Biology Reference
In-Depth Information
(A) TTL
(B) TLT
1
1
0.5
0.5
0
200
0
200
1
1
100
100
0.5
0.5
0
aTc
0
IPTG
aTc
0
0
IPTG
(C) LTT
(D) LTL
1
1
0.5
0.5
0
200
0
200
1
1
100
100
0.5
0.5
0
aTc
0
IPTG
aTc
0
0
IPTG
FIGURE 7.2
AND gate behavior. (A) TTL AND gate. AND-logic behavior is observed for the TTL AND gate as expression only occurs with
aTc and IPTG. (B, C) TLT and LTT AND gates. AND-logic is less apparent as the lacO operator site is moved farther
upstream in the synthetic promoter. Background expression is high even in the absence of IPTG. (D) LTL AND gate. Very low
induction is observed from promoters with two lacO operator sites even with high aTc and IPTG concentrations.
126
It has been previously demonstrated that transcription regulation by TetR:
tetO
is tight, while
regulation by LacI:
lacO
is significantly leakier in the absence of inducer molecules.
Additionally, regulation by LacI:
lacO
has been shown to be largely affected by its location
within the promoter.
35
Unfortunately, the AND gates with two
lacO
operator sites were
minimally inducible even at high IPTG concentrations. This was an artifact of very high
intracellular LacI concentrations in the experimental systems (
E. coli
). However, the
promoters with two
tetO
operator sites showed informative GFP expression dynamics. These
promoters allowed very little background GFP expression in the absence of aTc, while they
did exhibit a range of background GFP in the absence of IPTG. These behaviors capture this
leakiness and position dependence of LacI:
lacO.
Taken together, the behaviors of the AND
gates further support previous observations which proposed that the location of
lacO
significantly influences the promoters
leakiness, while
tetO
robustly regulates expression
from multiple locations.
35
These characteristics highlight the modularity limitations of
biomolecules and parts of biomolecules. However, despite their limitations, the AND gates
are applicable new molecular biology tools with which one can regulate target gene
expression given two input signals. They have also provided the field with new insights
regarding the modularity of biomolecules and part of biomolecules. As mentioned above,
the dynamics of the AND gates were also characterized using computer simulations. These
simulations are briefly discussed in the next section.
'
proTeOn and proTeOff
The proTeOn and proTeOff systems were also built to control target gene expression in
E. coli
. These systems were built and characterized both experimentally and
computationally.
15
In contrast to the AND gates, both the promoter sequence and the
protein molecules of proTeOn and proTeOff were engineered. Additionally, for both
