Difference between revisions of "Part:BBa K1189018:Experience"

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Applications of BBa_K1189018

The main purpose of this page is to display the characterization data of Prussian blue chemically modified commercial horse spleen ferritin as a catalyst. This ferritin is extremely similar to our constructed ferritin and we do not anticipate any differences in their properties. Next we show how our own constructed ferritin displays the same catalytic activity. At the end we show how the coil found in this part can bind to coils found on our other parts.

Kinetic Analysis of Prussian Blue Ferritin

This part was created by fusing the heavy chain and light chains (BBa_K1189024 BBa_K1189025) of human ferritin together (sequences from: P02794 and P02792 [UniParc]). It is expressed under the lacI promoter (BBa_J04500) and has a his-tag for protein purification. An E-coil (BBa_K1189011) is included in order to allow binding of parts containing the respective K-coil (BBa_K1189010). Characterization of this part was done primarily with commercially purchased ferritin, which is structurally very similar to this recombinant ferritin (Figure 1).

This construct can be used as a reporter through a modification of the iron core to form Prussian Blue (Figure 2). The resulting molecule can then catalyze the formation of radicals from hydrogen peroxide, which can then cause a colour change in substrates such as TMB or ABTS (Figure 3) (Zhang et al., 2013).

This protein is a highly robust protein, remaining stable under extreme pH, temperature, and denaturing conditions. It is also highly accepting of fusion proteins, as it continues to form the nanoparticle despite fusions to both N-terminus and C-terminus. In addition, proteins fused to this protein have been found to be stabilized due to the fusion.

Ferritin — **Figure 1.** Ribbon visualization of a fully assembled ferritin protein.

Substrate Colours — **Figure 2.** Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.

Prussian Blue Synthesis — **Figure 3.** Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period.

Applications of BBa_K1189018

The main purpose of this page is to display the characterization data of Prussian blue chemically modified commercial horse spleen ferritin as a catalyst. This ferritin is extremely similar to our constructed ferritin and we do not anticipate any differences in their properties. Next we show how our own constructed ferritin displays the same catalytic activity. At the end we show how the coil found in this part can bind to coils found on our other parts.

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 and 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 and 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	K_m (mM)	V_max (Ms^-1)	K_cat (s^-1)	K_cat/K_m (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	1.44 x 10^-9	H₂O₂ (TMB)	0.0176	1.45 x 10^-8	10.1	5.71 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 and 7). The results show TMB to be a much more robust substrate, showing high catalytic activity across a more broad range of pH compared to ABTS.

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 and 9). This showed us that the Prussian blue reporter has a much higher catalytic activity at higher temperatures.

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 and 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 10.** Blots of Prussian blue ferritin on nitrocellulose (5 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 11.** Blots of Prussian blue ferritin on nitrocellulose (5 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.

Making Prussian Blue out of This Part

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.

Creating Prussian Blue Ferritin out of our Own Ferritin — **Figure 12.** 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.

How does the use of Coils versus Direct Fusions of TALEs Affect our Prussian Blue reporter?

After successfully confirming that we could convert our own ferritin proteins that were produced from the parts we constructed (BBa_K1189018, BBa_K1189021) into Prussian blue ferritin the next step was to evaluate how the design of our parts could potentially affect the reporter activity of our Prussian blue ferritin. Based on the spatial modelling performed by our team it was suggested that assembly of the ferritin nanoparticle with TALE proteins directly fused was highly unlikely. This is because the TALE proteins are significantly larger than the ferritin subunits. Their size would likely result in steric hindrance and prevent the assembly of the full ferritin protein. In order to test the predictions put forward by our modelling we ensured that our protein samples were balanced in order to have the same number ferritin cores in each sample. The catalytic activity of these proteins was then compared. From the data gathered we saw that the Prussian blue ferritin with fused coils (even if TALES are additionally bound to the ferritin via coils) was more effective as a reporter than having the TALE proteins directly fused to the ferritin nanoparticle (Figure 13). The results from this experiment suggest that the predictions made by our model were correct. Using coils however alleviates this issue as these coils are small and would not interfere in the ferritin self-assembly but can be used to attach our TALES to create the FerriTALE.

Recombinant Prussian Blue FerritinMole Balanced — **Figure 12.** Samples of our parts that were converted to Prussian Blue ferritin were mole balanced in order to ensure that the same number of effective ferritin cores are present in every sample. Additionally the ferritin-coil fusion was incubated with the TALE-coil fusion part in order to allow their binding for a separate trial. Negative controls include unconverted recombinant ferritin, bovine serum albumin and a substrate only control. Samples were incubated with a TMB substrate solution for 10 minutes at a pH of 5.6. Absorbance readings were taken at the 10 minute time-point at a wavelength of 650 nm. An ANOVA (analysis of variants) was performed upon the values to determine that there was statistical difference in the data gathered (based off of three replicates). A t-test was then performed which determined that the * columns are significantly different from the ** column (p=0.0012). Neither * column is significantly different from each other (p=0.67).

