Project Design
The phage-bacteria interaction
The jumbobacteriophage 201 phi 2-1 infects P.chlororaphis by injecting its DNA into the bacteria. Immediately, a protein shell forms around the DNA, with the main shell protein being the phage protein gp105. At the same time, phage nucleases degrade the bacterial DNA. Thus, the only functional DNA left is the DNA protected by the compartment. As a side note: the compartment also protects DNA against the CRISPR/Cas system.DNA replication takes place within the compartment, while translation takes place outside of the compartment. It has been observed that proteins can enter and exit the compartment. After enough DNA has been replicated, virions are filled with DNA. This packaging happens at the compartment. After about 4 hours, the bacterial cell undergoes lysis, releasing the next phage generation.
Figure 4 in "The Phage Nucleus and Tubulin Spindle Are Conserved among Large Pseudomonas Phages" by Chaikeeratisak et al. (DOI: 10.1016/j.celrep.2017.07.064)
We selected 9 proteins with unknown function that we thought may play a role in forming the compartment. Additionally, there was one protein, gp105, that was proposed to be the main shell proteins by earlier papers by Chaikeeratisak which we used as a positive control for all our steps. We also added 3 other proteins with known localization to our pool of proteins, leading to a total of 13 proteins.
To be able to conclude a possible involvement with the compartment, we visualized the proteins in-vivo by tagging them with a fluorescent protein. This was done with our new best friend, Peterli4, our favorite plasmid.
Here you see a sketch of the plasmid Peterli4 with gp105, the main shell protein.
On here, you can see the all the important parts of our plasmid:
-- Resistance gene (SmR) which allows the selection for transformed bacteria (Spectomycin for DH5alpha and Streptomycin for P.chlororaphis)
-- Induction system (LacI) which allows induction of protein production with IPTG
-- eGFP which is connected to the protein of interest and allows
-- Protein of interest, here it's gp105, the main shell protein
A plasmid like this was constructed for all 13 proteins with the following workflow:
For more detailed protocols, check out our protocol page or the registry description of our Peterli4_egfp_gp105 part (BBa_K3265027).
Once we had all our plasmids transformed into P.chlororaphis, we induced the bacteria with IPTG and infected them with our phage. We could then check under the microscope where our proteins localize after infection.
In uninfected bacteria, the DNA, stained for by Dapi, and the tagged protein are uniformly distributed over the cell. If a cell is infected, the DNA outside of the compartment gets destroyed, so the remaining DNA is visible as a centralized circle. If the tagged protein localizes with the DNA, as gp105 does here, then we infer a possible interaction with the compartment.
gp105 is our positive control, which is proposed to be the main shell protein.
Our battleplan for the lab
To find the proteins responsible for forming the compartment, we compared genes from our phage with genes from two other related phages that also form the compartment. This was done with BLAST. If a gene is similar in all phages, and does not exist in other phages, then we concluded it's probable that the gene could play a role in forming the compartment. Additionally, we checked how the protein expression changes 30 minutes past infection (Supplementary Materials for "Assembly of a nucleus like structure during viral replication in bacteria", Chaikeeratisak, Jan 2017, Science).We selected 9 proteins with unknown function that we thought may play a role in forming the compartment. Additionally, there was one protein, gp105, that was proposed to be the main shell proteins by earlier papers by Chaikeeratisak which we used as a positive control for all our steps. We also added 3 other proteins with known localization to our pool of proteins, leading to a total of 13 proteins.
To be able to conclude a possible involvement with the compartment, we visualized the proteins in-vivo by tagging them with a fluorescent protein. This was done with our new best friend, Peterli4, our favorite plasmid.
Here you see a sketch of the plasmid Peterli4 with gp105, the main shell protein.
On here, you can see the all the important parts of our plasmid:
-- Resistance gene (SmR) which allows the selection for transformed bacteria (Spectomycin for DH5alpha and Streptomycin for P.chlororaphis)
-- Induction system (LacI) which allows induction of protein production with IPTG
-- eGFP which is connected to the protein of interest and allows
-- Protein of interest, here it's gp105, the main shell protein
A plasmid like this was constructed for all 13 proteins with the following workflow:
For more detailed protocols, check out our protocol page or the registry description of our Peterli4_egfp_gp105 part (BBa_K3265027).
Once we had all our plasmids transformed into P.chlororaphis, we induced the bacteria with IPTG and infected them with our phage. We could then check under the microscope where our proteins localize after infection.
In uninfected bacteria, the DNA, stained for by Dapi, and the tagged protein are uniformly distributed over the cell. If a cell is infected, the DNA outside of the compartment gets destroyed, so the remaining DNA is visible as a centralized circle. If the tagged protein localizes with the DNA, as gp105 does here, then we infer a possible interaction with the compartment.
gp105 is our positive control, which is proposed to be the main shell protein.