iGEM 2019 — Team UZurich

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# Team:UZurich/Notebook

iGEM UZH 2019

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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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# Team:UZurich/Experiments

iGEM UZH 2019

# [![](./images/UZH_iGEM_Logo_transp cropped.png)  iGEM UZH 2019](index.html)

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:UZurich/Results

iGEM UZH 2019

# [iGEM UZurich 2019](https://2019.igem.org/Team%3AUZurich)

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## Results

## Overview

**In order you will find the results for the different parts of our project:**

- Imaging (in-vivo approach)
- Sequence analysis (suggestion from Hilvert group at ETH)
- Compartment properties (suggestion from Hilvert group at ETH)
- protein purification (in-vitro approach)

## Imaging

The phage proteins were tagged with eGFP and visualized in-vivo in *P.chlororaphis*. As the bacteria were additionally infected with the phage, the formation of the compartment can be observed and thus, a co-localization of the DNA and the protein can be checked for.

Upon infection, the phage's DNA get enveloped in a protein shell. The bacterial DNA outside of the compartment gets degraded upon infection by the phage's nuclease while the DNA of the phage in the compartment is preserved. Therefore, DAPI staining affects only the DNA of the phage inside the compartment once a cell has been successfully infected.

![]( https://static.igem.org/mediawiki/2019/7/75/T--UZurich--InfvsUninf_test.png)

This allows for identification of infected bacteria: if the DAPI signal is uniformly spread over the cell, the cell has not been infected (left), but if the DAPI signal is shaped in a circle about the width of the cell (about 1µm), it has been infected (right). Not all bacteria show eGFP signal, but for the cells that do show eGFP signal and that do show the infected DAPI phenotype, one can compare the distribution of the eGFP signal and the DAPI signal.

## The different classes

#### Uninvolved

![](https://static.igem.org/mediawiki/2019/6/69/T--UZurich--Uninvolved_test.png)

If - in infected cells - the eGFP signal is universally spread, we propose no involvement with the compartment. It was also observed that the protein is uniformly spread but for the area where the compartment is localized.

#### Uninvolved but Localized

![]( https://static.igem.org/mediawiki/2019/6/6c/T--UZurich--Uninvolved_but_localized_test.png )

If - in infected cells - the eGFP signal is not universally spread, but also doesn't co-localize with the compartment, we propose no direct involvement with the compartment. The protein may be involved in other processes of the infection but not in structurally forming the compartment.

#### Involved

![]( https://static.igem.org/mediawiki/2019/8/8b/T--UZurich--Involved_test.png)

If - in infected cells - the eGFP signal co-localizes with the compartment, we propose possible involvement with the compartment. The protein is either directly and structurally involved with the formation of the compartment or it may also be involved in DNA processes like transcription. With images of good enough quality, one can differ between these two options: Either the protein is within the shell and the eGFP would fill out the circle (upper sketch), or the protein is localized with the shell, and the eGFP would then form a ring (lower sketch). We take the latter as strong evidence that the protein is involved with forming the compartment.

## Results

![]( https://static.igem.org/mediawiki/2019/9/9d/T--UZurich--Inf_uninf_example.jpg)

The image above shows an uninfected cell with an eGFP-tagged protein spread uniformly across the cell. Unless otherwise stated, this is the typical distribution of the protein in uninfected cells. Next to it is a typical DAPI staining for infected cells.

As mentioned in "Assembly of a nucleus like structure during viral replication in bacteria" (Chaikeeratisak, Jan 2017, Science), gp105 is the main shell protein of the compartment. It thus functions as a positive control to check if our general approach worked.
Note the ring-like structure that gp105 forms. It indicates that the protein is part of the shell and not located within the compartment.

![]( https://static.igem.org/mediawiki/2019/d/d0/T--UZurich--gp105_4.jpg)

As an example what it would look like if the protein was spread uniformly outside the compartment but not inside the compartment, we tagged gp287 with eGFP as well. gp287 is a phage protein that is involved in nucleotide synthesis.

![]( https://static.igem.org/mediawiki/2019/f/fd/T--UZurich--gp287_1.jpg)

#### Uninvolved

The proteins gp287, gp26, gp88, gp205 and gp311 did not co-localize with the compartment. Therefore, they are not structural proteins of the compartment but fulfil other functions in the phage's life cycle. For most of these proteins, the function remains unknown. gp189 also doesn't show co-localization with the compartment, but no image has been uploaded on this wiki.
Interestingly, the bacteria which carried the gene for gp137 grew a lot slower than the other bacteria, for both the YFP and the eGFP plasmids. No images are displayed here. Although the function of gp137 remains unknown, we suggest this protein may be toxic for the cell.
Due to technical problems, we were not able to visualize the localization of gp396.
Our controls gp130 and gp237, which both are supposed to localize within the compartment, gave contrary results that lead us to a new selectivity hypothesis. Check out the specifics below at "Compartment Properties".

![]( https://static.igem.org/mediawiki/2019/f/fd/T--UZurich--gp287_1.jpg )

![]( https://static.igem.org/mediawiki/2019/9/99/T--UZurich--gp26_2.jpg)

![]( https://static.igem.org/mediawiki/2019/0/06/T--UZurich--gp88_2.jpg)

![]( https://static.igem.org/mediawiki/2019/3/33/T--UZurich--gp205_1.jpg)

![]( https://static.igem.org/mediawiki/2019/f/f4/T--UZurich--gp311_1.jpg)

Note the localization of gp311 in uninfected cells. Somehow, these proteins already aggregate before infection, suggesting for example an interaction with bacterial proteins.

