- To have good sample runs clean the wells carefully. Using a
pipette set at 100μl and fitted with 1–200μl gel-loading tip,
pipette in electrophoresis buffer into wells (3–5 times) to
remove any residual polymerized acrylamide on the loading
well walls that might stick to sample and create a drag during
sample run. - Under the given growth conditions forV. harveyiBB120,
dilutions ranged from 10^6 -fold at start (0 h) to 5 107 -fold
at 10 h and later. It is recommended that a pilot experiment be
conducted to determine the dilution series required for the
strain of bacteria you are working with before you take up the
biosensor quantification. - Filtering and centrifugal devices fitted with Omega™and HT
Tuffryn membranes (Pall Life Sciences) were found to have less
interference with BAI-2 ligand sticking to membrane. So care
must be taken with the choice of filtration/centrifugation
device to identify the best membrane for this application. - If later time point cultures have cell debris that clogs the 3 kD
membrane, try including an additional step of passing the cell
supernatant through 0.2μm HT Tuffryn®membrane syringe
filter before 3 kD membrane filtration. - Care must be taken not to vortex the contents; this may result
in CLPY protein denaturation and loss of biosensor
functionality. - The assay has also been successfully adapted for use in a 96-well
plate format [18]. Such adaptation could increase the number
of assays that can be run from a single CLPY biosensor prepa-
ration and has potential for use in high-throughput assays. - In case the bacterial strain you are working with has noluxS
deletion derivative, try using borate-free AB medium-grown
culture filtrate as control. - Chloride ions (Cl) can affect YFP fluorescence [19]. You will
note that the FRET ratios are lower in the presence of chloride
due to YFP fluorescence (CFP remains unaffected). So care
must be taken to minimize changes in buffer and media con-
centrations between samples. FRET ratio of mature CLPY with
the choice of bacterial media can be initially determined as your
reference FRET ratio for the experiments involving culture
supernatants. For spent media experiments, to achieve same
chloride ion concentrations in all dilutions of test samples, use
desired volumes of fresh media with spent media and monitor-
ing the biosensor response.
Quorum Sensing Biosensor for AI-2 Detection and Quantification 87