iGEM 2018 — Team Lethbridge

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# Team:Lethbridge/Notebook

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* Project
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* [Parts](https://2018.igem.org/Team%3ALethbridge/Parts)
  + [Overview](https://2018.igem.org/Team%3ALethbridge/Parts)
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  + [Improved Parts](https://2018.igem.org/Team%3ALethbridge/Improve)
  + [Basic](https://2018.igem.org/Team%3ALethbridge/Basic_Part)
* Human Practices
  + [Human Practices](https://2018.igem.org/Team%3ALethbridge/Human_Practices)
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* Lab
  + [Experiments](https://2018.igem.org/Team%3ALethbridge/Experiments)
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![Notebook Banner Image](https://static.igem.org/mediawiki/2018/d/d6/T--Lethbridge--banner_Notebook.png)

# [May 2018](https://static.igem.org/mediawiki/2018/a/a2/T--Lethbridge--maylabbook.pdf)

# [July 2018](https://static.igem.org/mediawiki/2018/6/6d/T--Lethbridge--julylabbook.pdf)

# [August 2018](https://static.igem.org/mediawiki/2018/5/5b/T--Lethbridge--Auglabbook.pdf)

# [September 2018](https://static.igem.org/mediawiki/2018/5/57/T--Lethbridge--september.pdf)

# [October 2018](https://static.igem.org/mediawiki/2018/b/bc/T--Lethbridge--Octoberlabbookupdated.pdf)


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# Team:Lethbridge/Experiments

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![Experiments Banner Image](https://static.igem.org/mediawiki/2018/b/bf/T--Lethbridge--banner_Experiments.png)

# Mass Spectrometry

Mass spectroscopy is a quantitative analysis method that is based on the mass and the charge of the molecule in question. The sample is analyzed by first being transferred to the gas phase which is passed through the analyzer. The signals from the analyzer then sorts the sample ions of fragments by mass and charge (Urban, 2016). From there samples can be identified and formed into recognizable species. This technique is highly specific and selective, making it a great characterization tool for our PNC toolkit. It will be able to prove that the sequences we have used to form our constructs match when comparing this to the mass spectroscopy data we recieve Our mass spectroscopy data for the P22 capsid can be found [here](https://2018.igem.org/Team%3ALethbridge/Results).

# Analytical Ultracentrifugation

AUC is a way for analyzing macromolecules and interactions quantitatively. There are many advantages of this technology as the is able to work with samples in solution. This technology makes use of three main analytical features: Fluorescence, absorbance and interference. Fluorescence allows for analysis of complex mixtures and interactions of proteins giving distinct data. These techniques coupled with ultracentrifugation can also aid analysis of macromolecular interactions, sizes of complexes/individual molecules and how it travels through solutions without the need for tags or other types of modifications (Cole  *et al.,* 2009). In order to highly characterize our protein nano compartments we are using AUC to look at size, capsid formation and how these PNC interact with their cargos. This data will inform our project in terms of encapsulation kinetics and the stoichiometry of PNC to cargo.
This technology is versatile and highly specific which is perfect for characterizing our PNC toolkit, especially our novel PNC, the Arc minimal construct that is derived from HIV homology. Results on our AUC experiment with Arc minimal can be found [here](https://2018.igem.org/Team%3ALethbridge/Results). The AUC works by running the samples at a certain temperature and centrifugal force, much like a regular ultracentrifuge. The integrated analysis tools take in the data over time, giving dynamic data as the sample moves through solution.

![](https://static.igem.org/mediawiki/2018/d/d1/T--Lethbridge--auc_fig.jpg)

**Figure 1 .** Schematic of analytical ultracentrifugation (Wilkinson Group, Universite de Montreal)

# Transmission Electron Microscopy (TEM)

Transmission electron microscopy is an advanced form of microscopy used for resolving very small molecular structures or cells (Winey *et al.,* 2014). Due to the inability for structures seen by the naked eye, TEM is crucial for visualizing our ability to form capsids and or see encapsulation of cargos. TEM works by passing an electron beam through a thin layer of sample trapped on a plastic surface which then creates a contrast image formed by the absorbed and scattered light patterns from the beam (Winey *et al.,* 2014). The high amount of resolution and simple methods of sample preparation makes this technology an easy way to visualize and confirm capsid formation of our constructs. Our TEM results for Arc minimal and P22 with cargo SPCas9 can be found [here](https://2018.igem.org/Team%3ALethbridge/Results).

