Project Description and Inspiration
What was our goal?
The goal of our project was to create a self assembling protein cage in bacteria that allows the compartmentalization of biochemical reactions.
We wanted to achieve this by replicating an interaction between Pseudomonas chlororaphis and the jumbo-phage 201 phi 2-1. This phage encapsulates its DNA in a protein shell within the infected bacteria, where transcription occurs inside this compartment and translation takes place in the cytosol of the bacteria.
Our goal was to find the minimal required components for this structure and induce its formation in an uninfected P.chlororaphis bacteria.
Take a look
Check out the video below to see the natural assembly of the phage compartment we are trying to replicate! Credit for the awesome video goes to Elizabeth Villa and Joe Pogliano @ UC San Diego.Our approach:
Main approach
Since there are three phages known so far that induce this compartment upon infection of a particular Pseudomonas species, we have narrowed down genes that might be involved in the formation of this structure. We used existing data on the protein expression profile of the phage (the compartment begins to form immediately after infection) and sequence comparison to the other two phages to narrow down the number of genes that are potentially important for the formation of the protein shell.
With that approach, we identified ten candidate genes that we wanted to investigate.
We had two main focuses: first we expressed ten egfp-phage gene constructs (one for each candidate gene) in the P. chlororaphis bacteria. We then infected those cells with the 201 phi 2-1 phage and visualized the localization of the egfp-phage gene fusion proteins via confocal microscopy. In this paper here , the same approach was used to identify the protein that seems to be the main component of the compartment, gp105.
The idea is simple, if a phage-gene X is a part of the phage protein shell, the recombinant protein will also be incorporated into the shell. This would then be visible under the microscope thanks to the linkage to egfp.
With this approach we try to further narrow down the number of genes that are necessary to form the compartment in vivo.
As a second approach, we purified the protein fusions and phage DNA to assess whether or not there is an interaction between any of the proteins of interest and the phage DNA in-vitro.
We did this not only to assess if phage DNA is necessary to form the compartment, but also to further narrow down the genes that are potentially important.
Here we can also test more combinations of genes more easily than in vivo.
Once we had identified which of the ten proteins are involved in forming the compartment we would coexpress these in P.chlororaphis without infecting them with 201 phi2-1 later.
A self assembly of the structure would be the ultimate success.
A beautiful visualization of the compartment: GFP-gp105 is located in a shell around the DNA in infected cells.
(Figure 1A in "Assembly of a nucleus-like structure during viral replication in bacteria" by Chaikeeratisak et al., DOI: 10.1126/science.aal2130.)
If we were successful in recreating this protein shell, the next step would be to find out how to control the localization of enzymes, substrates or other proteins inside the structure, more generally, to investigate the selectivity and permeability mechanisms of the compartment.
With this side approach we wanted to provide some interesting nuggets of information that could be very useful later on. Again, we had two approaches:
First, we did a sequence analysis of the ten genes of interest to see if we could find common sequences in the proteins that could be important for permeability. Furthermore we looked for particularly charged/hydrophobic stretches that could give us hints to the function, localization or involvement of the gene in the structure.
Second, we also tried to fuse a phage protein that is only localized outside of the compartment with a strongly positively charged gfp-variant (+36gfp) to see if that could cause the protein to localize inside of the compartment.
No matter the results, the information gathered from this experiment would be very informative.
When we started brainstorming for projects, our teams host, Prof. Lucas Pelkmans who leads a research group in quantitative and systems biology, suggested we take a look at one of their recently finished projects in addition to brainstorming for ideas of our own. Said project looked at the interaction of P. chlororaphis and Phage 201 phi2-1 and how the system could be used to study how compartmentalization of cells can buffer intrinsic noise. We then learnt that there were some issues with the project, especially with their “noise reporter system”, which is simply said, a plasmid. Evidently, they needed to work with infected cells, which will go into lysis after the phage has propagated itself. As this happens about 4h after infection, not much time is left for studying whatever phenomena researchers are interested in. Additionally, it proved to be difficult to introduce a plasmid into this compartment in vivo. Since we were captivated by this phage-bacteria interaction we decided to continue on with this project and expand on the original concept. So the idea in the end became the following: We wanted to get the compartment into a cell without actually needing an infection by the phage.
If we could achieve this goal, it would speed up experiments with this compartment, since phage propagation and infection is not necessary anymore. Additionally, the bacterial genome will not get degraded and interactions between compartment and bacterial DNA could be studied.
The next step would be to study the selectivity rules of the compartment, once they are known it would be more obvious what kind of application the compartment would allow and of course, one could start to modify it, enabling applications otherwise infeasible.
What would be really interesting to us is the study of the selectivity rules for this compartment. Some data on selectivity has been collected already, but it would be worthwhile to see whether we can get plasmids and specific enzymes inside of the compartment. Not only would this be useful for noise modeling, but potentially for the production of biomolecules in the compartment.
