PROTEIN PURIFICATION

One of the major aspects of our system utilizes metal binding proteins, so it was imperative that we purify the protein in order to move forward and complete our Copper Binding Assay. Our team successfully purified the Cut A protein through Nickel Affinity Chromatography (click here ) and Size Exclusion Chromatography (click here ).

The CutA protein was expressed in BL21 E.coli cells, and those cells were lysed then centrifuged to separate the supernatant and cell pellet. The lysate was then introduced to a Nickel Sepharose affinity column to isolate the CutA protein as it was bound to the Nickel Sepharose by its histidine tag. Then after washing to remove the unwanted proteins and cell debris, the CutA protein was eluted from the Nickel Sepharose.

To further purify CutA, we ran the partially purified protein solution through a column, that contained beads. The beads have small crevices and this causes proteins of different sizes to pass through during different times.

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.

ELASTIN-LIKE POLYMER SYNTHESIS

Our team completed elastin-like polymer (ELP) synthesis because the repeating amino acid sequence is very long and difficult to order in one large sequence. Therefore we ordered the sequence in smaller pieces and completed extension PCR to overlap and add on another piece of the ELP. In the future, we hope to experiment and test the properties of the ELP. We successfully extended the ELP by connecting the first, second and third parts. The procedure of this experiment will be elaborated on in the design page.

CLONING RESULTS

This season our team successfully PCR amplified the Cup1 and CutA genes from the PSB1C3 plasmid.