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To make a definitive conclusion if an iron NP core can form in our mutant ferritin <a href="http://parts.igem.org/Part:BBa_K2638612">BBa_K2638612</a> we went to a higher resolution TEM performing close up images of the iron core. The iron core resolution is enhanced but contrast is weaker than the images acquired by the 80 kV TEM due to the 200kV TEM used for making those images having a higher energy beam. A second factor which influences the visibility in the TEM images is the composition of the metal core. Fe(III) oxide shown under the TEM is less visible compared to elemental metal. How elemental metal looks under the TEM can be seen on our page on gold and silver forming ferritin. | To make a definitive conclusion if an iron NP core can form in our mutant ferritin <a href="http://parts.igem.org/Part:BBa_K2638612">BBa_K2638612</a> we went to a higher resolution TEM performing close up images of the iron core. The iron core resolution is enhanced but contrast is weaker than the images acquired by the 80 kV TEM due to the 200kV TEM used for making those images having a higher energy beam. A second factor which influences the visibility in the TEM images is the composition of the metal core. Fe(III) oxide shown under the TEM is less visible compared to elemental metal. How elemental metal looks under the TEM can be seen on our page on gold and silver forming ferritin. | ||
− | To confirm that the particles consist of iron we performed an EDX measurement using the TEM. EDX measurements where however not precise enough to give a conclusive answer about the composition of the metal core. The electron beam is bigger than the size of the 8 nm metal core therefore it is not possible to measure a single NP directly but only large clusters of NP. Measuring large clusters has the disadvantage of not being able to see the exact shape and size of the NP which could lead to wrong conclusions when other particles than the one formed inside the ferritin are being measured. The precise size of | + | To confirm that the particles consist of iron we performed an EDX measurement using the TEM. EDX measurements where however not precise enough to give a conclusive answer about the composition of the metal core. The electron beam is bigger than the size of the 8 nm metal core therefore it is not possible to measure a single NP directly but only large clusters of NP. Measuring large clusters has the disadvantage of not being able to see the exact shape and size of the NP which could lead to wrong conclusions when other particles than the one formed inside the ferritin are being measured. The precise size of 8 nm or less and the round shape are the best indicators for NP formed by ferritin. |
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− | <a href="http://parts.igem.org/Part:BBa_E1010">mRFP</a> was overexpressed in psB3t5, the cells where ribolysed and purified using a 0. | + | <a href="http://parts.igem.org/Part:BBa_E1010">mRFP</a> was overexpressed in psB3t5, the cells where ribolysed and purified using a 0.5 mL protein filter (Amicon Ultra). Synthetized fluorescein was used which our team member Jakob Zubek also synthetized himself in one of his chemistry practical courses. |
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We therefore exposed the wild type and the mutant ferritin to a pH of below 1 and to roughly 3 using HCL. Afterwards we added purified mRFP or fluorescein in the highest possible concentration so that passive enclosure could take place. After returning the pH back to 8 we characterized the probe via FCS. | We therefore exposed the wild type and the mutant ferritin to a pH of below 1 and to roughly 3 using HCL. Afterwards we added purified mRFP or fluorescein in the highest possible concentration so that passive enclosure could take place. After returning the pH back to 8 we characterized the probe via FCS. | ||
− | Initially we wanted to look directly for overlapping fluorescence signals of the BFP tagged ferritin (BBa_K592100) and the mRFP enclosed inside. But the effects of brownian motion and the small size (~29 nm) of our fusion ferritin makes this kind of visualization impractical, which is why we turned to fluorescence correlation spectroscopy (FCS) as an alternative. | + | Initially we wanted to look directly for overlapping fluorescence signals of the BFP tagged ferritin (<a href="http://parts.igem.org/Part:BBa_K592100">BBa_K592100</a>) and the mRFP enclosed inside. But the effects of brownian motion and the small size (~29 nm) of our fusion ferritin makes this kind of visualization impractical, which is why we turned to fluorescence correlation spectroscopy (FCS) as an alternative. |
FCS uses a laser to excite single fluorescent molecules passing in front of it through a very small volume. A detector then measures the intensity and time of the fluorescent signal which can be used to estimate the mass and number of molecules in front of the laser. | FCS uses a laser to excite single fluorescent molecules passing in front of it through a very small volume. A detector then measures the intensity and time of the fluorescent signal which can be used to estimate the mass and number of molecules in front of the laser. | ||
Since free fluorophores are much smaller than ferritin encapsulated fluorophores they move through the small detection volume at a much higher speed. Thus the two populations of small free fluorophores and larger slower ferritin-associated fluorophores should be discernable by FCS. | Since free fluorophores are much smaller than ferritin encapsulated fluorophores they move through the small detection volume at a much higher speed. Thus the two populations of small free fluorophores and larger slower ferritin-associated fluorophores should be discernable by FCS. |
Revision as of 18:50, 7 December 2018
Nanoparticles Results
Short Summary
Enhanced Stability and Reassembly of Mutant Ferritin
Correct Folding And Assembly Of Mutant Ferritin
Analysis of Enclosed Fluorophores by Fluorescence Correlation Spectroscopy (FCS) using Zeiss LSM780
Human Ferritin engineering
Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
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