Native N-3-Oxo-Hexanoyl-l-Homoserine Lactone tests using the Tecan Infinite M200 PRO™ plate reader

File:Gfp od sigmoid1.png

In order to select mutated pLuxR promoters we need to know about the sensivity of the Wild-Type type. The test range was inspired form the paper about Evaluation of a focused libraryu of N-Acryl L-Homoserine lactone reveals a new set of potent quorum sensing modulators.The paper shows sensivities of the pLuxR promoter to different sets of OHHL molecules in Vibrio fisheri. We need to readjuste the ranges for our E.coli DH5alpha strain in a second run and finally get our sensivity curve over the linear range of [0 nM],[0.25 nM],[0.5 nM],[1 nM],[2 nM],[3 nM],[4 nM],[5 nM],[10nM],[20 nM],[30 nM],[40 nM]and[50 nM],.

The experimental set-up included a 96-well plate filled with 180 uL LB media , 10uL of receiver cells and 10 uL of different OHHL concentrations, everything in triplicates. (+ blank and negative control). The experiment runs for 16 hours in order to monitor the evolution of fluorescence and later choose the steady state to create the sensivity curve.The datas are analyzed with GraphPad Prism 6.0.

PluxR random mutagenesis PCR

PluxR promoter with different sensitivity analyzed using the single cell analyzer BD LSRFortessa™ Flow Cytometer System

In order to obtain more precise data out of our fluorescence analysis to better characterize the different promoters we use the single analysis.
The protocol was the following :
From overnight cultures we inoculate 50mL Falcon™ tubes containing 5mL LB media, 5uL OHHL and the antibiotic. We evaluate a range of 10 different concentrations from
10^-4nM OHHL to 10⁵nM OHHL in duplicates according to the results of the plate reader analysis.

Figure 2. Wild type sensitivity curve using the BD LSRFortessa™ Flow Cytometer System

The first graph (on the left) is the sensivity curve of the J08955-GFP construct with the wild type R0062 promoter. The EC₅₀ indicates the half maximal effective concentratio and is 0.02nM for the wild type. We had a bit background noise during the fluorescence measurements of the wild type construct, that's why the R²=0.8.
The second graph (on the right) are the fluorescence in function of the side scatter. The side scatter is a measurement of refracted light that occurs at each interface in the cell. The refracted light is collected and amplified in a photomultiplier tube. The SSC gives information about the cellular granularity and complexity (e.g. dead cells).

Figure 3. PluxR1 (G1) mutated promoter sensitivity curve using the BD LSRFortessa™ Flow Cytometer System

Figure 4. Comparison of the Wild Type and PluxR1 mutant sensitivity curve using the BD LSRFortessa™ Flow Cytometer System

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