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Applications of BBa_C0160

Characterization by ETH Zurich 2015

We wanted to know the effect of constitutively expressed aiiA versus no aiiA upon artificial addition of AHL onto P_lux.

Genetic design

We designed two plasmids:

Medium copy backbone with P_lux-GFP and BBa_J23118-BBa_B0032-luxR-BBa_B0012
Medium copy backbone with P_lux-GFP, BBa_J23118-BBa_B0032-luxR-BBa_B0012 and BBa_J23114-BBa_B0034-aiiA

Plate reader

E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (50 µg/mL) and chloramphenicol (13 µg/mL) at 37°C and 200 rpm. Cultures were diluted 1:50 in fresh LB with the corresponding antibiotic and transferred to a 96-well plate (200 µL/well). Cultures were grown for 90 min to arrive to exponential phase and then different concentrations of AHL were added. Samples were always made in triplicates and a blank of LB with the corresponding lactate concentration was done. During 7 h the absorbance at OD600 and fluorescence (excitation 488 nm and emission 530 nm) were measured with intervals of 7 min. The plate was always kept at 37°C in a Tecan Infinite M200 Pro Plate Reader. We calculated dose-response curves from the exponential phase of the bacteria using fluorescence divided by OD.

Results

In Figures 1 and 2 you can see that the K_M for the system is shifted when AiiA is present in the system, making it less sensitive to AHL.

Figure 2. Response to AHL of a plasmid not containing AiiA

Figure 1. Response to AHL of a plasmid containing AiiA

Modeling

Each experimental data set was fitted to a Hill function using the Least Absolute Residual method (Fitting Toolbox in MatLab).

Where:

al: leakiness in µM/min
mu: constant in µM/min
K_M: half-maximum effective concentration (a representation of sensitivity) in µM
n: Hill coefficient (a representation of cooperativity), unitless

	PresentAii	Absent AiiA (lumped parameters)	Absent AiiA (literature parameters)
Coefficient values	K_M=1e+06 (fixed at bound)	K_M=1e+06 (fixed at bound)	K_M = 9.983 (8.929, 11.04)
	al = 6134 (5222, 7046)	al = 5711 (-2.36e+04, 3.502e+04)	al = 2876 (1633, 4119)
	mu = 2e+04 (fixed at bound)	mu = 2e+05 (fixed at bound)	mu = 3.484e+04 (3.338e+04, 3.63e+04)
	n1 = 1 (fixed at bound)	n1 = 1.646 (-5.203, 8.494)	n1 = 1.089 (0.8608, 1.317)
Goodness to fit	sse: 5.2051e+09	sse: 3.8192e+12	sse: 1.4366e+09
	rsquare: 0.8768	rsquare: -89.3659	rsquare: 0.9986
	fe: 10	dfe: 9	dfe: 7
	adjrsquare: 0.8768	adjrsquare: -99.4066	adjrsquare: 0.9980
	rmse: 2.2815e+04	rmse: 6.5142e+05	rmse: 1.4326e+04

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