IGEM:IMPERIAL/2007/Projects/Biofilm Detector/TestingValidation

Infector Detector: Testing/Validation

• Introduction
• Specifications
• Design
• Modelling
• Fabrication
• Testing
• Data Analysis
• Validation
• Notes
• References

Testing on Simulated Biofilms (Agar with AHL)

Materials and Method

Fluorometer plate arrangement for the agar film tests.

It is necessary to test the Infector Detector system in an environment similar to that of its intended operation, if not in the environment itself. It was found that biofilms can be modelled by a gel with pore size of 80nm and fiber thickness of 0.6nm[1]. Agar gel, prepared at a concentration of 2% by weight has approximately these properties[2].

Therefore, an agar gel was prepared at a concentration of 2% (by weight) and with AHL dissolved at different concentrations (3nM, 5nM, 7nM, 10nM). 40μl of these gel samples were then pipetted into wells in a 96-well fluorometer plate, according to the diagram on the left. Three of the 10nM AHL agar containing wells were further treated with 10μl of 10x diluted stock GFP solution. Additionally, several control wells were set up, containing: agar without AHL; agar without AHL and with GFP; GFP only; and blanks (empty wells).

The surface tension between plastic and molten agar solution is difficult to pipette and spread at the bottom of the wells. Therefore, at least two wells were loaded with each sample, in order to determine the variability of measurements as a result of the well-loading procedure.

Similarly, three wells were loaded with 10μl of GFP solution, because the small volume of the solution makes it difficult to spread the solution evenly at the bottom of the plate. Finally, several empty wells were also measured, in order to look at the background fluorescence in them.

The 96-well plate was then loaded onto the fluorometer and fluorescence was measured with a counting time of 0.6s, with four repeats per well, using a filter for insert wavelength.

Results

Logarithmic plot of average fluorescence (with maximum and minimum shown as error bars) for each of the agar film tests and controls. Note that because the plot is logarithmic, the error bars are as well - the error for GFP only is smaller than that for both Agar with GFP samples.

References

1. Measurement of local diffusion coefficients in biofilms by microinjection and confocal microscopy Dirk de Beer, Paul Stoodley, Zbigniew Lewandowski 1997 Biotechnology and Bioengineering: Volume 53, Issue 2 , Pages 151 - 158

2. Determination of agarose gel pore size: Absorbance measurements vis a vis other techniques Janaky Narayanan, Jun-Ying Xiong and Xiang-Yang Liu 2006 J. Phys.: Conf. Ser. 28 83-86