Part:BBa_K4268020

S-TIP 37 Ghost Phagemid with Biotin-tagged Capsid

Usage and Biology

S-TIP 37 is a T7-like cyanophage that infects its host via a lytic life cycle (Shitrit et al., 2021). T7-like phages are characterized by a complex symmetrical capsid structure, which includes an icosahedral head that houses the phage's genome, an internal core region that stabilizes the packaged DNA inside the head, a neck region that facilitates DNA delivery into a host, and six tail fibers used for attachment to its host. (Raytcheva et al., 2011).

Figure 1: A labeled visual detailing the various structures of a T7-like phage (Kemp et al., 2005)

This part belongs to a collection that codes for a "ghost" phage. This ghost phage is a capsid-only, empty viral shell that could be modified to immobilize Cyanobacteria recognized by the viral tail fibers or used with modification to deliver substances to a chassis Cyanobacteria.

However, it will infect Synechococcus sp WH 8109, the cyanobacteria strain that is the natural host of S-Tip37. Further modeling will be needed to determine if the "ghost" phage could effectively target other strains of Cyanobacteria that are used in synthetic biology, such as Synechococcus sp PCC 11901.

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The S-TIP 37 Ghost Phagemid with Biotin-tagged Capsid codes for the putative proteins that make the icosahedral head (Internal Core, Biotin-tagged Capsid, and Capsid Assembly Protein), neck region (Tail Tubular A, B, and the Head-Tail Connector protein), and tail fibers (Tail Fiber-like Protein) of S-TIP 37. This construct, when introduced into E. coli, is expected to lead to the production of phage proteins and the assembly of those proteins into a ghost phage – that is, an empty phage capsid. This phage will contain a biotin-tagged icosahedral head region, which can be used to detect or immobilize the ghost phage for downstream applications in conjunction with streptavidin.

With modification, this phage could be utilized for a number of purposes, for example:

• Addition of the S-TIP 37 putative DNA packaging protein NCBI Gene ID: 54998430, to the phagemid to permit incorporation of a DNA construct into the phage, which could then be used to deliver the construct to a host cell for engineering purposes.

• Modification of the viral capsid to enable use of the virus as a nanomaterial for delivery of other materials to Cyanobacteria – for example, an antimicrobial. 

• Modification of tail-fiber sequences to create viruses that can specifically recognize different species of Cyanobacteria for tagging purposes or for immobilization. The presence of the biotin tag on the viral capsid simplifies this process as streptavidin-biotin interactions can be used for both purposes.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 3810
  Illegal PstI site found at 7523
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 6161
  Illegal NheI site found at 10697
  Illegal PstI site found at 3810
  Illegal PstI site found at 7523
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5174
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 3810
  Illegal PstI site found at 7523
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 3810
  Illegal PstI site found at 7523
  Illegal NgoMIV site found at 3450
  Illegal NgoMIV site found at 3589
  Illegal NgoMIV site found at 4545
  Illegal NgoMIV site found at 12194
  Illegal AgeI site found at 14225
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Shitrit D, Hackl T, Laurenceau R, Raho N, Carlson MCG, Sabehi G, Schwartz DA, Chisholm SW, Lindell D. Genetic engineering of marine cyanophages reveals integration but not lysogeny in T7-like cyanophages. ISME J. 2022 Feb;16(2):488-499. doi: 10.1038/s41396-021-01085-8. Epub 2021 Aug 24. PMID: 34429521; PMCID: PMC8776855.

Raytcheva DA, Haase-Pettingell C, Piret JM, King JA. Intracellular assembly of cyanophage Syn5 proceeds through a scaffold-containing procapsid. J Virol. 2011 Mar;85(5):2406-15. doi: 10.1128/JVI.01601-10. Epub 2010 Dec 22. PMID: 21177804; PMCID: PMC3067778.

Kemp P, Garcia LR, Molineux IJ. Changes in bacteriophage T7 virion structure at the initiation of infection. Virology. 2005 Sep 30;340(2):307-17. doi: 10.1016/j.virol.2005.06.039. PMID: 16054667.