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	==Characterization of the Hydrocarbon Solubility Parts==		==Characterization of the Hydrocarbon Solubility Parts==
-	===Emulsifier Production===	+	===Protein Production Analysis===
-	The ''alna'' gene was induced when the culture reached a density of about 10<sup>8</sup> 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.	+	The ''alna'' gene was induced when the culture reached a density of about 10<sup>8</sup> 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.
	===Emulsifier Assay===		===Emulsifier Assay===

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Revision as of 09:13, 20 October 2010

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.

Emulsifier Assay

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. In our opinion measuring turbidity turned out to be rather arbitrary.

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. Meanwhile it has a nice absorbance peak at 493 nm, which makes it convenient to measure.

Calibration and Background

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.