Since BMC’s come along with many restrictions we saw that our project had the potential to overcome two of the main restrictions of BMC’s.
First, our compartment is larger which would allow simply more product to accumulate within a compartmentalized space. Second, the selectivity rules for our compartment seem less strict as for the BMC’s, so far it's only been possible to encapsulate two different enzyme types at a time in a BMC. With our compartment, if the selectivity rules can be elucidated, many more could be imported.
Another important motivation for us is the potential our project has for synthetic biology and iGEM in areas that we haven’t imagined. If we can recreate the phage compartment, we are certain that teams will pick up on the project and try to use and modify it for all kinds of amazing applications. This really is the spirit of iGEM to us.
Last but not least, the compartment isn’t studied well, so our project will definitely help with its characterization.
(Figure 1A in "Assembly of a nucleus-like structure during viral replication in bacteria" by Chaikeeratisak et al., DOI: 10.1126/science.aal2130.)
Side-approach: Studying compartment selectivity
While we were trying to find out what it takes to create this compartment, we did two tests to see if we could gather some information about the permeability of the compartment.If we were successful in recreating this protein shell, the next step would be to find out how to control the localization of enzymes, substrates or other proteins inside the structure, more generally, to investigate the selectivity and permeability mechanisms of the compartment.
With this side approach we wanted to provide some interesting nuggets of information that could be very useful later on. Again, we had two approaches:
First, we did a sequence analysis of the ten genes of interest to see if we could find common sequences in the proteins that could be important for permeability. Furthermore we looked for particularly charged/hydrophobic stretches that could give us hints to the function, localization or involvement of the gene in the structure.
Second, we also tried to fuse a phage protein that is only localized outside of the compartment with a strongly positively charged gfp-variant (+36gfp) to see if that could cause the protein to localize inside of the compartment.
No matter the results, the information gathered from this experiment would be very informative.
Our Inspiration
When we started brainstorming for projects, our teams host, Prof. Lucas Pelkmans who leads a research group in quantitative and systems biology, suggested we take a look at one of their recently finished projects in addition to brainstorming for ideas of our own. Said project looked at the interaction of P. chlororaphis and Phage 201 phi2-1 and how the system could be used to study how compartmentalization of cells can buffer intrinsic noise. We then learnt that there were some issues with the project, especially with their “noise reporter system”, which is simply said, a plasmid. Evidently, they needed to work with infected cells, which will go into lysis after the phage has propagated itself. As this happens about 4h after infection, not much time is left for studying whatever phenomena researchers are interested in. Additionally, it proved to be difficult to introduce a plasmid into this compartment in vivo. Since we were captivated by this phage-bacteria interaction we decided to continue on with this project and expand on the original concept. So the idea in the end became the following: We wanted to get the compartment into a cell without actually needing an infection by the phage.
If we could achieve this goal, it would speed up experiments with this compartment, since phage propagation and infection is not necessary anymore. Additionally, the bacterial genome will not get degraded and interactions between compartment and bacterial DNA could be studied.
The next step would be to study the selectivity rules of the compartment, once they are known it would be more obvious what kind of application the compartment would allow and of course, one could start to modify it, enabling applications otherwise infeasible.
What would be really interesting to us is the study of the selectivity rules for this compartment. Some data on selectivity has been collected already, but it would be worthwhile to see whether we can get plasmids and specific enzymes inside of the compartment. Not only would this be useful for noise modeling, but potentially for the production of biomolecules in the compartment.
Expanding on our original application
As we were working on the project we got additional inspiration for our project. When we discovered the research on bacterial microcompartments (BMC’s), we realized that our system could be used in a similar way as BMC’s. Most groups working on BMC’s today try to encapsulate enzymes in BMC’s with the end goal of engineering reactions inside these “protein cages”.Since BMC’s come along with many restrictions we saw that our project had the potential to overcome two of the main restrictions of BMC’s.
First, our compartment is larger which would allow simply more product to accumulate within a compartmentalized space. Second, the selectivity rules for our compartment seem less strict as for the BMC’s, so far it's only been possible to encapsulate two different enzyme types at a time in a BMC. With our compartment, if the selectivity rules can be elucidated, many more could be imported.
Another important motivation for us is the potential our project has for synthetic biology and iGEM in areas that we haven’t imagined. If we can recreate the phage compartment, we are certain that teams will pick up on the project and try to use and modify it for all kinds of amazing applications. This really is the spirit of iGEM to us.
Last but not least, the compartment isn’t studied well, so our project will definitely help with its characterization.
References
- Discovery of the ability of the 201 phi 2-1 phage to form this compartment:
DOI: 10.1126/science.aal2130 - Characterization of 201 phi 2-1 and its compartment:
DOI: 10.1016/j.virol.2008.04.004. - Comparison of 201 phi 2-1 to two other jumbophages:
DOI: 10.1016/j.celrep.2017.07.064. - Engineering BMC's for potential use in nanoreactors:
https://doi.org/10.1038/srep24359