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| − | __NOTOC__
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| − | <partinfo>BBa_K1189037 short</partinfo>
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| − | <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®ion=CA">commercially purchased ferritin</a></html>, which is structurally very similar to this recombinant ferritin (Figure 1). </p>
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| − | <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>
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| − | <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.
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| − | </p>
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| | <html> | | <html> |
| − | <br>
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/1/18/UCalgary2013TRFerritinrender2png.png" alt="Ferritin" width="300" height="300">
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| − | <figcaption>
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| − | <p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p>
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| − | </figcaption>
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| − | </figure>
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| − | <figure> | + | <h1>Ferrtin fusions</h1> |
| − | <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>
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| − | </figcaption>
| + | |
| − | </figure>
| + | |
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| − | <figure> | + | <p>Ferritin is an protein shelled nanoparticle and is composed of a mixture of 24 light (x) and heavy (y) subunits. It is ubiquitous across eukaryotic and prokaryotic systems and is used to sequester intracellular iron. The <a href=”http://2013.igem.org/Team:Calgary”>2013 iGEM Calgary</a> used ferritin’s iron core as a reporter and its protein shell to scaffold <a href=”http://2013.igem.org/Team:Calgary/Project/OurSensor/Detector”>DNA sensing TALEs</a> (see Figure 1).</p> |
| − | <img src="https://static.igem.org/mediawiki/2013/c/c7/UCalgary2013TRPrussianblueferritinsynthesis.png" alt="Prussian Blue Synthesis" width="400" height="200"> | + | |
| − | <figcaption>
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| − | <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>
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| − | </figcaption> | + | |
| − | </figure> | + | |
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| − | </html> | + | <p>FIGURE ONE</p> |
| | | | |
| − | ===Applications of BBa_K1189037===
| + | <p>This particular version is a fusion of heavy and light ferritin subunits, such that nanoparticles are formed from 12 di-subunits. The rationale for this design is that it reduces the number of N-termini on ferritin to which proteins can be fused by half, which is important for lessening potential steric hindrances among fused proteins in the 3D sphere surrounding ferritin. Additionally, di-subunits ensure a 1:1 ratio of heavy and light subunits which ensures consistency in ferritin’s ability to uptake iron. Moreover, these fusions have been shown stable in engineered applications with other proteins scaffolded to ferritin (cite).</p> |
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| − | <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> | + | <h1>Design features</h1> |
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| − | <b>Kinetic Analysis of Prussian Blue Ferritin</b> | + | <p>This part has an N-terminal fusion to an E coil connected to ferritin by a GS linker. The coil system is of utility to other iGEM teams because they can express K coils on their own proteins of interest, and bind them to the complementary E coil on ferritin. Such a coiled-coil linker system reduces potential for large protein fusions to harm ferritin formation, allowing user to build intricate nanoparticle devices with myriad proteins. See Figures 2 application examples.</p> |
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| − | <html>
| + | <p>FIGURE TWO</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> | + | |
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| − | <figure> | + | <p>This part is identical to <a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189018>BBa_1189018</a>, with the exception of a his-tag for purification.</p> |
| − | <img src="https://static.igem.org/mediawiki/2013/3/36/UCalgary2013TRTmb6ulgraph.png" alt="Prussian Blue Ferritin and TMB" width="800" height="439"> | + | |
| − | <figcaption>
| + | |
| − | <p><b>Figure 1.</b> 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. </p>
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| − | </figcaption>
| + | |
| − | </figure>
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| − | <figure> | + | <h1>Results</h1> |
| − | <img src="https://static.igem.org/mediawiki/2013/1/15/UCalgary2013TRABTS8ulgraph.png" alt="Prussian Blue Ferritin and ABTS" width="800" height="433"> | + | |
| − | <figcaption>
| + | |
| − | <p><b>Figure 2.</b> 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.</p>
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| − | </figcaption>
| + | |
| − | </figure>
| + | |
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| − | <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> | + | <p>The <a href=”http://2013.igem.org/Team:Calgary”>2013 iGEM Calgary</a> successfully expressed and purified this protein in pSB1C3 and per this part sequence exactly using and FPLC and metal affinity purification of the his tag. Please see the <a href=https://parts.igem.org/Part:BBa_K1189037:Experience>experience page</a> for data on another expression vector.</p> |
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| − | <figure>
| + | <p>GEL</p> |
