Difference between revisions of "Part:BBa K1189037"
 
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<partinfo>BBa_K1189037 short</partinfo>
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<h1>A fusion of two ferrtin subunits</h1>
  
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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&region=CA">commercially purchased ferritin</a></html>, which is structurally very similar to this recombinant ferritin (Figure 1). </p>
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<p>Ferritin is a protein shelled nanoparticle and is composed of a mixture of 24 <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189024">light (BBa_K1189024)</a> and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189025">heavy (BBa_K1189025)</a> subunits. It is ubiquitous across eukaryotic and prokaryotic systems and is used to sequester intracellular iron (Chasteen <i>et al.</i>, 1991). 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> as part of their project, the <a href="http://2013.igem.org/Team:Calgary/Project/OurSensor">FerriTALE</a> (see 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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<img src="https://static.igem.org/mediawiki/2013/1/18/UCalgary2013TRFerritinrender2png.png" alt="Ferritin" width="300" height="300">
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<img src="https://static.igem.org/mediawiki/2013/thumb/3/34/Assembled_FerriTALE.png/463px-Assembled_FerriTALE.png" alt="BBa_K1189037 joined with DNA sensing TALEs" width="400" height="500">
 
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<p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p>
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<p><b>Figure 1.</b> 3D <i>in silico</i> rendering of BBa_K1189037 formed into functional nanoparticles bound to DNA sensing TALEs. The iron core is chemically modified and use to show when TALEs are bound to DNA. The TALEs are one specific, particular application of the ferritin E coil di-subunit fusion. This nanoparticle is the molecular basis of a DNA lateral flow strip biosensor pursued by the 2013 iGEM Calgary team.</p>
 
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<p><a href="https://parts.igem.org/Part:BBa_K1189037">BBa_K1189037</a> is a fusion of heavy and light ferritin subunits, such that ferritin 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 mandate 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 (Dehal <i>et al.</i>, 2010).</p>
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<img src="https://static.igem.org/mediawiki/2013/a/a9/UCalgary2013TRSubstratecolour.png" alt="Substrate Colours" width="250" height="300">
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<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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<h1>Design features</h1>
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<img src="https://static.igem.org/mediawiki/2013/c/c7/UCalgary2013TRPrussianblueferritinsynthesis.png" alt="Prussian Blue Synthesis" width="400" height="200">
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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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<p>This part has an N-terminal fusion to an E coil connected to ferritin by a GS linker (Figure 2). 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 3 application examples.</p>
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===Applications of BBa_K1189037===
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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>
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<b>Kinetic Analysis of Prussian Blue Ferritin</b>
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<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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<img src="https://static.igem.org/mediawiki/2013/3/36/UCalgary2013TRTmb6ulgraph.png" alt="Prussian Blue Ferritin and TMB" width="800" height="439">
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<img src="https://static.igem.org/mediawiki/2013/thumb/d/de/BBa_K1189037_SBOL.png/800px-BBa_K1189037_SBOL.png" alt="BBa_K1189037 SBOL part figure" width="600" height="100">
 
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<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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<p><b>Figure 2.</b> Ferritin di-subunit fused to an E coil and his tag. The E coil allows binding to other proteins expressing a complementary K coil.</p>
 
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<img src="https://static.igem.org/mediawiki/2013/1/15/UCalgary2013TRABTS8ulgraph.png" alt="Prussian Blue Ferritin and ABTS" width="800" height="433">
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<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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<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>
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<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">
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<img src="https://static.igem.org/mediawiki/2013/0/07/UCalgary2013TRCoilflexibility.png" alt="FerriTALE Scaffold Modularity" width="800" height="219" >
 
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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>
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<p><b>Figure 3.</b> Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.</a></p>
 
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<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>
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<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">
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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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<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">
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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>
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<center><b>Table 1.</b> Catalytic constants for our Prussian blue ferritin</center>
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<center><table width="800" border="1">
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<a name="Results"></a>
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<h1>Results</h1>
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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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    <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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    <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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    <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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</table></center>
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<b>pH Optimization of Prussian blue Ferritin</b>
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<a name="pSB1C3 expression"></a>
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<h3>Expression from pSB1C3</h3>
  
