Human Practices
Overview
In order you will find the different aspects of our silver and gold human practices:
Silver human practices
Road to human practices
Since our project is very fundamental with no short term applications outside of a lab, the societal and ethical implications of our project were unclear at first.Prof. Anna Deplzaes, whom we’ve met initially to talk about the ethics of our project, admitted that it’s still at such an early stage and since we “only” want to modify a bacteria there are no ethical or societal concerns with what we are doing, except maybe the more philosophical question as to whether it is ethical to change and also recreate life.
We decided not to pursue this interesting but not so practical philosophical question, as this is not the point of human practices.
Something to go on
Instead we took another look at the definition of human practices:On the main page it says to consider whether your project is responsible and “conducted with care and foresight.'' We decided to focus on this definition throughout our silver and gold human practices.
By reading through many past iGEM wikis and talking to iGEM alumnis we came to understand the importance of thinking ahead. Some things may work for your group in that specific lab and you might be comfortable with how you do your research, but if you make an application it needs to work for everyone, not just yourself or your team.
The next question for us was; for whom are we doing our project and how can we make it easy and safe to use for them? - To consider this question, is foresight.
Getting concrete
We identified two target groups of our project. First, researchers in Life Sciences and second, students. Whether it’s University students, iGEMers or even high-school students, we would need to make our parts user friendly for students to experiment, research and create new applications with our parts.There are many aspects that could be addressed when taking this approach, however, we think that there is one central one: Safety.
Safety
The first concern when working in a lab with organisms should be safety. This is especially important for students. Our goal was that our system can be worked with in a BSL 1 lab.To ensure this, we met with Dr. Silvio Hemmi, who is the safety officer of the Institute of Molecular Life Sciences at UZH where our lab is located.
For our project we try to replicate the natural interaction between certain phages and Pseudomonas bacteria. There are five phage-bacteria pairs known where, upon infection of the bacteria, the phage envelops its DNA into a protein cage to protect it from the bacterial defense system.
Some of the Pseudomonas belong in the risk group 2 category, f.e P. aeruginosa. Luckily, there is one Pseudomonas-phage pair where both organisms belong into risk group 1 category, which we confirmed with Dr. Hemmi.
Through our meeting with Dr. Hemmi we determined that Lab-coats, gloves, eye protection and regular cleaning and disinfection the workbenches should be sufficient safety measures when working with our organisms or derivatives thereof.
Silvio together with Alexander and Lynn!
This was the start of our silver human practices. The second part was not planned at first, which made it even better.
Does our system work for others?
When we did outreach at local high-schools to talk about our project, a teacher approached us with two students that were very interested in doing their “Maturaarbeit” with the topic of genetic engineering. The Maturaarbeit is a sort of bachelor thesis but for high-schoolers, which they must complete to graduate.We saw that this would be the perfect opportunity for us to continue our human practices.
We offered that they join us to do a part of our project with us for a week. We would be able to see how quickly they could understand the theory behind our project and conduct the same experiments as us.
First we want to state that we think it’s very important to make sure that in such a case high-school students are not unnecessarily exposed to any very hazardous substances or organisms that are above risk group 1. This should be a general point to consider for everyone, but for high-school students it carries more weight. In our case there were no obstacles that made it more difficult to adhere to this standard.
Here are the two students, Stephanie and Marton!
The work of the students
Marton and Stephanie are two very motivated and dedicated students at the KZU in Zurich. They were very interested and invested in the theory and research behind the phage-bacteria interaction and picked up on it very quickly.This was also true for the experiments.
After giving them a proper safety introduction to the lab and equipping them with their personal safety gear, we performed an amplification step and subsequent cloning of gene gp204 with Stefanie, including the imaging. This was our in-vivo approach.
With Marton we went through the amplification and subsequent cloning of gp204 into our His-tag vector for protein purification including the Ni-column affinity purification step with subsequent imaging. This was our in-vitro approach.
A week before they joined us in the lab we brought them up to speed with the fundamentals of our project and they were surprisingly quick in understanding the basics and were interested in a lot of details that we were looking into on the side.
During the lab-week, we supervised them conducting their experiments and it was great to see that at the end of the week they were getting more independent and needed less assistance from our side.
Conclusions
This experience showed clearly that the concept of our project is simple to understand, the laboratory procedures are safe and mostly straightforward.For the analysis of the bacteria a confocal microscope is needed which, unfortunately, would make working on our project difficult in developing countries without access to such instruments/facilities.
The in-vivo part (cloning) is much simpler than the in-vitro part (protein purification), which needs more background information, time and knowledge of the laboratory procedures which are also more complex.
