Biology Reference
In-Depth Information
(A)
TTL
tetO
-35
tetO
-10
lacO
gfp
TLT
tetO
-35
lacO
-10
tetO
gfp
LT T
lacO
-35
tetO
-10
tetO
gfp
LT L
lacO
-35
tetO
-10
lacO
gfp
(B)
Uninduced TTL AND-gate
RNApol
TetR
TetR
Lacl
tetO
-35
tetO
-10
lacO
gfp
Induced TTL and-gate
+aTc AND IPTG
IPTG
aTc
(C)
RNApol
tetO
-35
tetO
-10
lacO
gfp
TetR
TetR
Lacl
FIGURE 7.1
AND gates system design. (A) Illustration of the TTL, TLT, LTT, and LTL AND gates controlling gfp expression. The tetO and
lacO operator sites are located upstream of the 35, between the 35 and 10, and downstream of the 10 consensus
sequences. (B) Uninduced TTL AND gate. In the absence of inducers (IPTG and aTc), both TetR and LacI bind their operators
(tetO and lacO ) and prevent gfp transcription by RNApol. (C) Induced TTL AND gate. In the presence of IPTG and aTc,
inducer-bound TetR and LacI proteins dissociate from their operators. RNApol can access the 35 and 10 binding sites of
the free promoter and transcribe downstream gfp.
125
character of inducible gene expression. Endogenous expression of the LacI and TetR
repressor proteins was exploited for transcription repression (via binding their
complementary operator sequences
lacO
and
tetO
, respectively) in the absence of inducer
molecules. In the presence of the appropriate inducers, LacI and TetR undergo a
conformational change and dissociate from their operator sequences. While expression from
the lactose operon is induced by Isopropyl
-D-1-thiogalactopyranoside (IPTG), and
expression from the tetracycline operon is induced by anhydrotetracycline (aTc), the
character of these synthetic promoters allows transcription to occur only in the presence of
both inducers, IPTG and aTc. In the presence of only one inducer, only one type of
repressor protein dissociates from the promoters and, therefore, gene expression is still
repressed by the second repressor type. However, in the presence of both inducers, IPTG
binds LacI while aTc binds TetR. Both interactions result in a conformational change driving
the repressors to dissociate from their complementary operators. Upon the release of LacI
and aTc, gene transcription can occur.
β
Using these promoters, AND-logic gene expression control is achieved. While none of the
AND gates has a perfectly digital behavior, the promoters containing two
tetO
operator sites
(and one
lacO
) do produce an output signal, GFP expression, that increases in strength with
increasing aTc and IPTG concentrations. The robustness of the AND gate systems is largely
dependent upon both the placement of the operator sites relative to the
10
sequences, as well as the ratio of
tetO
to
lacO
sites. The results shown in
Figure 7.2
illustrate
these dependencies.
35 and
2
2
