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Characterization of the Receiver Module
The genetic circuit to receive messages (plasmid pRCV-3) was further charac-
terized by inducing the promoter with different levels of VAI extracted from cell
culture. Cultures of Vibrio fischeri and of E. coli containing the pTK1 plasmid
were grown overnight to stationary phase in Good Vibrio Medium (GVM)
broth or LB Amp, respectively, at 30°C, which allows evaluation of their bi-
oluminescence. After verification of light production, 100 ml of the cultures
were centrifuged at 3300 g , and the supernatant collected. The supernatant was
extracted with 10 ml of ethyl acetate by vigorous shaking in a separatory funnel
for 10 min. The ethyl acetate extract (upper fraction) was separated and dried
under vacuum. The resulting crude extract was redissolved in 1 ml of deionized
water to provide 100
VAI extract.
We analyzed the effectiveness of serial dilutions of the VAI extracts from
pTK1 and Vibrio fischeri in inducing GFP expression of the pRCV-3 cells.
Both the Vibrio fischeri and pTK1 extracts were about equally effective at
inducing expression of the pRCV-3 promoter, as measured by GFP production.
Cells with different levels of VAI were incubated at 37°C for 4 h, and the
maximum fluorescence achieved for each culture was recorded. Figure 7.18
shows that increasing levels of autoinducer yielded increasing GFP expression
by the receiver. High levels of the extract, however, were toxic to the cells and
resulted in relatively low fluorescence levels.
×
Maximum Fluorescence of pRCV-3
in Response to Different Levels of Autoinducer
1,200
1,000
800
600
400
200
0
0.1
1
10
Autoinducer Level
Figure 7.18 The effect of different autoinducer levels on the maximum fluorescence
attained by the receivers.
 
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