Difference between revisions of "Part:BBa K1189018:Experience"
TRemondini (Talk | contribs) |
TRemondini (Talk | contribs) |
||
Line 140: | Line 140: | ||
<figcaption> | <figcaption> | ||
<p><b>Figure 11.</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> | <p><b>Figure 11.</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> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | |||
+ | <b>Making Prussian Blue out of This Part</b> | ||
+ | |||
+ | <p>We applied a scaled down Prussian blue synthesis experiment to our own ferritinOur own Prussian blue ferritin was then exposed to the TMB substrate (Figure 12). From the results we can see that the ferritin with the E-coil attached had excellent catalytic activity.</p> | ||
+ | |||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/8/89/UCalgary2013TRRecombinantPrussianBlueFerritin.png" alt="Creating Prussian Blue Ferritin out of our Own Ferritin" width="800" height="511"> | ||
+ | <figcaption> | ||
+ | <p><b>Figure 12.</b> Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 µL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself. Due to limitations on the protein available only one replicate was performed. Zero time points do not have low absorbance as colour change was rapid and began before measurements started.</p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> |
Revision as of 05:05, 28 September 2013
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
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.
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).
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 |
We also performed a pH optimization of our Prussian blue ferritin using the substrates TMB and ABTS (Figure 6, 7).
figure>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.
Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8, 9).
Prussian Blue Ferritin on NitrocelluloseThe 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.
Making Prussian Blue out of This PartWe applied a scaled down Prussian blue synthesis experiment to our own ferritinOur own Prussian blue ferritin was then exposed to the TMB substrate (Figure 12). From the results we can see that the ferritin with the E-coil attached had excellent catalytic activity.
User Reviews
UNIQa8d2a23d020c0490-partinfo-00000001-QINU UNIQa8d2a23d020c0490-partinfo-00000002-QINU