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Template:Team:SydneyUni Australia/ProtocolHTML
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A method based on that of Bergmann and Sanik (Bergmann and Sanik, 1957) was used for quantitation of inorganic chloride, as follows. Resting cell supernatant (1 mL) was mixed with 200 µL Iron Reagent and 400 µL Mercury Reagent, incubated 10 min at room temperature, and the absorbance read at 460 nm. Chloride concentration was calculated via reference to a standard curve based on NaCl solutions in KP buffer (0, 0.1, 0.2, 0.5, 1.0 mM). Due to the time sensitivity of this assay, standards were prepared alongside every set of test samples, and processed at the same time. Test samples were diluted where necessary to bring them within the absorbance range of the standard curve (A460 approximately 0.045 – 1.080). The whole experiment was repeated three times, and the stoichiometry of chloride release calculated for each experiment as the number of moles Cl- produced per mole of chlorinated substrate degraded. The stoichiometry values from three independent experiments were averaged and statistical analysis were carried out using GraphPad Prism software using the one-way ANOVA (and nonparametric) test.
- We have a stock solution of NaCl in KP buffer at our bench in the lab (but this could easily be made up), and Hugh has also made up some different solutions for the reference curve.
- There are (up to) six vials that will be tested, these are in the cool room. Two are controls, which have E. Coli cells transformed with pBBR, two have E. Coli transformed with pBS-ToMO (toluene-o-xylene monooxygenase) and two have E. Coli transformed with pBS-TOM (toluene ortho-monooxygenase). There are two of each because one vial has DCA, the other doesn’t.
- From our GC results last week, it appeared that only ToMO attacked DCA. [RESULTS FROM GC - pBBR(DCA) and TOM(DCA) had an area of ~500,000 at 2.8 minutes, whereas ToMO(DCA) had an area of ~280,000 at 2.8 minutes. ToMO(control) had something like 30,000 at 2.1 minutes. The results are in the lab-book and the vials are in the cool room. Every run is saved with a new file name on the GC computer except the ToMO(control), which I accidentally overrode.]
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