| − | <img src="https://static.igem.org/mediawiki/2013/2/20/UCalgary2013TRPBFABTSmichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin with ABTS" width="800" height="436">
| + | |
| − | <figcaption>
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| − | <p> <b>Figure 3.</b> 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).</p > | + | |
| − | </figcaption>
| + | |
| − | </figure>
| + | |
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| − | <figure> | + | <p>This purified protein product was successfully converted into Prussian blue ferritin, a robust colourmetric reporter. Figure 4 shows that this part with coiled-coils performs better as a reporter than direct fusions to TALEs (<a href=https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189021>BBa_K118021</a>). It seems that large protein fusions reduce effectiveness of ferritin as a reporter. Figure 5 shows that ferritin with coiled-coils (BBa_1189037) maintains reporter functionality when TALEs are scaffolded using coiled-coil linkers.</p> |
| − | <img src="https://static.igem.org/mediawiki/2013/c/c7/UCalgary2013TRPBFTMBmichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin with TMB" width="800" height="435"> | + | |
| − | <figcaption>
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| − | <p><b>Figure 4.</b> 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).</p>
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| − | </figcaption>
| + | |
| − | </figure>
| + | |
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| − | <figure>
| + | <p>Figure 4 and 5</p> |
| − | <img src="https://static.igem.org/mediawiki/2013/e/e5/UCalgary2013TRPBFTMBGHydrogenperoxidemichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin Based on Hydrogen Peroxide (with TMB)" width="800" height="434">
| + | |
| − | <figcaption>
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| − | <p ><b>Figure 5 .</b> 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.</p > | + | |
| − | </figcaption>
| + | |
| − | </figure>
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| − | <center><b>Table 1.</b> Catalytic constants for our Prussian blue ferritin</center> | + | <p>Please see the experience page for a detailed analysis of how Prussian blue ferritin performs as a reporter.</p> |
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| − | <center><table width="800" border="1">
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| − | <tr>
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| − | <td>Catalyst</td>
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| − | <td>Enzyme Concentration (M)</td>
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| − | <td>Substrate</td>
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| − | <td>K<sub>m</sub> (mM)</td>
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| − | <td>V<sub>max</sub> (Ms<sup>-1</sup>)</td>
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| − | <td>K<sub>cat</sub> (s<sup>-1</sup>)</td>
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| − | <td>K<sub>cat</sub>/K<sub>m</sub> (M<sup>-1</sup>s<sup>-1</sup>)</td>
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| − | </tr>
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| − | <tr>
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| − | <td>Prussian Blue Ferritin</td>
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| − | <td>1.31 x 10<sup>-9</sup></td>
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| − | <td>ABTS</td>
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| − | <td>0.448</td>
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| − | <td>1.25 x 10<sup>-8</sup></td>
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| − | <td>9.51</td>
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| − | <td>2.12 x 10<sup>4</sup></td>
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| − | </tr>
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| − | <tr>
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| − | <td>Prussian Blue Ferritin</td>
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| − | <td>1.31 x 10<sup>-9</sup></td>
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| − | <td>TMB</td>
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| − | <td>0.0432</td>
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| − | <td>1.12 x 10<sup>-7</sup></td>
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| − | <td>85.3</td>
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| − | <td>1.97 x 10<sup>6</sup></td>
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| − | </tr>
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| − | <tr>
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| − | <td>Prussian Blue Ferritin</td>
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| − | <td>1.31 x 10<sup>-9</sup></td>
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| − | <td>H<sub>2</sub>O<sub>2 </sub> (TMB)</td>
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| − | <td>0.0176</td>
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| − | <td>1.31 x 10<sup>-8</sup></td>
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| − | <td>11.1</td>
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| − | <td>6.28 x 10<sup>5</sup></td>
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| − | </tr>
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| − | </table></center>
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| − |
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| − | <b>pH Optimization of Prussian blue Ferritin</b>
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| − | <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>
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/c/ca/UCalgary2013TRABTSphoptimization.png" alt="ABTS pH Optimization" width="800" height="431">
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| − | <figcaption>
