Part:BBa_K3265027
gp105-GFP fusion (Phage 201 phi2-1 main shell protein gp105)
This part can be used to express gp105, in a Pseudomonas chlororaphis bacteria. By infecting this transformed bacteria with the jumbophage 201 phi2-1 the compartment can be easily visualized via the egfp tag on the gp105. gp105 is the main shell protein of this compartment.
Background and Biology
Jumbo ϕKZ-like phages have been the object of many studies in the recent years. This is manly because of the fact that they are bacteriophages of the very lethal, opportunistic and multi antibiotic resistant bacteria Pseudomonas aeruginosa. The phages themselves not being harmfull to humans where considered in phage therapies for illneses that range from the serious lung infections to the less serious "swimmers ear".
During the rave of ϕKZ-like phage discovery and characterization, a new member of the genus was discovered : 201 phi2-1. This virus, although it infects a non pathogenic member of the Pseudomonas family (Pseudomonas Chlororaphis),it still garnered quite a bit of interest from the scientific community.
This interest was based on the fact that when pseudomonas chlororaphis is infected by the 201 phi2-1, a certain amount of the infected population starts showing a nucleus like structure approximately 40min psi (post infection). This nucleus like structure was then identified to be a compartment made by the virus to protect its self from the bacterias natural defences. Crisper cas9 enzymes were shown to have no effect against viral dna when it was in the compartement (insert relevant papers). The compartment was also shown to functionally separate transcription and translation, which forced researchers to belive that this compartment had a way to let transcripts out and let newly translated proteins in. Viral capsids after translation and self assembly are somehow docked onto the outer layer of said compartement, soon after packed with viral protein and dna.
Characterization
The gp105-GFP fusion was expressed through a plasmid. The backbone pSEVA44.1 was given to us by our advisor Adrian Tschan. It included a lacI promoter system and antibiotic resistance against Spectinomycin/Streptomycin. For easier reference, we called this plasmid Peterli4.
Peterli4_gp105 is a plasmid designed for use in P.chlororaphis. The Phage201phi2-1 protein gp105, which was isolated from the phage DNA via PCR, is linked with GFP, and can be expressed under the lacI promoter system (induction with IPTG). Antibiotic resistance against Spectinomycin / Streptomycin allows selection for bacteria which were successfully transformed with the plasmid.
This phage protein allows the visualization of the compartment that forms around the phage DNA after infection of P.chlororaphis.
P.chlororaphis was transformed with Peterli4_gp105 through electroporation, transformed bacteria were selected for through antibiotic resistance to streptomycin. Expression of gp105-GFP was induced with 10mM IPTG for 2 hours (28°C, shaking at 180rpm), the bacteria were infected for 1 hour with Phage201-phi2-1 with an MOI 20, stained with Dapi for 30 minutes, and then imaged under a confocal microscope.
For more detailed protocols, consult the protocol page of Team UZurich: https://2019.igem.org/Team:UZurich/Protocols
The DNA and gp105-GFP co-localize in an infected bacteria, with the gp105-GFP surrounding the DNA as a shell.
In uninfected cells, the GFP tagged protein is uniformly spread over the cell.
Results and Discussion
Part overview with snapviewer
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2616
Illegal NotI site found at 139
Illegal NotI site found at 1633 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2672
Illegal BamHI site found at 1658
Illegal XhoI site found at 3840 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2556
Illegal AgeI site found at 3849
Illegal AgeI site found at 4068 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3270
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