This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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

User Reviews

UNIQ1141f9e9a1dd9588-partinfo-00000000-QINU

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

UNIQ1141f9e9a1dd9588-partinfo-00000002-QINU