Signalling

Part:BBa_F2620:Specificity

Designed by: Barry Canton [bcanton@mit.edu] and Anna Labno [labnoa@mit.edu]   Group: MIT   (2004-08-09)
Input: 3OC6HSL
Output: PoPS

tetR
R0040
LuxR
I0462
lux pR
R0062
BBa F2620Icon.png

Part Main Page        Transfer Function        Specificity        Response time        Stability        Add Data       


Specificity describes the ability of a device to distinguish between its cognate input and other similar inputs. There are many AHL molecules with a structure similar to 3OC6HSL. Some of these might be used in a system alongside 3OC6HSL and BBa_F2620. Hence, it is necessary to characterize to what extent, if any, BBa_F2620 responds to alternate AHL molecules. Please see the part experience page for a general description of our characterization method.

Data

Figure 1 - Transfer function of BBa_F2620 for 8 different input molecules. The error bars represent 95% confidence intervals measured over three replicates.
Data Notes Date Uploaded
File:BC-Specificity-R1-P1.txt Raw data for C4HSL, C6HSL, and 3OC6HSL 03/28/07
File:BC-Specificity-R1-P2.txt Raw data for C7HSL, C8HSL, 3OC8HSL 03/28/07
File:BC-Specificity-R7-P1.txt Raw data for C10HSL, C12HSL 03/28/07

Protocol

  1. Two cultures, one MG1655 bearing BBa_T9002 and one of MG1655 bearing the BBa_T9002 mutant lacking a GFP expression device (see stability section) were prepared as described in steps 1 – 3 of the transfer function section above. However, in this case the overnight cultures were diluted into 20 ml of fresh medium in a 200 ml flask and shaken at 220 rpm during growth.
  2. Three of the eight AHL variants (see table below) were preloaded into a flat-bottom 96 well plate to eight different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 M, and 1E-4 M). Three wells were each filled with 200 μl of media to measure the absorbance background. Three further wells were filled with 200 µl of the mutant BBa_T9002 culture to measure the fluorescent background.
  3. Seventy-two 200 µl aliquots of the BBa_T9002 culture were transferred to the plate. Three replicate wells were filled for each concentration of each AHL.
  4. The plate was incubated in a Wallac Victor3 multi-well fluorimeter at 37°C and assayed with an automatically repeating protocol of absorbance measurements, fluorescence readings, and shaking (as described in the transfer function protocol). Time between repeated measurements was 2 min and 21 s.
  5. Steps 1 through 4 were repeated once with three more of the AHL variants and again with the final two AHL variants. The time between repeated measurements was kept fixed in each case.
  6. Data processing is described here. In Figure 1, snapshot transfer functions are plotted for each AHL variant at the 60 min time point similar to the transfer function experiment. The error bars represent the 95% confidence interval for the mean of the three replicate wells of each measurement.
Full Name Molecule abbreviation Species Notes Source Images (from Sigma Aldrich)
Butanoyl-homoserine lactone C4HSL P. aeruginosa Sigma Aldrich (#09945) Butryl-homoserine lactone.GIF
3-oxohexanoyl-homoserine lactone 3OC6HSL V. fischeri Lux system signaling molecule Sigma Aldrich (#K3007) 3-oxohexanoyl-homoserine lactone.GIF
Hexanoyl-homoserine lactone C6HSL C. violaceum Very similar to 3OC6HSL Sigma Aldrich (#09926) Hexanoyl-homoserine lactone.GIF
Heptanoyl-homoserine lactone C7HSL E. psidii R. IBSBF 435T Sigma Aldrich (#10939) Heptanoyl-homoserine lactone.GIF
Octanoyl-homoserine lactone C8HSL B. cepacia, V. fischeri Sigma Aldrich (#10940) Octanoyl-homoserine lactone.GIF
3-oxoctanoyl-homoserine lactone 3OC8HSL A. tumefaciens Sigma Aldrich (#O1764) 3-Oxooctanoyl-L-homoserine lactone2.GIF
Decanoyl-homoserine lactone C10HSL B. pseudomallei Sigma Aldrich (#17248) Decanoyl-homoserine lactone.GIF
Dodecanoyl-homoserine lactone C12HSL Synthetic Sigma Aldrich (#17247) Dodecanoyl-homoserine lactone.GIF