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| − | <p><b>Figure 6.</b> 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. </p>
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/1/1c/UCalgary2013TRTMBphoptimization.png" alt="TMB pH Optimization" width="800" height="444">
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| − | <figcaption>
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| − | <p><b>Figure 7.</b> 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. </p>
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| − | </figcaption>
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| − | </figure>
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| − | <b>Temperature Optimization of Prussian Blue Ferritin</b>
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| − | <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>
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/5/57/UCalgary2013TRABTStemperatureoptimization.png" alt="ABTS Temperature Optimization" width="800" height="437">
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| − | <figcaption>
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| − | <p><b>Figure 8.</b> 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. </p>
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/6/63/UCalgary2013TRTMBtemperatureoptimization.png" alt="TMB Temperature Optimization" width="800" height="440">
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| − | <figcaption>
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| − | <p><b>Figure 9.</b> 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. </p>
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| − | </figcaption>
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| − | </figure>
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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 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>
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| − | <figure>
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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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| − | <figcaption>
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| − | <p><b>Figure 10.</b> 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.</p>
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| − | </figcaption>
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| − | </figure>
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| − | <figure>
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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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| − | <figcaption>
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| − | <p><b>Figure 11.</b> 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.</p>
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| − | </figcaption>
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| − | </figure>
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| − | <b>Making Prussian Blue out of This Part</b>
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| − | <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>
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| − | <figure>
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| − | <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">
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| − | <figcaption>
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| − | <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>
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| − | </figcaption>
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| − | </figure>
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| − | <br>
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| | </html> | | </html> |
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| | <!-- --> | | <!-- --> |
Ferritin is an protein shelled nanoparticle and is composed of a mixture of 24 light (x) and heavy (y) subunits. It is ubiquitous across eukaryotic and prokaryotic systems and is used to sequester intracellular iron. The 2013 iGEM Calgary used ferritin’s iron core as a reporter and its protein shell to scaffold DNA sensing TALEs (see Figure 1).
This particular version is a fusion of heavy and light ferritin subunits, such that nanoparticles are formed from 12 di-subunits. The rationale for this design is that it reduces the number of N-termini on ferritin to which proteins can be fused by half, which is important for lessening potential steric hindrances among fused proteins in the 3D sphere surrounding ferritin. Additionally, di-subunits ensure a 1:1 ratio of heavy and light subunits which ensures consistency in ferritin’s ability to uptake iron. Moreover, these fusions have been shown stable in engineered applications with other proteins scaffolded to ferritin (cite).
This part has an N-terminal fusion to an E coil connected to ferritin by a GS linker. The coil system is of utility to other iGEM teams because they can express K coils on their own proteins of interest, and bind them to the complementary E coil on ferritin. Such a coiled-coil linker system reduces potential for large protein fusions to harm ferritin formation, allowing user to build intricate nanoparticle devices with myriad proteins. See Figures 2 application examples.
This purified protein product was successfully converted into Prussian blue ferritin, a robust colourmetric reporter. Figure 4 shows that this part with coiled-coils performs better as a reporter than direct fusions to TALEs (BBa_K118021). It seems that large protein fusions reduce effectiveness of ferritin as a reporter. Figure 5 shows that ferritin with coiled-coils (BBa_1189037) maintains reporter functionality when TALEs are scaffolded using coiled-coil linkers.
Please see the experience page for a detailed analysis of how Prussian blue ferritin performs as a reporter.
