Team:Lethbridge HS/Demonstrate



PROTEIN PURIFICATION

Protein purification of our binding peptides was an essential procedure in order for the Copper Binding Assay to be completed. This assay was completed using the purified CutA protein (purified by Nickel Affinity Chromatography (link) and Size-Exclusion Chromatography (link)). CutA purification was an important goal in order for further experimentation with the protein to be done to better understand its dynamics.

Figure 1 - 15% SDS-PAGE of Nickel Affinity batch purification of CutA.In the first lane we used the molecular weight marker RMR002 from GMbiolab. Lanes 2-5 show the elutions that contain our CutA protein. CutA protein runs at around 12kDA; however, in our SDS-PAGE gel it is seen at around 14kDa, this is likely because of the histidine tag. The remaining lanes are as follows: 6- Nickel Regeneration; 7- Cell Lysate Before Binding; 8- Cell Lysate After Binding; 9- Wash Sample; 10- Cell Pellet.

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.

Figure 2 - 15% SDS-PAGE of Size Exclusion Chromatography elution samples.The first column contains the molecular weight marker RMR002 from GMbiolab, and lanes 2-16 contain our elutions.

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.

Figure 3 - Chromatograph demonstrating the peak of CutA protein. This figure is a chromatograph of the Size Exclusion Chromatography purification of the CutA protein. The A280 absorbance was read over time as the sample was eluted off the column. The resulting peak shows the elution volumes containing the CutA protein.

COPPER BINDING ASSAY

The copper binding assay was used to determine the standard copper concentration curve from measurements of the average absorbance from copper solutions of varying concentrations. This assay allows us to quantify the efficiency of CutA copper binding, as well as the optimal initial concentration and time of binding.

Figure 4 - Graph of standard copper concentration curve. Measured standard concentrations of copper in solution to provide a frame of reference for future copper binding assay.
Figure 5 - Graph demonstrating the absorbance of the copper solution after CutA (copper binding protein) was introduced as a function of time.Measured absorbance over various time intervals and determined that the optimal time is 60 minutes, and the optimal concentration is 151mg/L.

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. (Link to 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.

Figure 6 - Table showing the number of bacteria cells in each well. Each well had a certain number of phage added so that the concentration of phage was 2.5x10^-9 PFU. The wells labeled LB are the blanks for background, and the wells labelled T4 are only T4 phage and contain no bacteria in order to observe the absorbance of only the phage over time.
Figure 7 - Graph demonstrating the bacterial growth over time when the same number of phage was introduced to varying concentrations of bacteria. In the graph we can see that the highest concentration of bacteria grew relatively slowly in comparison to the other concentrations, and this is likely because the phage had an abundance of bacteria to infect and more bacteria were dying.
Figure 8 - Graph showing the bacterial growth curve when varying concentrations of phage were introduced.The bacteria concentrations that were introduced to the most phage grew slightly slower than the other samples. This shows that the phage may be successfully infecting the bacteria.

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. (link to design page)

Figure 9 - Agarose gel of elastin-like polymer synthesis samples. In the second lane there is the 1Kb ladder from Bio Basic and the fifth lane there is the 100bp ladder from Gene Ruler. The third lane contains the first and second pieces of the ELP after being connected. The fourth lane contains the first, second and third pieces of the ELP after being connected.

CLONING RESULTS

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

Figure 10 - 2% Agarose gel demonstrating the CutA and Cup1 genes. The samples in the gel are in the following lanes: lane 1- digested PSB1C3; lane 2- PSB1C3; lane 3- CutA plasmid 1; lane 4- CutA plasmid 2; lane 5- CutA plasmid 3; lane 6- Cup1 plasmid 1; lane 7- Cup1 plasmid 2; lane 8- Cup1 plasmid 4; lane 9- Cup1 plasmid 5; lane 10- 1Kb ladder from Bio Basic; lane 11- CutA PCR 1; lane 12- CutA PCR 2; lane 13- CutA PCR 3; lane 14- Cup1 PCR 1; lane 15- Cup1 PCR 2; lane 16- Cup1 PCR 3; lane 17- Cup1 PCR 4.