# Protocols

* [Colony PCR](https://static.igem.org/mediawiki/2018/b/ba/T--Lethbridge--colony_pcr.pdf)
* [DNA Purification](https://static.igem.org/mediawiki/2018/b/b9/T--Lethbridge--DNA_purification.pdf)
* [LB Media](https://static.igem.org/mediawiki/2018/a/a7/T--Lethbridge--lb.pdf)
* [Ligation](https://static.igem.org/mediawiki/2018/f/fc/T--Lethbridge--ligation.pdf)
* [PCR](https://static.igem.org/mediawiki/2018/7/71/T--Lethbridge--pcr.pdf)
* [Phusion PCR](https://static.igem.org/mediawiki/2018/d/db/T--Lethbridge--phusion.pdf)
* [pJET Cloning](https://static.igem.org/mediawiki/2018/b/b0/T--Lethbridge--pjet.pdf)
* [Minipreps](https://static.igem.org/mediawiki/2018/0/01/T--Lethbridge--miniprep.pdf)
* [Protein Overexpression and SDS PAGES](https://static.igem.org/mediawiki/2018/1/1d/T--Lethbridge--overexpression.pdf)
* [Affinity Chromatography](https://static.igem.org/mediawiki/2018/4/41/T--Lethbridge--his-tagged.pdf)
* [Restriction Enzymes](https://static.igem.org/mediawiki/2018/0/03/T--Lethbridge--p22_purification.pdf%3E%20P22%20Purification%20%3C/a%3E%3C/li%3E%3Cli%3E%3Ca%20href%3D)
* [Transformations](https://static.igem.org/mediawiki/2018/c/cb/T--Lethbridge--transformationprotocol.pdf)
* [Methanol-Chloroform Protein Extraction](https://static.igem.org/mediawiki/2018/4/47/T--Lethbridge--methanol.pdf)
* [Trichloroacetic Acid Protein Extraction](https://static.igem.org/mediawiki/2018/1/1d/T--Lethbridge--TCA.pdf)
* [Arc minimal/Arc protein Purification](https://static.igem.org/mediawiki/2018/9/9c/T--Lethbridge--arcminpurification.pdf)
* [RNA Spinach in Vitro](https://static.igem.org/mediawiki/2018/2/29/T--Lethbridge--RNASpinach.pdf)

###### References

* Urban, P. (2016) Quantitative mass spectrometry: an overview. Philosophical transactions of the royal society A: mathematics, physical and engineering sciences. 13, 376.
* Cole, J., Lary, J., Moody, T and Laue, T. (2009) Analytical Ultracentrifugation: Sedimentation Velocity and sedimentation Equilibrium. Methods in cell biology. 84, 143-179
* Winey, M., Meehl, J., O’Toole, E., and Giddings Jr., T. (2014) Conventional transmission electron microscopy. Molecular biology of the cell. 25, 319-323
* Wilkinson, K. Universite de Montreal. https://www.webdepot.umontreal.ca/Usagers/wilkinsk/MonDepotPublic/analytical-ultracentrifugation-(auc).html

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# Team:Lethbridge/Results

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* [Parts](https://2018.igem.org/Team%3ALethbridge/Parts)
  + [Overview](https://2018.igem.org/Team%3ALethbridge/Parts)
  + [Composite](https://2018.igem.org/Team%3ALethbridge/Composite_Part)
  + [Improved Parts](https://2018.igem.org/Team%3ALethbridge/Improve)
  + [Basic](https://2018.igem.org/Team%3ALethbridge/Basic_Part)
* Human Practices
  + [Human Practices](https://2018.igem.org/Team%3ALethbridge/Human_Practices)
  + [Software](https://2018.igem.org/Team%3ALethbridge/Software)
  + [Public Engagement](https://2018.igem.org/Team%3ALethbridge/Public_Engagement)
  + [Entrepreneurship](https://2018.igem.org/Team%3ALethbridge/Entrepreneurship)
  + [Product Design](https://2018.igem.org/Team%3ALethbridge/Applied_Design)
* Lab
  + [Experiments](https://2018.igem.org/Team%3ALethbridge/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ALethbridge/Notebook)
  + [Safety](https://2018.igem.org/Team%3ALethbridge/Safety)
  + [InterLab](https://2018.igem.org/Team%3ALethbridge/InterLab)
* [Team](https://2018.igem.org/Team%3ALethbridge/Team)
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# Purification of Full-Length Arc and Minimal Arc Gag

![](https://static.igem.org/mediawiki/2018/4/4a/T--Lethbridge--arcmin_pur.png)

**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 in Figure 2.

![](https://static.igem.org/mediawiki/2018/c/c0/T--Lethbridge--arcfull_sec.png)

**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.

![](https://static.igem.org/mediawiki/2018/d/df/T--Lethbridge--arcmin_actual.png)

**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 2. 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.

![](https://static.igem.org/mediawiki/2018/3/3c/T--Lethbridge--arcmin_sec.png)

**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

![TEM Full](https://static.igem.org/mediawiki/2018/7/7a/T--Lethbridge--ArcFullTEM.png)

**Figure 5.** TEM image (60,000X magnification) of negatively-stained self-assembled full-length Arc PNCs. Scale bar = 200um.