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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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<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. See Figure 4 for an SDS-PAGE of this 42 kDa isolate. Please see the <a href="https://parts.igem.org/Part:BBa_K1189037:Experience#User reviews">user reviews section</a> for data on another expression vector which generated this protein with a higher yield.</p>
  
 
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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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<img src="https://static.igem.org/mediawiki/2013/3/3a/BBa_1189037_SDSPAGE_1c3_expression.png" alt="FerriTALE Scaffold Modularity" width="400" height="400" >
 
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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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<p><b>Figure 4.</b> FPLC purification fraction of <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189037">BBa_K1189037</a> as express per our <a href="http://2013.igem.org/Team:Calgary/Notebook/Protocols/LargeScaleProteinExpressionAndPurification">large scale expression protocol.</a> Were were disappointed with the yield of this 42 kDa protein from the pSB1C3 cloning vector. Please see the <a href="https://parts.igem.org/Part:BBa_K1189037:Experience#User reviews">user reviews section</a> for results from a proper expression vector.</p>
 
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<a name="Reporter data"></a>
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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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<h3>Conversion of BBa_K1189037 into a reporter</h3>
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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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<b>Temperature Optimization of Prussian Blue Ferritin</b>
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<p>This purified protein product was successfully converted into Prussian blue ferritin, a robust colourmetric reporter. Figure 5 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 6 shows that ferritin with coiled-coils (BBa_1189037) maintains reporter functionality when TALEs are scaffolded using coiled-coil linkers.</p>
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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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<img src="https://static.igem.org/mediawiki/2013/5/57/UCalgary2013TRABTStemperatureoptimization.png" alt="ABTS Temperature Optimization" width="800" height="437">
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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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<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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<p><b>Figure 5.</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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<img src="https://static.igem.org/mediawiki/2013/6/63/UCalgary2013TRTMBtemperatureoptimization.png" alt="TMB Temperature Optimization" width="800" height="440">
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<img src="https://static.igem.org/mediawiki/2013/0/0d/UCalgary2013TRPBFAssayNoColour.png" alt="Recombinant Prussian Blue FerritinMole Balanced" width="465" height="480">
 
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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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<p><b>Figure 6.</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>
 
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<b>Prussian Blue Ferritin on Nitrocellulose</b>
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<p>Please see the <a href="http://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin">Prussian blue ferritin Wiki</a> page for a detailed analysis of how Prussian blue ferritin, synthesized from commercially available ferritin, performs as a reporter. This data informs how <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189037">BBa_K1189037</a> is useful as a reporter.</p>
  
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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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<h3>TecCEM_HS and ferritin</h3>
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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 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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<p>[http://2015.igem.org/Team:TecCEM_HS TecCEM_HS team] HS wanted to see if there were any significant changes in the SDS binding to the molecule if there were any extra elements in the construction. That's why they used this biobrick to see if the SDS molecules were captured although there was an extra coil in the structure.
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The team made an induction to see if the protein was expressing and here is the result.
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We also made a chromatography to see the expression of the protein and to verify the open reading frame. </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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<img src="https://static.igem.org/mediawiki/2015/2/20/TecCEM_HS_RESULTS_Cromatogrtaf%C3%ADa2.png" alt="Ferritin cromatography" width="500" height=300">
 
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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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<p><b>Figure 7.</b> An induction of this Biobrick was made and a cromatography at hour 4 was performed (Nickel affinity chromatography). The overexpression can be seen in lane 5, were an approx. 42 kDa band can be seen, that means the cromatography was successful and the protein was actually expressing.</p>
 
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<b>Making Prussian Blue out of This Part</b>
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<h1>References</h1>
  
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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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<li>Chasteen, N. D., & Harrison, P. M. (1999). Mineralization in ferritin: an efficient means of iron storage. Journal of structural biology, 126(3), 182-194.</li>
  
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<li>Dehal, P. K., Livingston, C. F., Dunn, C. G., Buick, R., Luxton, R., & Pritchard, D. J. (2010). Magnetizable antibody‐like proteins. Biotechnology journal, 5(6), 596-604.</li>
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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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<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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Latest revision as of 03:34, 19 September 2015

A fusion of two ferrtin subunits

Ferritin is a protein shelled nanoparticle and is composed of a mixture of 24 light (BBa_K1189024) and heavy (BBa_K1189025) subunits. It is ubiquitous across eukaryotic and prokaryotic systems and is used to sequester intracellular iron (Chasteen et al., 1991). The 2013 iGEM Calgary used ferritin’s iron core as a reporter and its protein shell to scaffold DNA sensing TALEs as part of their project, the FerriTALE (see Figure 1).

