Difference between revisions of "Team:Lethbridge HS/Demonstrate"

 
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<p style="font-size: 3vw; font-family: 'Open Sans'">COPPER BINDING ASSAY</p>
 
<p style="font-size: 3vw; font-family: 'Open Sans'">COPPER BINDING ASSAY</p>
 
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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. </p>  
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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.</p>  
 
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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.  
 
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.  
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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)</p>
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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 <a href="https://2018.igem.org/Team:Lethbridge_HS/Model"> here </a>to view modelling page)</p>
 
<p style="font-size: 3vw; font-family: 'Open Sans'">BACTERIOPHAGE ASSAY</p>
 
<p style="font-size: 3vw; font-family: 'Open Sans'">BACTERIOPHAGE ASSAY</p>
 
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<figcaption><b>Figure 6 - Table showing the number of bacteria cells in each well.</b> 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.
 
<figcaption><b>Figure 6 - Table showing the number of bacteria cells in each well.</b> 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.
 
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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.</p>
 
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.</p>
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Latest revision as of 03:48, 18 October 2018



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.

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. (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.

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.