Biology Reference
In-Depth Information
+aTc
-aTc
(A)
proTeOn
PROTEON
GFP
PROTEON/aTc
GFP
LuxR
RNApol
LuxR
rTetR
rTetR
tetO
luxbox
gfp
tetO
luxbox
gfp
Synthetic promoter
(B)
proTeOff
PROTEOFF/aTc
GFP
PROTEOFF
GFP
LuxR
RNApol
LuxR
rTetR
rTetR
luxbox
gfp
tetO
tetO
luxbox
gfp
FIGURE 7.4
proTeOn and proTeOff behavior. (A) proTeOn behavior and logic. In the absence of aTc, rTetR does not bind tetO and
PROTEON does not upregulate gfp. Upon activation with aTc, rTetR binds the inducer, undergoes a conformational change
and binds tetO, bringing LuxR near its operator site to bind luxbox and up-regulate gfp transcription through RNApol
recruitment to the promoter. (B) proTeOff behavior and logic. In the absence of aTc, TetR binds to tetO, brings LuxR near its
operator site to bind luxbox and up-regulate gfp transcription through RNApol recruitment. After the addition of aTc, TetR
binds to the small molecule, undergoes a conformational change, and releases tetO. Upon dissociation of TetR:tetO, the
LuxR:luxbox interaction is destabilized and upregulation by RNApol recruitment terminated.
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the addition of aTc, TetR binds the small molecule, undergoes a conformational change and
releases
tetO.
Upon dissociation of TetR:
tetO
, the LuxR:
luxbox
interaction is destabilized and
up-regulation by RNApol recruitment is terminated.
proTeOn and proTeOff both up-regulate gene expression in the presence and absence of
aTc, respectively. Flow cytometry results illustrate the phenotypes of both systems in
Figure 7.5
. proTeOn achieves steady-state levels of gene overexpression, 20-fold above
uninduced samples, approximately 5 hours post-induction with aTc, and maintains them
through long periods. Also, the PROTEON protein is rapidly transcribed, translated, folded,
and becomes functional when under the control of a free promoter.
15
Given these
characteristics, applications of proTeOn would be appropriate in systems when quick bursts
or long-term gene up-regulation is desired. On the other hand, proTeOff readily activates
target gene expression in the absence of aTc, whereas upon administration of the latter, up-
regulation is significantly reduced. This decrease is achieved just 1 hour post-treatment with
aTc, and expression is maintained at a low level, 10-fold below untreated samples, through
long periods. The PROTEOFF protein is also rapidly transcribed, translated, folded, and
functional when under the control of a free promoter.
15
Thus, the proTeOff system can be
applied to up-regulate gene expression for both short and long periods. Continuous long-
term up-regulation can be achieved, as well as up-regulation with intermittent periods of
low expression, using a range of aTc concentrations. Taken together, the proTeOn and
proTeOff systems are new tools that can be used to robustly control gene expression in
prokaryotic systems. As mentioned above, stochastic simulations were performed to capture
the behavior of both systems and further characterize the governing interactions. These
computational analyses are detailed in the next section.
