Team:TU Delft/Project/solubility/characterization


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Characterization of the Hydrocarbon Solubility Parts

Protein Production Analysis

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. Because the IPTG inducable protomor is generally regarded as verey strong, we expect that the resulting gel wil show when the expression of AlnA begins and when it reaches its maximum.

Standard Emulsifier Assays

Figure 1
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, we were unable to reproduce them. In our opinion measuring turbidity turned out to be rather arbitrary.

Example from literature

Figure 1 on the right shows a calibration graph with 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

Our Emulsifier Assay

Ball-and-stick model of the Sudan II molecule. Source Wikipedia

That is why, instead of a colorless hydrocarbon, we choose to use the orange Sudan II dye. This molecule stains non-polar molecules and hardly dissolves water. It makes measuring the emulsification much easier, because it has an orange color that causes a nice absorbance peak at 493 nm.

Calibration and Background

File:TU Delft emulsification assay calibration curve.jpg
Emulsification Assay calibration curve
The assay was calibrated using SDS, because this detergent is widely used and makes the outcome of the assay comparable 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.