Team:Lethbridge/Results
Purification of Full-Length Arc and Minimal Arc Gag
Figure 1. A 15% SDS-PAGE gel of the Arc protein after purification with sucrose and cesium chloride gradients. The RMR002 protein ladder from genemark bio. was used. Lanes 5 and 6 are the purified fractions from the cesium Chloride gradient as seen around the 45kDa mark as expected, and Lanes 1 and 3 show the cell pellet and Lysate respectively. These samples were then used for transmission electron microscopy.
Purification was also done with a sucrose cushion with size exclusion chromatography afterwards. However concentrations were too low and the gel came up as negative. The chromatogram can be seen below.
Figure 2. Chromatogram of the Arc full Size exclusion chromatography purification on the superdex 75 column. Although fractions were taken from the experiment, the protein amount was too low to be seen on the gel.
Figure 3. A 15% SDS-PAGE gel of the purification of Arc minimal construct first using a sucrose cushion followed by dialysis in 1XPBS and Size Exclusion Chromatography. The confirmed band is at about 40kDa indicating that Arc minimal construct is likely dimerizing. From left to right Lanes 2-10 contain the elutions taken according to the histograms seen in figure {ADDDDD]. In Lane 11 there is the sample that was dialyzed. Lane 12 contains the cell pellet and Lane 13 contains the crude fraction from the sucrose cushion.
Figure 4. Chromatogram of Arc minimal construct size exclusion chromatography purification on the superdex 75 purification column. Samples were taken at Fractions 7-10 (whereby the two peaks were isolated), 11-13, 19-20 and 23.
Transmission Electron Microscopy
Transmission electron microscopy (TEM) confirmed the presence of spherical nanoparticles in both the full-length M. musculus Arc protein and minimal Arc Gag protein samples. Even in the absence of RNA cargo, the Arc-derived PNCs were capable of self-assembly in vitro. In both samples, the average PNC diameter was approximately 30nm +/- 10nm.
Full-Length Arc
Figure 5.(left) TEM image (60,000X magnification) of negatively-stained self-assembled full-length Arc PNCs. Scale bar = 200um.
Minimal Arc Gag
(right) TEM image (100,000X magnification) of negatively-stained self-assembled minimal Arc Gag PNCs. Scale bar = 100um.
TEM also confirmed the presence of spherical nanoparticles in the P22 protein sample (without added scaffolding proteins; SPs) and P22 + SP-Cas9 protein samples. In the absence of the scaffolding protein, P22 capsid proteins were capable of self-assembly but with a significant variation in size and stability (many collapsed structures were evident in the sample). The P22 + SP-Cas9 PNCs had a much more consistent architecture but had a diameter of 120 +/- 5nm, which is approximately twice that of typical P22 virus-like particles. The 1:1 capsid to cargo ratio may have caused overloading of the PNCs, which caused an increase in overall size.
P22 coat proteins
Figure 6. (left) TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids in the absence of scaffolding proteins. Scale bar = 500um.
P22-SP-Cas9
(right) TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids with the SP-Cas9 cargo. Scale bar = 200um.
Mass Spectrometry
Figure 7. For further characterization of our parts, we have run mass spectroscopy samples extracted from SDS-PAGE gels and digested with Trypsin.The data from the Mass spectroscopy analysis was then run on the software Thermo proteome Discoverer 2.0.0.802.
The above figure shows an alignment of the P22 sequence based against the spectroscopy data. Consensus between the Sequence and Mass spectroscopy data, is shown in the green and has a value of 68.56%. The average cover success is about 70% according to the biomolecular analysis facility at the University of Virginia School of Medicine. Therefore, our mass spectroscopy analysis was successful and can inform the P22 structure and chemical composition. More importantly it proves that from comparing to our peptide sequence data to this experimental data, we were able to express and purify P22 successfully.
Minimal Arc Gag
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P22 Capsid
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Analytical Ultracentrifugation of Minimal Arc Gag
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Cell Culture Transfection with Minimal Arc Gag
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Purification of P22 and SP-Cas9
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Collaboration with the University of Calgary iGEM Team
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Improved Parts
We characterized 15 parts from the 2017 Lethbridge iGEM team's "Next Vivo" project. Please visit the Improved Parts page for links to the new registry pages and all related data.