We therefore recommend high-schoolers to focus on the in-vivo part if there is interest to replicate our finding or continue investigating this phage-Pseudomonas interaction.
More experienced university students and researchers should go for both approaches if enough time is planned for the in-vitro approach.
These experiences are very useful for the future of the project. If other iGEM teams want to continue to investigate this special phage and its compartment with the end goal of compartmentalization it will not be difficult for them to pick it up. With a confocal microscope, a standard lab and some inexpensive reagents they only need our parts which are all available on the registry. We have provided solid groundwork and proofs of concept for the imaging based approach and protocols that work very well.
Lessons from collaboration
After a few weeks of having trouble with the imaging we made contact with the iGEM team from Warwick University through official Mentor Jack Lawrence. The team had heard from him that we were having trouble getting good images (and we did not know why), so they offered to confirm the results we had gotten at that point but more importantly to image the genes we hadn't at that point. So we sent them a few stabs of P. chlororaphis carrying plasmids for five different YFP-phage gene fusions.Unfortunately, we later identified that the main issue with our imaging results was the YFP. The signal from YFP was very weak and when we exchanged the YFP to eGFP in all our plasmids we got at least 10x more signal and therefore more high quality images than with YFP.
Therefore, the images we got through the collaboration did not have a very good quality. Had we thought more about the design of our parts before, we could have avoided this.
We are unsure why we never thought of the possibility of using eGFP until quite late in the project. It was certainly a mistake that could have easily been avoided.
We advise everyone to not use YFP for imaging in P. chlororaphis and instead use a fluorophore that has a higher excitability, like gfp, egfp or mcherry.
Ensuring the future of iGEM UZurich
Being the first iGEM team of the University of Zurich, we made sure that next year, there will again be a team. For this purpose we founded a student organisation, which is now officially recognized by the University: SynBio UZH.We will recruit the next team through this association as well as make sure that students interested in synthetic biology have a place to connect, socialize and exchange their thought and ideas.
We also want to offer ambitious students opportunities to partake in research or corporate internships to get first hand experience in a field they are interested in.
Visit our website to learn more.
Integrated human practices
The last and largest part of our human practices certainly had the strongest impact on the way we conducted our project. We had already thought carefully about how simple to use the system we work with is and gathered some first hand data from the students. We made sure beforehand that it would be as safe and as simple as possible to work with the organisms we used. What we had not yet considered, is how could we address potential issues with the design of our project.Namely, are we taking a too narrow approach to create the compartment? - Are there other paths to get to the compartment we did not think about? - Are there techniques or experiments to get more comprehensive data on the individual phage proteins and the compartment?
Could we improve the functionality of our parts? - Have we thought carefully about the next steps after our project is complete?
It was clear to us that to get answers to these questions, we needed to talk to researchers outside of our lab. Because the Pelkmans lab focuses mostly on mammalian cells we knew we needed a different perspective on our project.
Ideally we would not meet with molecular biologists, but a group that focused more on biochemistry or structural biology, they have totally different sets of tools, ways of thinking and approaches from which we could benefit maximally.
On our search for a lab at the ETH Zurich or our own University we were recommended to the research group of Prof. Donald Hilvert. They are located at the department of Chemistry and Applied Biosciences and work on self assembling protein cages!
We couldn't have found a better group to talk about our project.
Here we pose for a picture with some members of the Hilvert group:
Meeting the experts
We were aware that making our own protein cage could be easy (like in Eut-protein compartments, EutS alone is able to form a bacterial microcompartment) or very difficult. As stated before our main goal was to get a different perspective on the overall design of our project and ultimately, to also get more ideas we could try out in the lab. To be honest we did not know what to expect from the interaction, so here is how it went.The Hilvert group works on many types of self assembling protein cages. They were very happy to hear about our project and iGEM in general and felt that what we trying to do was really intriguing. They asked sharp questions and made us realize that we need to be very thorough and that we should look into many kinds of properties of the phage compartment.
We had two important takeaways from the discussion:
First, We should not only think about how to make the compartment, but also what its properties are and test some basic things. It is important to think ahead; What is the next step IF we can make the compartment? - Elucidating its properties! -Therefore, we should already gather as much data as we can handle about the properties of the phage compartment.
Second, our approach to try and find the minimal components of the phage compartment is somewhat narrow. We should also try different methods, for example knocking out phage genes that we identified with imaging to be co-localized with the compartment or using mass spec to analyze its composition.
Lastly, they also mentioned that we should hammer home harder when it comes to the future of our project; what will happen after we are done with iGEM? - They advised us to always consider this as it's very important to know the next steps because that will also influence your experiments and ways of thinking in the present. We tried to incorporate this advice into how we talk about our project, on the wiki and in person.
