Difference between revisions of "Part:BBa K3187021"

Revision as of 12:37, 16 October 2019 (view source) Lertu (Talk | contribs) ← Older edit		Revision as of 14:28, 17 October 2019 (view source) Lertu (Talk | contribs) Newer edit →
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	<h4>Assembly</h4>		<h4>Assembly</h4>
	<p> The assembly is tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with		<p> The assembly is tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with
−	ultracentrifugation  <a href="#"target="_blank">ultracentrifugatione</a> and are visualized with	+	ultracentrifugation  <a href="https://2019.igem.org/wiki/images/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">ultracentrifugatione</a> and are visualized with
−	<a href="#"target="_blank">TEM</a>. For more information look at our <a href="#"target="_blank">wiki</a>	+	<a href="https://2019.igem.org/wiki/images/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">TEM</a>.
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	contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually		contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually
	intact VLPs all over the sample measuring a diameter of 60 nm or less (Fig. 2). For more information about VLP assembly,		intact VLPs all over the sample measuring a diameter of 60 nm or less (Fig. 2). For more information about VLP assembly,
−	visit our <a href="#"target="_blank">wiki</a>.	+	visit our <a href="https://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP"target="_blank">wiki</a>.

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Revision as of 14:28, 17 October 2019

P22 Bacteriophage Scaffolding Protein

Profile

Name	Scaffold protein
Base pairs	489
Molecular weight	18 kDa
Origin	Enterobacteria phage P22
Properties	In combination with the coat protein (BBa_K3187017) this protein builds the virus capsid of the P22 phage.

Usage and Biology

The P22 scaffold protein (SP) is an important part of the Enterobacteria phage P22 capsid. The virus capsid is assembled with the help of up to 300 copies of the 18 kDa scaffold protein out of approx. 400 copies of the 47kDa coat protein ^{[1] [2]} .
After the assembly of the virus-capsid the SP is released into the capsid. In case of a functional P22 bacteriophage, this protein is extracted out of the capsid in vivo while the viral DNA is loaded into the capsid ^{[3] [4]} . Because the artificial capsid is not filled with DNA the SP remains in the capsid. By fusing the SP with a cargo-protein, one can load the capsid with said cargo ^[5] . This fusion has to occur at the N-Terminus of the SP, because the C-Terminus is important for mechanism of the assembly ^[6] .

Methods

Assembly

The assembly is tested in vivo and in vitro. The assembled VLPs are collected with ultracentrifugation ultracentrifugatione and are visualized with TEM.

Results

Assembly

The images of ultracentrifugation displays that monomeric proteins were separated from assembled capsids by ultracentrifugation at 150.000 x g in a sucrose cushion (35% w/v). After completion of the ultracentrifugation reatment, sediment was clearly visible in the centrifuge tube which we suspected to mainly contain VLPs. Transmission electron microscopy (TEM) was used to image capsids taken from the sediment. For increased contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually intact VLPs all over the sample measuring a diameter of 60 nm or less (Fig. 2). For more information about VLP assembly, visit our wiki.

References

1. ↑ W. Earnshaw, S. Casjens, S. C. Harrison, Assembly of the head of bacteriophage P22: X-ray diffraction from heads, proheads and related structures J. Mol. Biol. 1976, 104, 387. [1]
2. ↑ W. Jiang, Z. Li, Z. Zhang, M. L. Baker, P. E. Prevelige, W. Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nat. Struct. Biol. 2003, 10, 131. [2]
3. ↑ King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119. [3]
4. ↑ S. Casjens and R. Hendrix, (1988) "Control mechanisms in dsDNA bacteriophage assembly", in The Bacteriophages, volume 1, ed. R. Calendar, Plenum Press, p. 15-91. [4]
5. ↑ Dustin P. Patterson, Benjamin Schwarz, Ryan S. Waters, Tomas Gedeon, and Trevor Douglas, Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle ,ACS Chemical Biology 2014 9 (2), 359-365 [5]
6. ↑ P. R. Weigele, L. Sampson, D. Winn‐Stapley, S. R. Casjens, Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains , J. Mol. Biol. 2005, 348, 831. [6]

Sequence and Features