## Minimal Arc Gag

![TEM Arc Min](https://static.igem.org/mediawiki/2018/4/40/T--Lethbridge--TEM-ArcMin.png)

**Figure 6.** 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

![TEM P22](https://static.igem.org/mediawiki/2018/4/48/T--Lethbridge--TEM-P22-NoSP.png)

**Figure 7.** TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids in the absence of scaffolding proteins. Scale bar = 500um.

## P22-SP-Cas9

![TEM P22-SP-Cas9](https://static.igem.org/mediawiki/2018/0/0e/T--Lethbridge--TEM-P22-SP-Cas9.jpg)

**Figure 8.** TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids with the SP-Cas9 cargo. Scale bar = 200um.

# Mass Spectrometry

![](https://static.igem.org/mediawiki/2018/1/18/T--Lethbridge--p22_massspec.png)

**Figure 9.** Characterization of P22 purified P22 proteome using 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 figure above 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.

# Analytical Ultracentrifugation of Minimal Arc Gag

We attempted to characterize our purified Arc-min protein with the use of sedimentation velocity data gathered through analytical ultracentrifugation. We prepared samples from both Arc-min and Arc-full, but were only able to observe any sedimentation from Arc-min. This suggests that there were insufficient amounts of Arc-full protein to run on AUC. The data that we observed for Arc-min indicate that the protein monomers aggregated which can be observed in the aggregation boundary in Figure 10 ( top left, absorbance; top right, relative intensity).(Refer to our [Experiments page](https://2018.igem.org/Team%3ALethbridge/Experiments) for more details on AUC.)

![](https://static.igem.org/mediawiki/2018/9/9e/T--Lethbridge--AUC_data.png)

**Figure 10.** Analytical ultracentrifugation sedimentation results and simulations. **(top left)** Converted relative light intensity sedimentation rates into absorbance to compare to simulations. **(top right)** AUC sedimentation rates of Arc-min (top-right) measured in intensity of light at 217 nm and spun at 30,000 rpm. **(bottom left)** Modelled expected sedimentation rates of an Arc-min monomer and **(bottom right)** hexamer. Simulated data is based on estimated molar masses and globular anisotropies, using identical experimental conditions used in the actual experiment.

# Cell Culture Transfection with Minimal Arc Gag

In progress! Visit us at the iGEM Jamboree for more info.

# Purification of P22 and SP-Cas9

![](https://static.igem.org/mediawiki/2018/f/fb/T--Lethbridge--cas9_pur.png)

**Figure 11.** Nickel Affinity Chromatography Purification of SPCas9 using a gravity flow column shown on a 12% SDS-PAGE gel. Protein ladder is the 10-240kDa prestained ladder from Biobasic. The protein can be seen at the 245kDa mark. Lanes right to left show the stages of purification from Cell pellet, Lysate, supernatant flow through, column washes, elutions and the regeneration of the column.

The Purification of SPCas9 using nickel affinity chromatography was successful in terms of isolating the protein. Although yield is low compared to the levels of protein seen in the wash samples and lysate, protein was isolated. Further purification of smaller proteins was done by using a centrifugation spin column with a molecular weight cutoff filter of 50kDa. Loss of yield is likely due to overloading the column with supernatant and not enough nickel resin on the column compared to supernatant volumes. No further purification methods were done. These samples were used for both transmission electron microscopy imaging and experiments done by the University of Calgary as found [here](https://2018.igem.org/Team%3ACalgary).The cell expression and purification protocol used can be found [here](https://2018.igem.org/Team%3ALethbridge/Experiments).

![](https://static.igem.org/mediawiki/2018/f/f8/T--Lethbridge--p22_pur.png)

**Figure 12.** Purification of P22 capsid using 35% sucrose solution and ultracentrifugation. Samples are shown on a 15% SD-PAGE gel. Protein ladder is the 10-240kDa prestained ladder from Biobasic. From lane Right to left shows the stages of purification. Lane 2 and 3 show the initial cell pellet and supernatant respectfully, Lane 4 shows the proteins within the sucrose and lane 5 shows the resulting crude fraction of P22 which is seen at the point shown at the red arrow.

We were unable to continue the purification due to time constraints; however, we were able to isolate the protein in the crude fraction as seen in Lane 5. The sample was then dialyzed against 1XPBS to get rid of residual sucrose. The sample was used for the Transmission electron microscopy experiments and the experiments done by the University of Calgary iGEM team.

# Collaboration with the University of Calgary iGEM Team

In progress! Visit us at the iGEM Jamboree for more info.

# Improved Parts

We characterized 15 parts from the 2017 Lethbridge iGEM team's "[Next Vivo](https://2017.igem.org/Team%3ALethbridge)" project. Please visit the [Improved Parts](https://2018.igem.org/Team%3ALethbridge/Improve) page for links to the new registry pages and all related data.