Our goal is to enable a wide range of bioengineering and biomedical applications by making a collection of protein nanocompartments (PNCs) that are modular, specific, standardized, and safe. With that in mind, we designed a number of PNCs for encapsulation of various cargo types, as well as application-specific surface modifiers and cargo-loading approaches.

The surface modifiers typically act as a targeting mechanism, which directs the PNC to desired cell types or other targets. However, some surface modifiers may change other PNC attributes, such as tendency for aggregation, which makes each PNC specific for each application. Ultimately, VINCEnT is intended to be a diverse tool for research, drug and vaccine development, gene therapy delivery, and many other applications.

Proper encapsulation of small molecule, nucleic acid, or protein cargos relies on stable and predictable protein-protein and protein-chemical interactions between the PNC and the intended cargo. Of particular relevance to nucleic acid encapsulation, PNCS derived from viruses have naturally evolved to have specific interactions with the viral nucleic acid genome. Though we are NOT using viruses, we are still able to exploit the natural strategies that these systems use for encapsulation.

In total, we have designed nine new parts:

1. P22 coat protein (PNC)
2. P22 scaffolding protein fused to Cas9 (CRISPR-associated cargo)
3. P22 scaffolding protein fused to sfGFP (reporter cargo)
4. P22 decoration protein, DecS134C (surface modifier for P22 PNC aggregation)
5. MS2 coat protein (PNC)
6. Polyarginine cell penetrating peptide (surface modifier)
7. GFP protein with an anionically charged peptide (cargo modified for MS2)
8. Arc (full-length) protein (PNC for RNA cargo, specifically)
9. Minimal Arc Gag (PNC for RNA cargo, specifically)

All designed parts contain the same T7 promoter (BBa_I719005), medium strength ribosome binding site (RBS) (BBa_B0034), the coding sequence with special modifications (which will be explained further on), and double terminator (BBa_B0015).

The coat protein from the Salmonella typhimuriam P22 bacteriophage produces one of the largest known capsids. It assembles from 420 copies of the coat protein and 100-300 copies of a scaffolding protein (SP) to form a PNC with a diameter of approximately 60nm (O’Neil et al., 2011; Parent et al., 2010). P22 is being preferentially used for our toolkit because of its large, hollow interior space that can accommodate larger cargos. Although this capsid protein has the ability to self-assemble, efficiency and stability of assembly is enhanced with the addition of the P22 SP (O’Neil et al., 2012; 2011). For our purposes, because the SP interacts with the P22 coat protein on the C-terminus, the SP has been truncated on the N-terminus and can be fused to protein cargos or other modifiers (O’Neil et al., 2011). For VINCEnT, the P22 SP has been fused to our three cargos: Cas9, a super-folded GFP (sfGFP), and FitD to ensure encapsulation.

All designed parts contain the same T7 promoter (BBa_I719005), medium strength ribosome binding site (RBS) (BBa_B0034), the coding sequence with special modifications (which will be explained further on), and double terminator (BBa_B0015).

While virus-like particles are useful components of VINCEnT, we also wanted to develop a non-immunogenic PNC with RNA packaging capabilities. Such a tool could potentially enable simpler transfection of mammalian cell lines for fellow iGEMers and other researchers.

Arc is an activity-regulated cytoskeletal-associated protein that has recently been recognized as a repurposed Ty3/Gypsy retrotransposon. A bi-lobar domain within Arc has significant homology to Gag proteins, which are the major capsid proteins of many viruses including Human Immunodeficiency Virus type 1 (HIV-1) and Rous-Sarcoma Virus (RSV). In response to synaptic activity in neurons, Arc proteins self-assemble via this Gag domain (similar to the related viral particles) to encapsulate Arc mRNA and shuttle it to neighbouring cells (Pastuzyn et al., 2018; Ashley et al., 2018).

For RNA encapsulation, we take advantage of the self-mRNA encapsulation strategy employed by Arc proteins in vivo. Based on sequence homology with HIV-1 Gag, the predicted mRNA encapsulation sequence is located within the N-lobe of the Arc protein and so fusion of cargo RNA to the Arc mRNA (or this short predicted consensus sequence) should enable encapsulation (Clever et al., 1995). However, Arc PNCs also readily encapsulate nearby non-specific RNA molecules lacking this consensus sequence in vitro (Pastuzyn et al., 2018). To test RNA encapsulation efficiency, Clover and mRuby RNA with or without the predicted Arc encapsulation sequence was incubated with Arc proteins in vitro before application to various cell cultures.

To ensure we would not retain any native Arc functionality that might impact cellular activity in culture, we also designed a “minimal” Arc Gag protein based on homology with other known Gag domains, including HIV-1 and RSV. We used template-based structural predictions to model this minimal Arc Gag and its predicted assembly into higher-order structures. We expected this minimal Arc PNC to perform similarly to the full-length Arc PNC.