User Reviews

UNIQe78e9213688c5a3f-partinfo-00000008-QINU UNIQe78e9213688c5a3f-partinfo-00000009-QINU

@@ Line 2: / Line 2: @@
 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 how you used this part and how it worked out.
+===Applications of BBa_K1189018===
+<p>The main purpose of this page is to display the characterization data of Prussian blue chemically modified commercial horse spleen ferritin as a catalyst. This ferritin is extremely similar to our constructed ferritin and we do not anticipate any differences in their properties. Next we show how our own constructed ferritin displays the same catalytic activity. At the end we show how the coil found in this part can bind to coils found on our other parts. </p>
+<b>Kinetic Analysis of Prussian Blue Ferritin</b>
+<p>This part was created by fusing the heavy chain and light chains (<partinfo>BBa_K1189024</partinfo> <partinfo>BBa_K1189025</partinfo>) of human ferritin together (sequences from: P02794 and P02792 [UniParc]). It is expressed under the lacI promoter (<partinfo>BBa_J04500</partinfo>) and has a his-tag for protein purification. An E-coil (<partinfo>BBa_K1189011</partinfo>) is included in order to allow binding of parts containing the respective K-coil (<partinfo>BBa_K1189010</partinfo>). Characterization of this part was done primarily with <html><a href="http://www.sigmaaldrich.com/catalog/product/sigma/f4503?lang=en&region=CA">commercially purchased ferritin</a></html>, which is structurally very similar to this recombinant ferritin (Figure 1).  </p>
+	<p>This construct can be used as a reporter through a modification of the iron core to form Prussian Blue (Figure 2). The resulting molecule can then catalyze the formation of radicals from hydrogen peroxide, which can then cause a colour change in substrates such as TMB or ABTS (Figure 3) (Zhang <i> et al.,</i> 2013).</p>
+<p> This protein is a highly robust protein, remaining stable under extreme pH, temperature, and denaturing conditions. It is also highly accepting of fusion proteins, as it continues to form the nanoparticle despite fusions to both N-terminus and C-terminus. In addition, proteins fused to this protein have been found to be stabilized due to the fusion.
+</p>
+<html>
+<br>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/1/18/UCalgary2013TRFerritinrender2png.png" alt="Ferritin" width="300" height="300">
+<figcaption>
+	<p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/a/a9/UCalgary2013TRSubstratecolour.png" alt="Substrate Colours" width="250" height="300">
+<figcaption>
+	<p><b>Figure 2.</b> Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.</p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/c/c7/UCalgary2013TRPrussianblueferritinsynthesis.png" alt="Prussian Blue Synthesis" width="400" height="200">
+<figcaption>
+	<p><b>Figure 3.</b> Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period. </p>
+</figcaption>
+</figure>
+</html>
 ===Applications of BBa_K1189018===
@@ Line 10: / Line 46: @@
 <html>
-<p>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.</p>
+<p>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 and 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.</p>
 <figure>
@@ Line 26: / Line 62: @@
 </figure>
-<p>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).</p>
+<p>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 and 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).</p>
 <figure>
@@ Line 81: / Line 117: @@
    <tr>
      <td>Prussian Blue Ferritin</td>
-     <td>1.31 x 10<sup>-9</sup></td>
+     <td>1.44 x 10<sup>-9</sup></td>
      <td>H<sub>2</sub>O<sub>2 </sub> (TMB)</td>
      <td>0.0176</td>
-     <td>1.31 x 10<sup>-8</sup></td>
+     <td>1.45 x 10<sup>-8</sup></td>
-     <td>11.1</td>
+     <td>10.1</td>
-     <td>6.28 x 10<sup>5</sup></td>
+     <td>5.71 x 10<sup>5</sup></td>
    </tr>
 </table></center>
@@ Line 92: / Line 128: @@
 <b>pH Optimization of Prussian blue Ferritin</b>
-<p>We also performed a pH optimization of our Prussian blue ferritin using the substrates TMB and ABTS (Figure 6, 7).</p>
+<p>We also performed a pH optimization of our Prussian blue ferritin using the substrates TMB and ABTS (Figure 6 and 7). The results show TMB to be a much more robust substrate, showing high catalytic activity across a more broad range of pH compared to ABTS.</p>
-figure>
+<figure>
 <img src="https://static.igem.org/mediawiki/2013/c/ca/UCalgary2013TRABTSphoptimization.png" alt="ABTS pH Optimization" width="800" height="431">
 <figcaption>
@@ Line 110: / Line 146: @@
 <b>Temperature Optimization of Prussian Blue Ferritin</b>
-<p>Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8, 9).</p>
+<p>Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8 and 9). This showed us that the Prussian blue reporter has a much higher catalytic activity at higher temperatures.</p>
 <figure>
@@ Line 128: / Line 164: @@
 <b>Prussian Blue Ferritin on Nitrocellulose</b>
-<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>
+<p>The next aspect of our analysis was to see how Prussian blue ferritin would act in a catalytic sense on nitrocellulose (Figures 10 and 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>
 <figure>
@@ Line 156: / Line 192: @@
 </figure>
-<b>Does the coil bind?</b>
+<b>How does the use of Coils versus Direct Fusions of TALEs Affect our Prussian Blue reporter?</b>