As mentioned above they suggested many things we could try out, here we sum up the most important and feasible suggestion considering the time we had left for the project:
Their suggestions:
By their prompt, we analyzed the amino acid sequences of genes that are known to be involved in the compartment or can be found inside. The results can be viewed here
The compartment could then be analyzed in a mass spectrometer for composition of genes.
We ultimately decided against it, because the preparation and evaluation of the data from the mass-spec would take too much time. Since we are a small team it was hard to make time to go for this route, but after the jamboree or for the next team this is definitely an approach we would encourage.
Normally gfp does not enter the compartment and this would be a proof of concept for understanding how one could use the compartment for later applications.
They kindly provided us with a plasmid containing the +36 gfp variant so that we could express it in the infected Pseudomonas bacteria to test its localization.
Integrate and expand
We thought this last suggestion was a very intriguing idea and carried it out in the lab.More so, from their initial idea we came up with another interesting experiment.
Our goal was to fuse the (+)supercharged gfp variant to a phage protein that normally occurs only outside of the phage compartment, in our case, gp287. If we could show that the resulting fusion-protein would localize inside the compartment that would be a proof of concept for importing proteins of interest into the structure via positive charge. That alone would be a very useful finding if one wants to engineer a reaction in the protein cage we are trying to create.
The goal we had in mind with this test was to think ahead and get some sort of information on the selectivity rules of the compartment. No matter the results, the finding would be useful for the future of the project. If it is possible to create the compartment via self assembly, the next step would be to elucidate mechanisms of selectivity to which we could already add with this data.
Results
Sequence AnalysisWe analyzed the amino acid sequences of eight genes, six of which are expressed 20 minutes past infection (with unknown localization thus far). The other two are genes that have a known localization (inside and outside of the compartment). These eight genes are the ones were we had also gathered solid imaging data on. By running the amino acid sequences of proteins that have the same localization through protein blast we hoped to find common regions, maybe hinting to conserved stretches or even signaling peptides that are important for localization.
We did not find any significant, high quality alignments using pBlast between gp204, gp105 and gp460 (localize with compartment), gp26 and gp287 (localize outside, uniformly spread). For gp88 gp311 and gp205 (localize outside but not with the compartment) we also did not find any common amino acid stretches. We are not experts in these kinds of analyses, therefore we still suggest that a larger more thorough investigation could be carried out by someone who is into sequence analysis.
Compartment properties
Here you see the results from fusing the supercharged gfp variant, +36gfp to gp287, a phage protein that is always localized outside of the cell, and expressing this fusion protein in infected P. chlororaphis.
Interestingly, gp287 stays outside of the compartment even if it is tagged with a supercharged gfp variant. Charge alone can thus not be a single deciding factor for getting proteins into the compartment. There seems to be a more sophisticated mechanisms of selectivity when it comes to import and export with the compartment.
Due to technical problems we were not able to get images for the expression of the supercharged gfp variant NOT fused to gp287, we don't expect much, but this is something that can be done easily as a follow up on the project.
The sequence analysis did not reveal any conserved regions in proteins with similar localization. However the imaging of the supercharged gfp led to the hypothesis that positive charge alone is not enough to import a protein of interest into the phage compartment.
As a next step in this direction we suggest to express only the supercharged gfp variant in infected P. chlororaphis . Additionally, a more thorough sequence analysis of genes that have the same localization (outside, inside, shell) should be conducted.
Moreover individual knockouts of gp105, gp204 and gp460 (shell forming) should be carried out to see if one or multiple are essential to form the phage compartment.
The interaction with the Hilvert group changed the way we thought about our experiments and the project as a whole. The discussions also added to the overall design of our project in so far that we would also investigate properties of the compartment, rather than "just" trying to find the minimal components.
Conclusion
The data we gathered through our integrated human practices took us a little bit closer to understanding the properties of the compartment we are trying to build. It was an important first step in considering what comes after our project and is certainly something that should be pursued further.The sequence analysis did not reveal any conserved regions in proteins with similar localization. However the imaging of the supercharged gfp led to the hypothesis that positive charge alone is not enough to import a protein of interest into the phage compartment.
As a next step in this direction we suggest to express only the supercharged gfp variant in infected P. chlororaphis . Additionally, a more thorough sequence analysis of genes that have the same localization (outside, inside, shell) should be conducted.
Moreover individual knockouts of gp105, gp204 and gp460 (shell forming) should be carried out to see if one or multiple are essential to form the phage compartment.
The interaction with the Hilvert group changed the way we thought about our experiments and the project as a whole. The discussions also added to the overall design of our project in so far that we would also investigate properties of the compartment, rather than "just" trying to find the minimal components.