COPPER BINDING ASSAY

To begin, our team determined the standard copper concentration curve by measuring the average absorbance of various copper concentration solutions, and determined that the standard curve is linear. We can then relate the absorbance to the amount of copper left in solution; therefore, we can determine how efficient the metal binding proteins are, how many ions can be removed, the protein activity, optimal concentration of binding and optimal time of binding.

We added the CutA protein to the copper solutions and left the samples for several time intervals. After the given time intervals, the process of salting out was used to aggregate the remaining proteins in the solution while they remained bound to the copper. Then, following centrifugation, we measured the absorbance of the remaining solution, making sure to minimize capturing protein from the sample. At approximately an hour the absorbance was lowest in all samples demonstrating that the most copper was bound at that point. The concentration at 151mg/L seems to show the optimal amount of binding over time. However, our data does not show a significant change in absorbance and therefore there is not a significant change in the amount of copper ions being bound by the protein . This is likely because there was not enough protein being introduced into the reaction during our assay. To validate this idea we created a model demonstrating the binding events in our assay. This model showed that insufficient amounts proteins were indeed the issue and is explained more in depth on the modelling page. (Click here to view modelling page)

BACTERIOPHAGE ASSAY

The relationship between bacteriophage and bacteria is crucial to the implementation of our project. To demonstrate this relationship and help to improve our mathematical modelling, we completed a phage assay. We used a 96 well plate and filled various wells with a specific amounts of bacteria. Then introducing various concentrations of phage to specific wells, and using a plate reader we measured the absorbance during 23 hours.

The results demonstrate the growth curve of the bacteria, and while the bacteria are still growing, they grow at a slower rate than they would be without the phage present. In addition, the maximum amount of bacteria decreases when phage are present. The starting OD is higher in the graph than in the legend, and this is due to the bacteria reproducing during the time period between when the original dilutions were measured and when the sample was measured after the phage were added. Our phage assay demonstrates that it is likely the phage are infecting the bacteria, as a higher concentration of phage resulted in decreased bacterial growth. Furthermore, increased numbers in bacteria result in decreased growth, and it can be interpreted that this is as a result of the phage having an abundance of bacteria to infect. This increases the rate at which the bacteria are infected and results in a less extreme growth curve.