Team:TU Delft/Project/solubility/characterization


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Characterization of the Hydrocarbon Solubility Parts

Emulsifier Production

The alna gene was induced when the culture reached a density of about 108 bacteria per mL. Bacterial and medium samples were taken for sodium dodecyl sulfate (SDS)-gel electrophoresis to monitor AlnA production. The results shown in Fig. ? indicate that expression of AlnA begins ? min after induction and peaks after ? h.

Emulsifier Assay

Calibration graph showing a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate. Rajakumari et al (2006) Biochemical and Biophysical Methods
We developed a new assay to measure the emulsification caused by AlnA. Assays currently described in literature involve spectrophotometric measurements of turbidity after mixing. Although many articles show nice graphs (see textbox on the right), we were unable to reproduce them. Measuring turbidity turned out to be rather arbitrary.
Ball-and-stick model of the Sudan II molecule, an orange-red azo dye used for staining of non-polar substances. Source Wikipedia

Instead of a colorless hydrocarbon we choose to use the orange Sudan II dye. It stains non-polar molecules and hardly dissolves water. Meanwhile it has a nice absorbance peak at 493 nm, which makes it convenient to measure.

Calibration and Background

File:TU Delft emulsification assay calibration curve.jpg
Emulsification Assay calibration curve
The assay was calibrated using SDS to be able to compare it to existing assays. Our measurements are shown in the graph on right. It shows that the assay is very sensible at low SDS concentrations.

Next, the influence of the culture media was determined. We used LB medium for the initial growth of the cells, and induced the production of AlnA in M9 medium. The background influence of the media on the assay were measured, as shown below. It shows that LB medium already has some emulsification effect, but M9 shows very little influence.