Our switch
was sensitive to leaky expression of Hin and FimB, so we required a very tightly
controlled expression system. Currently, there are only a few tightly-controlled,
regulatable expression systems available. One of the best is the P BAD system, but
this system cannot be used with a lac promoter, as they have significant crosstalk
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