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simulations show how reductions in the binding affinity of the repressor to the
operator reshape the transfer curve of the inverter upward and outward.
To reduce the repressor/operator affinity, we constructed three new plas-
mids with modified
O
R
1 sequences using site-directed mutagenesis to have the
following sequences:
orig:
TACCTCTGGCGGCGGTGATA
mut4:
TAC
A
TCTGGCGGCGGTGATA
mut5:
TAC
A
T
A
TGGCGGCGGTGATA
mut6:
TAC
AGA
TGGCGGCGGTGATA
cI
's amino domain is folded into five successive stretches of
α
helix, where
α
helix-3 lies exposed along the surface of the molecule [10]. This
α
helix
recognizes the
operators and binds the repressor to those particular DNA
sequences. The two
λ
helix-3 motifs of the repressor's dimer complex are
separated by the same distance as the one separating successive segments of
the major groove along one face of the DNA. These motifs efficiently bind
the repressor dimer to the mostly symmetric
α
operator regions, where each
operator consists of two half-sites. The following is the consensus sequence for
the 12 operator half-sites in the wild-type bacteriopage
λ
λ
(subscripts correspond
to the frequency of the basepair in the given position):
T
9
A
12
T
6
C
12
A
9
C
11
C
7
G
9
C
5
C
2
C
3
T
2
T
1
T
4
T
2
T
1
A
1
A
1
C
1
G
1
C
1
In choosing mutations to perform, we conjectured that bases with high
frequency in the consensus sequence would be significant to strong repres-
sor/operator binding. Mut4 is a one-basepair mutation
C
A
of the fourth
O
R
1 position; mut5 is a two-basepair mutation that also modifies the sixth
O
R
1
position
C
→
A
; and mut6 is a three-basepair mutation that also modifies the
fifth
O
R
1 position
T
→
G
.
The experimental results in Figure 7.11 demonstrate the effect of coupling
the three
→
λ
P(R
−
O
12
)
O
R
1 operator mutations with the weakest RBS from above.
The two- and three-basepair
O
R
1 mutations, coupled with the weak RBS,
produce a circuit where the highest levels of
cI
cannot repress the output of
the
cI
/
λ
P(R
−
O
12
)
gate. A one-basepair mutation to
O
R
1 in plamids pINV-107-
mut4/pINV-112-R3 yields a circuit with a well-behaved response to the IPTG
signal and is a good gate candidate for other biocircuits.
As described in this section, using genetic process engineering we first ex-
amined the behavioral characteristics of the
cI
/
λ
P(R
−
O
12
)
inverter and then
genetically modified the gate until we produced a version with the desired
inverse sigmoidal behavior. The design and experimental results illustrate how
