Part:BBa_K150000:Experience

Characterization of 3OC₆HSL sender device (BBa_K150000)

Quantification of 3OC₆HSL (AHL) production

For AHL quantification the amount of AHL in supernatant of producing cells (E. coli TOP 10 containing BBa_K150000) was determined. Therefore eight samples containing TB-media were inoculated with 2 µl/ml of an AHL producing cell overnight culture at 37 °C. Sterile filtered supernatant of these samples was produced hourly for 7 hours and stored at 4 °C. To estimate the amount of AHL producing cells the optical density was measured before. (Figure 1, top left).

Figure 1: Results of sender activity test. Top left: Growth curve of sender culture. Top right: GFP/OD values of sender supernatants at t = 10 h. Bottom left: GFP/OD – AHL calibration curve. Dotted line visualized regression of an exponential growth curve. Bottom right: Produced AHL concentrations of the sender cells.

For the AHL determination 1 ml supernatant was diluted with 1 ml TB-media and inoculated with a constant amount of AHL inducible cells which produce GFP after induction (BBa_T9002 in E. coli TOP 10). The OD and GFP intensities were measured at 37 °C half-hourly for about 28 hours. For calibration the same test were performed with distinct AHL concentrations from 0M to 50 nM (SIGMA). Figure 2 shows the GFP/OD development over the time depending on the sender supernatants (left panel) or the preassigned AHL concentrations (right panel). The values increase and reach a maximum after 6 hours. Then they decrease slightly.

Figure 2: GFP/OD results of sender activity measurements. Left panel shows the GFP/OD values for induction with sender supernatants. Right panel shows the GFP/OD values for predefined AHL inductions. During the first six hours the values increase and reach a maximum until the decrease slightly. The values at t = 10 h were used for calculations of AHL concentrations in supernatants.

A GFP/OD – AHL calibration curve (Figure 1 bottom left) was calculated using the GFP/OD values at t = 10 h. An exponential growth curve was fitted to these data (blue dotted line; y = 4325 * e^0.00346*x). The AHL concentrations of the sender supernatants were determined using the GFP/OD values at t = 10 h and the exponential regression. In the course of the time the AHL concentration increase until it reaches a certain maximum before a slight decrease (see Figure 1, bottom right, Table 1). The maximum is reached at the end of the logarithmic phase. Several tests have shown that the maximal concentration that can be produced by part BBa_K150000, is approximately 1100 and 1300 pM.

Table 1: Results of AHL production determination

time [h]	GFP/OD	GFP/OD STD	c(AHL) [pM]	c(AHL) STD [pM]
0	5427,847	48,01405	135,7623515	5,310956899
1	8713,655	235,8338	418,6964705	16,40039009
2	11849,3	296,7075	602,4247781	15,1577546
3	15982,94	1123,325	781,2969295	43,55933826
4	25196,72	720,799	1053,378175	17,34848139
5	36745,75	652,7182	1278,906415	10,71294138
6	28636,28	2980,17	1129,8639	65,68601047
7	33271,21	2281,635	1219,533962	42,46359884

Applications of BBa_K150000

This 3OC₆HSL sender was succesfully tested in an prey killer system with part BBa_K150009 of iGEM Team Heidelberg 2008 ([http://2008.igem.org/Team:Heidelberg/Project/Killing_II click for more details...]).

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