-<p>In the case of the coils we were interested to see if the K-coil fused to TALE proteins (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1189029"><span class="Green"><b>BBa_K1189029</b></span></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1189030"><span class="Green"><b>BBa_K1189030</b></span></a>) could bind to the E-coil found on one of our Prussian blue ferritin constructs (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1189018"><span class="Green"><b>BBa_K1189018</b></span></a>). To complete this task we placed the TALE on the membrane, washed and blocked the membrane. The ferritin protein with the complimentary coil was then added to the membrane. If this coil successfully binds to the other coil then the ferritin will not be washed off during the next wash step. We can then see if Prussian blue ferritin is bound by adding a TMB substrate solution that will cause a colour change. To this extent we saw a blue ring in this trial indicating a positive result. This suggests that our coils are actually binding in an <i>in vitro</i> system.</p>
+<p>After successfully confirming that we could convert our own ferritin proteins that were produced from the parts we constructed (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1189018">BBa_K1189018,</a> <a
+href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1189021">BBa_K1189021</a>) into Prussian blue ferritin the next step was to evaluate how the design of our parts could potentially affect the reporter activity of our Prussian blue ferritin. Based on the spatial modelling performed by our team it was suggested that assembly of the ferritin nanoparticle with TALE proteins directly fused was highly unlikely. This is because the TALE proteins are significantly larger than the ferritin subunits. Their size would likely result in steric hindrance and prevent the assembly of the full ferritin protein. In order to test the predictions put forward by our modelling we ensured that our protein samples were balanced in order to have the same number ferritin cores in each sample. The catalytic activity of these proteins was then compared. From the data gathered we saw that the Prussian blue ferritin with fused coils (even if TALES are additionally bound to the ferritin via coils) was more effective as a reporter than having the TALE proteins directly fused to the ferritin nanoparticle (Figure 13). The results from this experiment suggest that the predictions made by our model were correct. Using <a
-<p>Another interesting element of this assay is why we used two variants of the TALE K-coil negative control.  A blue ring on our TALE negative control confirmed our fear that during the  second protein application and wash step that some of the ferritin with coil proteins would drift over and bind to the TALE K-coils on the nitrocellulose. This did not occur for our separate negative control (Figure 13).</p>
+href="http://2013.igem.org/Team:Calgary/Project/OurSensor/Linker">coils</a> however alleviates this issue as these coils are small and would not interfere in the ferritin self-assembly but can be used to attach our TALES to create the FerriTALE. </p>
 <figure>
-<img src="https://static.igem.org/mediawiki/2013/e/e3/UCalgary2013TRCoilbindingpreliminary.png" alt="Preliminary Coil Binding" width="757" height="751">
+<img src="https://static.igem.org/mediawiki/2013/0/0d/UCalgary2013TRPBFAssayNoColour.png" alt="Recombinant Prussian Blue FerritinMole Balanced" width="465" height="480">
 <figcaption>
-	<p><b>Figure 13.</b> This basic qualitative assay was used to inform us whether certain elements of our system are able to bind to each other. Our TALE proteins were mounted to the membrane along with positive controls of three Prussian blue variants; two recombinant ferritins and one commercial protein. The membranes were then washed and blocked. Prussian blue ferritin with a coil was added to our TALE protein containing a coil. Prussian blue ferritin with a TALE that could bind to the DNA held by another TALE on the membrane was also added. A TMB substrate solution was added to cause a colourimetric change over 5 minutes. Positive results are indicated by dark rings of colour. Negative controls include a TALE with a coil on the same membrane and the same TALE and bovine serum albumin on separate membranes that were treated separately. Image contrast was altered to make the results more clear on a digital monitor; the same changes were applied to each element of the figure.</p>
+	<p><b>Figure 12.</b> Samples of our parts that were converted to Prussian Blue ferritin were mole balanced in order to ensure that the same number of effective ferritin cores are present in every sample. Additionally the ferritin-coil fusion was incubated with the TALE-coil fusion part in order to allow their binding for a separate trial. Negative controls include unconverted recombinant ferritin, bovine serum albumin and a substrate only control. Samples were incubated with a TMB substrate solution for 10 minutes at a pH of 5.6. Absorbance readings were taken at the 10 minute time-point at a wavelength of 650 nm. An ANOVA (analysis of variants) was performed upon the values to determine that there was statistical difference in the data gathered (based off of three replicates). A t-test was then performed which determined that the * columns are significantly different from the ** column (p=0.0012). Neither * column is significantly different from each other (p=0.67).</p>
+</figcaption>
+</figure>
 <br>
 </html>
 ===User Reviews===
 <!-- DON'T DELETE --><partinfo>BBa_K1189018 StartReviews</partinfo>