We have employed several methods of purification. The methods used for our non-tagged PNCs are based on those found in the literature (Qazi et al., 2016; Pastuzyn et al., 2018).

This iGEM season, we were only able to clone one part (the novel minimal Arc Gag construct) and begin characterizing it. We intend to finish characterizing our toolkit, which includes collecting additional data from transmission electron microscopy imaging, analytical ultracentrifugation, mass spectroscopy, and application experiments (including eukaryotic cell transfection, CRISPR system delivery, and protein delivery).

We would like for our system to be accessible to other iGEM teams and researchers who may want to use these parts for their own projects. Therefore, we hope to eventually submit all nine of our designed parts to the registry (and more!).

As most of our PNC purification methods include high speed centrifugation and specialized equipment, we intend to add His tags (or other purification tags) to all of our PNCs to make purifications easier and enable higher yield.

This year, because we only expressed parts in E. coli, our system was limited as to what types of cell-penetrating peptides could be tested due to lack of specificity and because the peptides often killed the host during expression. In the future, we would like to characterize PNCs with antimicrobial surface peptides as a possible novel antibiotic using yeast models.

• Ashley, J., Cordy, B., Lucia, D., Fradkin, L. G., Budnik, V., & Thomson, T. (2018). Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons. Cell, 172, 262-274.
• Baldwin, B., Mayer, M., Dayton, J., Pau, N., Mendila, J., Sullivan, M., Moore, A., & Mills, E. (2002) Comparative growth and feeding in zebra and quagga mussels (Dreissena Polymorpha and Dreissena bugensis): implication for North American Lakes. Canadian Journal of Aquatic Science, 59, 680-694.
• Bartnicki, F., Bonarek, P., Kowalska, & Strzalka, W. (2017) The argi-system: one-step purification of protein tagged with arginine-rich cell-penetrating peptides. Science Reports, 7, 2619.
• Clever, J., Sassetti, C., & Parslow, T. G. (1995) RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1. J Virol, 69, 2101-2109.
• Glasgow, J., Capehart, S., Francis, M., & Tullman-Ercek, D. (2012) Osmolyte-mediated encapsulation of proteins inside MS2 viral capsids. ACS Nano, 6, 8658-8664.
• Mazzone, H. (1998) CRC Handbook of Viruses: Mass-Molecular Weight Value and Related Properties. Boca Raton (FL): CRC Press LLC.
• Molloy, D., Mayer, D., Giamberini, L., & Gaylo, M. (2013) Mode of action of Pseudomonas fluorescens strain CL145A, a lethal control agent of Dreissend mussels (Bivalvaia: Dreissenidae). Journal of Invertebrate Pathology, 113, 115-121.
• O’Neil, A., Prevelidge, P., Basu, G., & Douglas, T. (2012) Coconfinement of fluorescent proteins: spatially enforced communication of GFP and mCherry encapsulated within the P22 capsid. Biomacromolecules, 13, 3902-3907.
• O’Neil, A., Reichhardt, C., Johnson, B., Prevelige, P., & Douglas, T. (2011) Genetically programmed in vivo packaging of protein cargo and its controlled release from bacteriophage P22. Angewandte Chemie International Edition, 50, 7425-7428.
• Parent, K. N., Khayat, R., Tu, L. H., Suhanovsky, M. M., Cortines, J. R., Teschke, C. M., Johnson, J. E., & Baker, T. S. (2010) P22 coat protein structures reveal a novel mechanism for capsid maturation: stability without auxiliary proteins or chemical cross-links. Structure, 18, 390-401.
• Pastuzyn, E., Da, C., Kearns, R., Kyrke-Smith, M., Taibi, A., McCormick, J., Yoder, N., Belnap, D., Erlendsson, S., Morado, D., Briggs, J., Feschotte, C., & Shepherd, D. (2018) The neuronal gene Arc encodes a repurposed retrotransposon gag protein that mediates intercellular RNA transfer. Cell. 172, 275-288.
• Qazi, S., Miettinen, H., Wilkinson, R., McCoy, K., Douglas, T., & Wiedenheft, B. (2016) Programmed self-assembly of an active P22-Cas9 nanocarrier system. Molecular Pharmaceutics, 13, 1191-1196.
• Schmidt, N., Mishra, A., Lai, G., & Wong, G. (2010) Arginine-rich cell-penetrating peptides. FEBS Letters, 584, 1806-1813.
• Sprung, M., & Rose, U. (1988) Influence of food size and food quantity on the feeding of the mussel Driessena Polymorpha. Oncologia, 77, 526-532.
• Uchida, M., LaFrance, B., Broomwell, C., Prevelige Jr., P., & Douglas, T. (2015) Higher order assembly of virus-like particles (VLPs) mediated by multi-valent protein linkers. Small, 13, 1562-1570.