Information Technology Reference
In-Depth Information
Sender Cells
Receiver Cells
VAI
VAI
lux P(R)
GFP(LVA)
LuxR
lux P(L)
rrnB T1
rrnB T1
P(LtetO-1)
LuxI
T1
Fragment of pRCV-3
2038 bp (molecule 4149 bp)
*
Fragment of pLuxI-Tet-8
1052 bp (molecule 2801 bp)
* E. coli strain expresses TetR (not shown)
Sender Cells
Receiver Cells
LuxR
0
GFP
tetR
0
luxI
VA I
VAI
aTc
aTc
pLuxI-Tet-8
pRCV-3
Figure 7.19
Genetic and logic circuits for pLuxI-Tet-8 sender and pRCV-3 receiver.
Sending Controlled Cell-to-Cell Signals
The third experiment characterized the response of the receivers to variations in
the strength of the message transmitted by the senders. For this, the
LuxI
gene
was placed under the control of the Tet promoter from the Clontech pPROTet
system. Figure 7.19 provides a schematic representation of the experiment.
In one cell, the pLuxI-Tet-8 plasmid exerts controlled expression of the LuxI
autoinducer synthesase using the Tet operon. The synthesase catalyzes the con-
version of normal cellular metabolic products into VAI; thus, controlling the
LuxI
expression level controls the VAI production in the cells. The VAI pro-
duced within the cells migrates though the cell membrane of the sender, into the
culture medium, and through the membrane of the receiver—a cell containing
the pRCV-3 plasmid. There, it interacts with the N-terminal domain of the
LuxR DNA-binding protein product, disabling it from binding to the Lux box
binding site. The expression of the GFP reporter gene is enhanced, resulting in
high levels of fluorescence (Figure 7.20).
The experiment involved the incubation of similar mixed-cell cultures on
96-well, clear-bottom plates. One important difference was the culture medium:
the pPROTET cells carry spectinomycin and chloramphenicol resistance, while
the pRCV-3 cells carry ampicillin resistance. The experiments were carried out
by growing overnight cultures of both types of cells in the appropriate antibiotic
containing medium, followed by centrifugation at 4000
g
to remove the medium
and resuspension to similar cell density in LB containing no antibiotics, so that
both cell types could grow.
Three rows of pRCV-3 cells were loaded on a microplate, and two of these
rows were also loaded with pLuxI-Tet-8 cells. The sender cells in the var-
