Biology Reference
In-Depth Information
between populations. Once secreted into the culture, the enzymes from each population
can act in concert to allow a cooperative task to be completed. The advantage of an
enzyme-based system is that it can combine communication and production of a useful
molecule via the secreted enzymes.
ENGINEERING UNIDIRECTIONAL COMMUNICATION
Early examples of gene circuits that employ QS elements have focused on communication
between cells within a single population,
18
or unidirectional communication between
two populations. In 2004, Basu et al. implemented a QS-based circuit to control the
spatiotemporal expression of genes.
19
Their system consisted of two
E. coli
populations: a
sender population and a receiver population. In the sender population, the LuxI protein,
which synthesizes 3-oxohexanoyl-homoserine lactone (3OC6HSL), is under the control of
a TetR-repressible promoter (P
LtetO-1
). Upon induction of P
LtetO-1
by anhydrotetracycline
(aTc), 3OC6HSL is synthesized and accumulates in the culture. The receiver population
constitutively expresses a 3OC6HSL receptor protein, LuxR. At a sufficiently high
concentration, 3OC6HSL activates LuxR, which in turn activates expression of the CI
repressor protein from
phage, which is under control of the cognate promoter (luxP
R
)of
LuxR. Finally, the circuit readout, a green fluorescent protein (GFP), is under the regulation
of a dual promoter (luxP
R
cI-O
R
1), which is repressed by the CI repressor protein, but
activated by active LuxR.
λ
The logic of this circuit is as follows: addition of aTc leads to the synthesis of 3OC6HSL
from the sender population. As the concentration of 3OC6HSL builds up in the culture,
it binds to LuxR and activates expression of both CI and GFP in the receiver population.
However, once CI reaches a sufficiently high concentration in the cell, it represses GFP
expression. Overall, this circuit is expected to generate a transient, pulsatile response in
GFP expression in the receiver population.
245
To test the receiver circuit function, the authors inoculated the receiver cells in a liquid
culture, applied various concentrations of exogenously added 3OC6HSL, and examined
GFP expression using cell sorting. Indeed, the receiver cells generated a transient pulse of
GFP. The amplitude and length of the GFP expression could be modulated by the amount
of exogenously added 3OC6HSL or the rate increase of 3OC6HSL. In the latter property,
increasing the rate of 3OC6HSL concentration led to higher peak expression of GFP.
As such, the authors surmised that, when the sender and receiver cells were coinoculated
on solid medium (i.e. the spatial domain), the distance between the sender and
receiver populations would lead to a differential response in GFP expression. Here, the
accumulation rate of 3OC6HSL in the culture would depend upon the distance from the
sender population. Indeed, when both populations were seeded on an agar plate, the
authors demonstrated that receiver cells that were closer to the sender population had a
stronger pulse of GFP expression than the receiver cells farther away from the sender
population.
In 2005, Basu et al. used the same sender population and a redesigned receiver population
to generate a consortium able to generate robust spatial patterns.
20
Again, the receiver
cells constitutively express LuxR. They also express the CI repressor and a modified LacI
repressor (LacI
M1
), both driven by 3OC6HSL responsive P
luxR
promoter, as well as the wild-
type LacI repressor under control of a CI-repressible promoter (
P(R-O12)). As an output,
the expression of GFP is driven by a P
lac
promoter, which is repressed by either LacI
M1
or LacI.
λ
Using a mathematical model, the authors predicted that the circuit could generate ring patterns
of fluorescent protein expression in receiver cells in response to an AHL gradient generated
