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Figure 7.3
Genetic circuit to set protein expression levels. Isopropylthio-
β
-galacto-
side (IPTG) concentration controls the level of the output protein, enhanced yellow
fluorescent protein.
IMPLIES gate. We constructed two such circuits, one on plasmid pINV-102
with the enhanced yellow fluorescent protein (EYFP) output protein (Figure
7.3) and another circuit on a similar plasmid named pINV-112-R1 with the
enhanced cyan fluorescent protein (ECFP) as the output protein. The fluorescent
proteins are both from Clontech [7]. The inverter that comprises the constitutive
promoter p(lacIq) has an input that is always set to low because the cell does
not contain a repressor for the p(lacIq) promoter. Therefore, the output of the
inverter,
lacI
, is constantly high. Then, since the
lacI
repressor input to the
p(lac) IMPLIES gate is constantly high, the level of the inducer molecule input,
IPTG (isopropylthio-
-galactoside), is positively correlated with the level of the
output. The researcher controls the level of the output signal with this circuit
by externally setting the level of IPTG, which freely diffuses into the cell.
In Figure 7.3, the double-stranded plasmid layout includes the following
genetic elements: the p15A origin controlling the copy number of the plasmid
in the cell, kanamycin antibiotic resistance for selective growth, promoters
represented by short arrows, protein-coding sequences downstream of specific
promoters, and transcription terminators such as T1 term. The promoters and
protein-coding sequences are either clockwise or counterclockwise depending
on the direction of the DNA strand that encodes them.
Figure 7.4 shows data from
Escherichia coli
cells with the pINV-102 and
pINV-112-R1 plasmids grown for approximately5hinculture until they
reached steady state. The construction of all plasmids and experimental con-
ditions in this chapter are described in detail elsewhere [15]. The data include
median fluorescence values obtained using fluorescence-activated cell sorting
(FACS) [12] of the different cell populations induced with a range of IPTG
β
