Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory - Genetic Engineering: Recent Developments in Applications - page 176

[28]. Thus, Ptet was used along with the P BAD system on a single vector con-
taining both (Fig. 2A), to express the recombinases independently. However, P Te t
did exhibit leaky expression and required remedy prior to use with our sensitive
switch.
In addition to promoter optimization, translational efficiency was optimized.
Seven different ribosome binding sites (RBSs) across a range of translational ef-
ficiencies were tested with the hin and fimB genes [16]. The optimization criteria
were level of expression (does it cause inversion when expressed) and leakiness
(does it cause inversion when not expressed). As our PCR based reporting method
was very sensitive, the selection was biased against leakiness rather than towards
high expression. In addition, degradation tags (LVA) or LacI binding sites were
tested for their effects and possible efficacy at reducing background leaky expres-
sion (data not shown). The pTSH68 vector (containing hin with RBS sequence
“AGGGACAGGATA” plus the LVA tag driven by PTet and fimB with RBS se-
quence “GAAGGTTCCTCA” driven by PBAD) was chosen as the recombinase
expression vector.
target device optimization
The two recombinase recognition sites in the middle flanking the terminator
needed to be constructed in such a way that they form an inverted repeat even
after a recombination event. As such, they had to be mirrored; that is, the bind-
ing site is repeated immediately adjacent, but in the opposite strand orientation,
to maintain the inverted repeat orientation (Fig. 2B). The design for the switch
is finalized by placing the necessary elements of both the fim inversion (2 IHF
elements, LRP sites, the inverted repeats), and hin inversion (hin enhancer site,
inverted repeats) along with generated sequences that separate the components
(Fig. 2B). The enhancer elements are somewhat flexible in their location [29],
[30], [31], so they were placed in similar places as the original switches. The entire
switch, excluding gfp and rfp, is approximately 1 kbp.
Predicted and Measured Function of the switch
The heritable switch functions as shown in (Fig. 1C) and briefly outlined above.
Figure 2B shows the actually constructed state 0. Hin plays the role of invertase B
and FimB plays the role of invertase A. State 3 is only reached and GFP expressed
when Hin is induced following FimB. State 4 is only reached, and RFP expressed
when FimB is induced following Hin.
Because our inversion recombinases are reversible, two deviations from ide-
ality are immediately expected. First given a long enough induction time, the