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80,000
60,000
BL21-LuxITet
DH5a-LuxITet
RCV Only
40,000
20,000
0
aTc concentration (ng / ml)
Figure 7.21
Controlling the sender's signal strength: maximum response of receivers
to different anhydrotetracycline (aTc) induction of senders. The 10
×
V. fischeri
auto-
inducer (VAI) positive control column shows the results of introducing the VAI ex-
tract into the wells. The rest of the columns illustrate the response of the receivers
from induction of the senders with various levels of aTc.
the cells to constitutively express
luxI
, resulting in constant transmission of the
message.
Second, pRCV-3 and pPROLar.A122 were transformed into
E. coli
JM2.300
cells to create receiver cells. The pRCV-3 plasmid instructs the cell to express
GFP(LVA) when VAI enters the cytoplasm. The pPROLar.A122 plasmid con-
fers kanamycin resistance to the cell. Therefore, these sender and receiver cells
have both kanamycin and ampicillin resistance.
In the experiments reported here we used a Nikon Eclipse E800 fluores-
cence microscope equipped with a Hamamatsu C4742 ORCA I CCD camera
controlled by QED Imaging software. The cyan fluorescence filter is chroma
cyan GFP (excitation: 436/20 nm, emission: 480/40 nm), the green fluores-
cence filter is a chroma fluoroscein isothiocyanate/EGFP (excitation: 480/40
nm, emission: 535/50 nm), and the yellow fluorescence filter is a chroma yellow
GFP (excitation: 500/20 nm, emission: 535/30 nm).
In the first visual observation experiment, sender and receiver cells were
grown separately overnight at 37°C, shaking at 250 rpm, each in 2 ml LB
Amp/kanamyain (Kan) and 1 mM IPTG inside 14 ml Falcon polystyrene
tubes (352051). Then, the JM2.300[pSND-1/pINV-112-R3] cells were pel-
leted at 6800 rpm and resuspended in fresh 50
l LB Amp/Kan 1 mM IPTG.
The JM2.300[pRCV-3/pPROLar.A122] cells were pelleted at 6800 rpm and
µ