BBa_K1189037 joined with DNA sensing TALEs

Figure 1. 3D in silico rendering of BBa_K1189037 formed into functional nanoparticles bound to DNA sensing TALEs. The iron core is chemically modified and use to show when TALEs are bound to DNA. The TALEs are one specific, particular application of the ferritin E coil di-subunit fusion. This nanoparticle is the molecular basis of a DNA lateral flow strip biosensor pursued by the 2013 iGEM Calgary team.

BBa_K1189037 is a fusion of heavy and light ferritin subunits, such that ferritin 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 mandate 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 (Dehal et al., 2010).

Design features

This part has an N-terminal fusion to an E coil connected to ferritin by a GS linker (Figure 2). 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 3 application examples.

BBa_K1189037 SBOL part figure

Figure 2. Ferritin di-subunit fused to an E coil and his tag. The E coil allows binding to other proteins expressing a complementary K coil.



FerriTALE Scaffold Modularity

Figure 3. Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.

This part is identical to BBa_1189018, with the exception of a his-tag for purification.

Results

Expression from pSB1C3

The 2013 iGEM Calgary 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. See Figure 4 for an SDS-PAGE of this 42 kDa isolate. Please see the user reviews section for data on another expression vector which generated this protein with a higher yield.

FerriTALE Scaffold Modularity

Figure 4. FPLC purification fraction of BBa_K1189037 as express per our large scale expression protocol. Were were disappointed with the yield of this 42 kDa protein from the pSB1C3 cloning vector. Please see the user reviews section for results from a proper expression vector.

Conversion of BBa_K1189037 into a reporter

This purified protein product was successfully converted into Prussian blue ferritin, a robust colourmetric reporter. Figure 5 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 6 shows that ferritin with coiled-coils (BBa_1189037) maintains reporter functionality when TALEs are scaffolded using coiled-coil linkers.

Creating Prussian Blue Ferritin out of our Own Ferritin

Figure 5. 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.

Recombinant Prussian Blue FerritinMole Balanced

Figure 6. 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).

Please see the Prussian blue ferritin Wiki page for a detailed analysis of how Prussian blue ferritin, synthesized from commercially available ferritin, performs as a reporter. This data informs how BBa_K1189037 is useful as a reporter.



TecCEM_HS and ferritin

[http://2015.igem.org/Team:TecCEM_HS TecCEM_HS team] HS wanted to see if there were any significant changes in the SDS binding to the molecule if there were any extra elements in the construction. That's why they used this biobrick to see if the SDS molecules were captured although there was an extra coil in the structure. The team made an induction to see if the protein was expressing and here is the result.
We also made a chromatography to see the expression of the protein and to verify the open reading frame.


Ferritin cromatography

Figure 7. An induction of this Biobrick was made and a cromatography at hour 4 was performed (Nickel affinity chromatography). The overexpression can be seen in lane 5, were an approx. 42 kDa band can be seen, that means the cromatography was successful and the protein was actually expressing.

References

  • Chasteen, N. D., & Harrison, P. M. (1999). Mineralization in ferritin: an efficient means of iron storage. Journal of structural biology, 126(3), 182-194.
  • Dehal, P. K., Livingston, C. F., Dunn, C. G., Buick, R., Luxton, R., & Pritchard, D. J. (2010). Magnetizable antibody‐like proteins. Biotechnology journal, 5(6), 596-604.


    • Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      COMPATIBLE WITH RFC[12]
    • 21
      COMPATIBLE WITH RFC[21]
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 1307