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Figure 7.26
Time-series fluorescence images illustrating the response of smaller
colonies of receivers to communication from nearby senders on a plate.
microscope using a Plan Fluor 1
×
objective. The physical width of each image
is approximately 7 mm.
First, a brightfield image was captured to record the location of the droplets.
Then, a series of fluorescence images was captured at 1-min intervals using
the green filter. The cyan semicircle in each fluorescence image is an artifi-
cial marker of the location of the sender droplet, superimposed based on the
brightfield image.
The first fluorescence image was captured 10 min after spotting the droplets.
It shows that the receiver cells closest to the senders have already started re-
sponding to the VAI message. The two subsequent images display an increase
in fluorescence intensity due to the diffusion and accumulation of VAI. Based
on the fluorescence response, VAI appears to diffuse at approximately 1 cm/h
through the agar.
The third visual observation experiment captured a time-lapse series of im-
ages with smaller colonies of receivers and a smaller droplet of servers (Figure
7.26). Sender cells were grown overnight at 37°C, shaking at 250 rpm, in 2
ml LB Amp/Kan inside 14 ml Falcon polystyrene tubes. Receiver cells were
picked from
−
80°C cell stock into 1000
µ
l LB Amp/Kan medium, then 20
µ
l
was plated on LB Amp/Kan and incubated at 37°C for 7 h. Then 0.3
lof
sender cell were spotted, and brightfield and fluorescence images were cap-
tured as above. The three images show the communication gradient over time
across the different receiver colonies.
µ
Similar Signaling Systems
This section describes work that successfully isolates an important intercellular
communication mechanism from a naturally occurring bacterial system, ana-
lyzes its components, and engineers its interfaces with standard genetic control
and reporter mechanisms. While the work reported in this section captures