#### Involved

We found 3 proteins that co-localized with the compartment. One was the positive control gp105, which is known to be a shell protein of the compartment. The other two proteins that co-localized with the compartment were gp460 and gp204.
This co-localization is evidence that these two proteins are also structurally involved in forming the compartment. For stronger evidence, one could aim to obtain images that allow visualization of the ring-like distribution or proceed with the next steps mentioned below.

![](https://static.igem.org/mediawiki/2019/d/d0/T--UZurich--gp105_4.jpg)

![]( https://static.igem.org/mediawiki/2019/6/6c/T--UZurich--gp460_2.jpg )

![]( https://static.igem.org/mediawiki/2019/8/85/T--UZurich--gp204_1.jpg )

#### Additional Imaging by Team Warwick

As a collaboration, we sent some of our plasmids and transformed bacteria to Team Warwick to support our findings. At the timepoint when we decided on this collaboration, we were still working with YFP in our plasmids and were struggling to visualize it on our microscopes. We later on decided to switch to GFP, which lead to our findings above.
The images we received from Team Warwick showed that YFP signal was detectable, although it still seemed rather weak. This showed us that our problems with the YFP may have been due to our microscope and not due to our project design. It is possible that the YFP signal was too low for detection on our microscope.
They additionally hypothesized that the substantial amount of background fluorescence they experienced may have come from lysed cells and the aggregated proteins visible from time to time may support our findings of these proteins are structurally involved in the compartment.

There were no infections detectable in their images so we could not use them to support our findings of the localizations of the respective proteins.

![]( https://static.igem.org/mediawiki/2019/3/3b/T--UZurich--YFP105_1.jpg )

The cells visualized here were transformed with a plasmid expressing YFP-gp105. It is visible that the protein is produced and uniformly spread in uninfected cells. Note that we visualized the YFP signal in green, this was done because yellow coloring was harder to see on these images.

---

## Sequence Analysis

We analyzed the amino acid sequences of twelve genes, ten of which are expressed 20 minutes past infection. These were the ten genes we did our imaging analysis with. The other two are genes that have a known localization (inside and outside of the compartment).
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.

---

## Compartment properties

We tested whether a supercharged gfp variant could localize inside of the compartment. The +36 gfp variant has a 36X higher positive charge than the classical gfp and because the phage DNA inside the compartment is strongly negatively charged we thought this could lead to a transfer of the gfp inside of it.
We got this idea from the Donald Hilvert group at ETH Zurich, check out our integrated human practices to learn more:  [Human Practices](https://2019.igem.org/Team%3AUZurich/Human_Practices)

Furthermore we also fused this supercharged gfp to gp287 which is known to localize outside of the compartment. We wanted to see if positive charge alone would be enough to import a protein into the phage compartment in-vivo.

![]( https://static.igem.org/mediawiki/2019/7/7a/T--UZurich--ch287_1.jpg )

We found that 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.

We got some additional insight into potential compartment properties through our controls gp130 and gp237. Both of these proteins are supposed to localize within the compartment - which was shown in prior publications - but when we imaged them, they were diffused outside of the compartment and excluded. After some initial confusion and additional research, we realized we fused GFP at the C-terminal of the protein while they used the N-terminal, thus leading to a new hypothesis that the sequence at the C-terminal of these proteins may be of importance for the import into the compartment. This should be researched.

![]( https://static.igem.org/mediawiki/2019/8/85/T--UZurich--gp130_2.jpg )

![]( https://static.igem.org/mediawiki/2019/1/18/T--UZurich--gp237_1.jpg )

---

## Protein purification (in-vitro approach)

Our original plan included purifying proteins that seem to be involved in the compartment: gp105, gp204 and gp460. Since it took us a while to get information on these three genes due to technical problems with the microscope we only prepared the protein purification for gp105.
We hoped to test interactions between the proteins and the phage DNA in-vitro.

Unfortunately we had massive problems throughout the preparations. First a protein purificaiton vector we got from another lab was missing an RBS (which we were not told), when we got a commercial vector our SDS page indicated that our protein was not produced. In the end we needed to clone gp105 into a third vector at which point it was too late in the project to continue with the protein purification.
If one has experience in protein purification it's a good thing to try, inexperienced students and high-school students should rather stick to the more straightforward imaging. It can be very time consuming and the laboratory methods are sometimes very complicated.

---

## Outlook: Where will the project go after iGEM?

**Here we lay out what the next steps in the project should be:**
With the 3 genes where we found evidence that they take part in forming the compartment, we propose some in-vitro experiments. It is possible that DNA, possibly specifically phage DNA, is an important factor for assembly. Thus, it would be interesting to check for any interaction of these proteins with DNA and phage DNA.
Additionally, it would be interesting to generally check the interaction of these proteins with each other. This would need to be checked in at least 3 different settings: just the purified proteins together, the purified proteins with (phage) DNA, and a "dirty" setting where bacterial proteins are also present. Looking at protein interactions in these 3 settings not only allows to distinguish the proteins that form the compartment together, but also allows the assessment if DNA and bacterial proteins are involved as well.
Then one should also express the involved proteins with a single plasmid in P.chlororaphis to test the formation of the compartment without an infection. If this works, this can also be tried in other bacterial cells.

In parallel it would be very informative to knock out gp105, gp204 and gp460 in the phage individually to see if they are essential components to form the compartment.
Pooling this information with a DNA interaction assay could reveal more insight into the minimal components necessary to form the compartment.

If with these approaches it's still not possible to induce the formation of the compartment without the phage, things get more complicated. The genome of phage 201 phi2-1 is very large and encompasses 450 genes, many of which are completely unknown. A large knock out screen could give more information on crucial genes. More so, we can not be certain that certain lipids or carbohydrates are necessary for the assembly of the phage compartment, which would make the goal of our project very difficult to achieve.

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