Information Technology Reference
In-Depth Information
1,000.00
pINV-107/pINV-112-R1
pINV-107/pINV-112-R2
pINV-107/pINV-112-R3
100.00
10.00
1.00
0.1
1.0
10.0
100.0
1,000.0
IPTG (uM)
Figure 7.10
The effect of weaker ribosome binding sites on the behavior of the
cI
/
λ
P(R
−
O
12
)
circuit. Here the output, YFP, exhibits the desired inverse sigmoidal
relationship to the isopropylthio-
β
-galactoside (IPTG) input.
level, which pushes the entire transfer curve upward and outward. BioSPICE
simulations serve as an abstract model to study the effects of changing reaction
kinetics of specific genetic components in the logic computation.
The sequences for the original highly efficient
cI
RBS used in the circuit
above, as well as three other less efficient RBS's from the literature [6] are:
orig:
ATTAAAGAGGAGAAATTAAGC
ATG
(strongest)
RBS-1:
TCACACAGGAAACCGGTTCG
ATG
RBS-2:
TCACACAGGAAAGGCCTCG
ATG
RBS-3:
TCACACAGGACGGCCGG
ATG
(weakest)
Starting from pINV-110, we constructed three new plasmids (pINV-112-R1,
pINV-112-R2, pINV-112-R3) where the three weaker RBS's replace the original
RBS of the
cI
. pINV-112-R1 contains the strongest RBS, while pINV-112-R3
contains the weakest RBS.
Figure 7.10 shows the dramatic effect of the RBS change on the behavior of
the circuit, where now the output YFP exhibits the desired inverse sigmoidal
relationship to the IPTG input. The circuit with the strongest RBS (pINV-
107/pINV-112-R1) shows a moderate sensitivity to IPTG, while the circuits with
the other two RBSs display a more pronounced response to variations in IPTG.
Modifying Repressor/Operator Affinity
Replacing the strong RBS with weaker sites converted a nonfunctional circuit
into a functional one and demonstrated the utility of genetic process engineer-
ing. This subsection describes further modifications to the repressor/operator
affinity that yield additional improvements in the performance of the circuit.
These modifications are also motivated by BioSPICE simulations [15, 16]. The
