Difference between revisions of "Part:BBa K1189018:Experience"
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<b>Temperature Optimization of Prussian blue Ferritin</b>
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<b>Temperature Optimization of Prussian Blue Ferritin</b>
  
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Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8, 9).
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<p>Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8, 9).</p>
  
 
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<b>Prussian Blue Ferritin on Nitrocellulose</b>
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<p>The next aspect of our analysis was to see how Prussian blue ferritin would act in a catalytic sense on nitrocellulose (Figures 10,11). From these results we can that TMB is a better substrate on for use on nitrocellulose(Figure 11). With this substrate we saw a result from only 5 ng of Prussian blue ferritin present on the nitrocellulose.</p>
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<img src="https://static.igem.org/mediawiki/2013/5/53/UCalgary2013TRABTSnitrocellulose.png" alt="Prussian Blue Ferritin and ABTS on Nitrocellulose" width="701" height="600">
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<p><b>Figure 15.</b> Blots of Prussian blue ferritin on nitrocellulose (20 µL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.</p>
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<img src="https://static.igem.org/mediawiki/2013/c/cd/UCalgary2013TRTMBnitrocellulose.png" alt="Prussian Blue Ferritin and TMB on Nitrocellulose" width="693" height="600">
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<p><b>Figure 16.</b> Blots of Prussian blue ferritin on nitrocellulose (20 µL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.</p>
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Revision as of 04:58, 28 September 2013

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Applications of BBa_K1189018

Kinetic Analysis of Prussian Blue Ferritin

We performed a kinetic analysis of our Prussian blue ferritin. We included a comparison of Prussian blue horse spleen ferritin to regular horse spleen ferritin for both TMB and ABTS (Figures 1, 2). For both of the substrates we can see that normal ferritin has a very low catalytic activity compared to our modified ferritin. Using this data were able to determine the Michaelis-Menten catalytic constants for Prussian blue ferritin with different substrates.

Prussian Blue Ferritin and TMB

Figure 1. Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 µL of 10 mg/mL substrate was used in a 242 µL reaction volume.Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data.

Prussian Blue Ferritin and ABTS

Figure 2. Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 µL of 10 mg/mL substrate was used in a 242 µL reaction volume. Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.

In order to complete our kinetic analysis we had to determine the catalytic properties of our Prussian blue ferritin according to the Michaelis-Menten kinetic model. For these tests we varied the colourimetric substrate concentrations (ABTS and TMB) (Figures 3,4). We also varied the hydrogen peroxide concentration in association with TMB as this the first chemical compound that will react in the system (Figure 5).

Michaelis-Menten Plot for Prussian Blue Ferritin with ABTS

Figure 3. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of ABTS. Absorbance readings were taken at 415 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 6).

Michaelis-Menten Plot for Prussian Blue Ferritin with TMB

Figure 4. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of TMB. Absorbance readings were taken at 650 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 5).

Michaelis-Menten Plot for Prussian Blue Ferritin Based on Hydrogen Peroxide (with TMB)

Figure 5. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of hydrogen peroxide. Absorbance readings were taken at 650 nm which measure the breakdown of TMB. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data.

Table 1. Catalytic constants for our Prussian blue ferritin
Catalyst Enzyme Concentration (M) Substrate Km (mM) Vmax (Ms-1) Kcat (s-1) Kcat/Km (M-1s-1)
Prussian Blue Ferritin 1.31 x 10-9 ABTS 0.448 1.25 x 10-8 9.51 2.12 x 104
Prussian Blue Ferritin 1.31 x 10-9 TMB 0.0432 1.12 x 10-7 85.3 1.97 x 106
Prussian Blue Ferritin 1.31 x 10-9 H2O2 (TMB) 0.0176 1.31 x 10-8 11.1 6.28 x 105
pH Optimization of Prussian blue Ferritin

We also performed a pH optimization of our Prussian blue ferritin using the substrates TMB and ABTS (Figure 6, 7).

figure> ABTS pH Optimization

Figure 6. pH optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

TMB pH Optimization

Figure 7. pH optimization of commercial Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

Temperature Optimization of Prussian Blue Ferritin

Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8, 9).

ABTS Temperature Optimization

Figure 8. Temperature optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

TMB Temperature Optimization

Figure 9. Temperature optimization of commerical Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

Prussian Blue Ferritin on Nitrocellulose

The next aspect of our analysis was to see how Prussian blue ferritin would act in a catalytic sense on nitrocellulose (Figures 10,11). From these results we can that TMB is a better substrate on for use on nitrocellulose(Figure 11). With this substrate we saw a result from only 5 ng of Prussian blue ferritin present on the nitrocellulose.

Prussian Blue Ferritin and ABTS on Nitrocellulose

Figure 15. Blots of Prussian blue ferritin on nitrocellulose (20 µL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.

Prussian Blue Ferritin and TMB on Nitrocellulose

Figure 16. Blots of Prussian blue ferritin on nitrocellulose (20 µL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.


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