Difference between revisions of "Part:BBa K1189020"
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| − | <p>This light ferritin chain comes from humans. This part along with heavy ferritin (<partinfo>BBa_K1189025</partinfo>), form the ferritin nanoparticle, an iron-storage particle made up of 24 subunits. The formed nanoparticle is highly robust, remaining stable at extreme pHs and temperatures. | + | <p>This light ferritin chain comes from humans. This part along with heavy ferritin (<partinfo>BBa_K1189025</partinfo>), form the ferritin nanoparticle, an iron-storage particle made up of 24 subunits <Lawson <i>et al.</i>, 1991). The formed nanoparticle is highly robust, remaining stable at extreme pHs and temperatures. |
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| − | This nanoparticle can also be used as a reporter when the iron core is modified with potassium ferrocyanide to form Prussian Blue. The Prussian Blue ferritin can then act as a peroxidase mimic, similar to horseradish peroxidase, resulting in colour changes in the presence of hydrogen peroxide, and TMB or ABTS. | + | This nanoparticle can also be used as a reporter when the iron core is modified with potassium ferrocyanide to form Prussian Blue. The Prussian Blue ferritin can then act as a peroxidase mimic, similar to horseradish peroxidase, resulting in colour changes in the presence of hydrogen peroxide, and TMB or ABTS (Zhang <i>et al.</i>, 2013). |
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Revision as of 01:51, 26 October 2013
Light chain human ferritin with his-tag and E-coil under the lacI inducible promoter
This light ferritin chain comes from humans. This part along with heavy ferritin (BBa_K1189025), form the ferritin nanoparticle, an iron-storage particle made up of 24 subunits <Lawson et al., 1991). The formed nanoparticle is highly robust, remaining stable at extreme pHs and temperatures.
This nanoparticle can also be used as a reporter when the iron core is modified with potassium ferrocyanide to form Prussian Blue. The Prussian Blue ferritin can then act as a peroxidase mimic, similar to horseradish peroxidase, resulting in colour changes in the presence of hydrogen peroxide, and TMB or ABTS (Zhang et al., 2013).
We added the lacI promoter (BBa_J04500), double terminator (BBa_B0010, & BBa_B0012) and a his-tag in order for us to induce protein expression as well as purify it.
Applications of BBa_K1189020
We evaluated the binding of our coils using other constructs that make use of the E and K coil parts submitted. In the case of the coils we were interested to see if the K-coil fused to TALE proteins (BBa_K1189029, BBa_K1189030) could bind to the E-coil found on one of our Prussian blue ferritin constructs (BBa_K1189018). 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 in vitro system.
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 3).
Figure 3. 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.
We also performed an immunoprecipitation assay to demonstrate the binding of the E/K coils (Figure 4).
Figure 4. Assay showing coiled-coil interaction in vitro. Crude lysates from a negative control (RFP), GFP-Ecoil and His-Kcoil were combined together to investigate interaction and immunoprecipitated with GFP or an isotype control and then further probed with α-His antibody. Only in the presence of both GFP and a His tag we see a band indicating interaction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 230
Illegal AgeI site found at 881 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 746