Information Technology Reference
In-Depth Information
Sender Cells
Receiver Cells
VAI
VAI
lux P(R)
GFP(LVA)
rrnB T1
p(LAC-const)
LuxI
rrnB T1
LuxR
rrnB T1
lux P(L)
rrnB T1
Fragment of pSND-1
1937 bp (molecule 3052 bp)
Fragment of pRCV-3
2038 bp (molecule 4149 bp)
Sender Cells
Receiver Cells
LuxR
0
GFP
luxI
VA I
0
VAI
pSND-1
pRCV-3
Figure 7.15
Genetic and logic circuits for pSND-1 sender and pRCV-3 receiver. The
sender cells constitutively express
luxI
, which catalyzes the formation of
V. fischeri
autoinducer
(VAI). VAI diffuses into the environment and neighboring cells, which
detect VAI through the transcriptional activation of lux P(R).
directs the cell to continuously send the VAI message. The pRCV-3 plasmid
encodes a circuit that directs the cell to express GFP(LVA), a variant of the
green fluorescent protein from Clontech, when VAI enters the cell (Figure 7.15).
Cultures of
E. coli
DH5
α
transformed with the pRCV-3 plasmid and cultures
of
E. coli
DH5
transformed with the pSND-1 plasmid were grown separately
overnight at 37°C in Luria-Bertani ampicillin (LB AMP). A 96-well clear-
bottom plate was loaded with 200
α
µ
l of LB AMP in each well. We loaded
10
l of pSND-1 cells horizontally to each well, along with controls consisting
of cells expressing GFP(LVA) constitutively with the pRW-LPR-2 plasmid,
E.
coli
DH5
µ
containing pUC19 to serve as a negative control, and a series of
wells containing extracted VAI (see below).
Vertically, 10
α
l of cells containing the pRCV-3 construct were also loaded
into each well. Thus, each well contained a variety of senders and a uniform set
of receivers. The plate was grown in a Biotek FL-600 fluorescent plate reader
for 2 h, and fluorescence at the GFP(LVA) peak (excitation filter 485/20 nm,
emission filter 516/20 nm) was measured every 2 min. Figure 7.17 shows the
time-series response of the different cultures. Wells containing only the pRCV-
3 cells, or with added pUC19 cells, showed no increase in fluorescence. The
well containing pRCV-3 cells and pRW-LPR-2 cells (which express GFP[LVA])
served as a positive control for high levels of fluorescence. Wells containing
the pRCV-3 cells plus extracted pTK1 autoinducer showed high and increasing
levels of fluorescence. Cells with pRCV-3 and pSND-1 showed the expected
increase in fluorescence demonstrating successful cell-to-cell signaling.
µ
