iGEM 2025 — Team tu-dresden

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# Timeline

![Notebook](https://static.igem.wiki/teams/5690/siteheader/headers-timeline.webp)
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**Take a look at our timeline,** where we showcase all the events from our entire iGEM journey 2025. You’ll find detailed insights into everything we did along the way.

iGEM team formation and first project ideas

In the early stages of TRAPS there was a lot of brainstorming on different project ideas. Back then neither the name TRAPS nor Conny existed. There were many ideas like an optogenetic CAR-T cell based tumor recognition or a project to overcome metabolic burden due to toxic intermediates within an enzyme cascade by enzyme condensation. Throughout the brainstorming process the idea of using condensates as a cellular platform became increasingly appealing.

Introducing the ideas to our PI

Our PI, Prof. Mascher, was in favor of using condensates as platform for our iGEM journey. He discouraged the further pursuit of the CAR-T cell project, since it was out of proportion for an iGEM project. From this time on the direction of our project was set. We will do something with cellular condensates. To be honest, when you live in the condensate capital, you are kind of obligated to do something with them.

Core team formed

In late January the core team began to form. Subgroups like lab, dry lab, awareness person, finance, wiki & graphics were set up and members could decide where to join. Additionally group leaders were elected.

Brainstorming ideas what to do with condensates

Following the decision of using condensates, we invested a lot of elbow grease into brainstorming what system we could utilize in condensates. Firstly, we thought a lot about enzyme cascades which could potentially be improved when they occur within a condensate. Ultimately, we decided on a RNA detection system.
So, the whole team began to research how to engineer such a system. We decided on a SH3 domain as high valency unit and a pumby domain as low valency unit which get brought together by the target RNA, resulting in a condensate.

First expert meeting, discussing the project ideas

Our general system received positive feedback from our secondary PI Prof. Alberti, Dr. Franzmann and Prof. Honigmann. They helped us answer raised questions, like which vectors we should use, how we should design our linker, and if we should use mCherry as test target and GFP as reporter fluorophores. But most of all, the granted as access to their lab space, which was our home for the following iGEM-journey.

Second expert meeting

In the second meeting, with the same experts we got more advice like using a network formation (multiple RNA-protein connections) instead of a single RNA-protein connection. Additionally, they brought our attention to further investigate the potential detection of non-coding RNAs with our system. Even though, the most important take away of this meeting was the discussion of using Cas13 as RNA binding protein instead of Pumbys. This was due to the background, that previous literature of Pumby testing was only done in *in vitro*, making it hard to adapt it to our wanted, *in vivo* use case.

Uni stem day

On this year’s Uni Steam Day, an outreach event, TRAPS got 3rd place and won a 60€ price.

Team photoshoot and name brainstorming

On this Tuesday, the day for our weekly meeting, we did a team photoshoot and brainstormed different team name ideas (like LillyPhase, DRIPS, Condensia… and TRAPS)

Instagram launch

On this day our first Instagram post went online! It was our Group foto.

Frankfurt preparation meetup

The poster and presentation for Frankfurt was assessed in this meeting. We gave the presenting persons feedback to achieve the best results possible.

Expert meeting (Bioinformatics, Dr. Friedrich)

She gave us advice on our bioinformatic problems. For preventing off-target effects we should have ≥3 mismatches.

Team vote

TRAPS wins the team name election!! Additionally, we planned to participate at the Elbe cleanup event.

Lab subgroup meeting in the cafeteria

The wet lab subgroup met over lunch to discuss RNA target choices and possible gRNA designs.

Elbwiesen clean-up

This was a team bonding event, while also cleaning up nature.

Organisation of wetlab start

Starting on this date we began building our DNA constructs. We further discussed the cloning strategy, deciding to go with a gateway-system.

Expert meeting (Prof. Honigmann, Dr. Franzmann, Yu Wei (Elsa) )

The experts advised us to use GFP as a stable reporter and mCherry as target RNA, since it is a fast-degrading model. They also suggest the use of mini-Cas13 variants potentially combined or as alternatives to Pumbys. The goal was to start with wet lab work in 2-3 weeks from this date.

Safety briefing

On this day we had our official safety briefing in the Biotec-Lab. We got a really nice tour and our Keys! We came the lab work an important step closer!

Team research meeting discussing cloning strategy

On May 6th a research meeting was held to discuss and refine experimental planning. Gateway cloning was chosen as the main cloning strategy and using d.Cas13X.1 in yeast was considered.

First lab work

At this point the first plasmid isolations and clonings were done. mCherry was first tried to be induced.

Research meeting discussing the use of the "Heidenreich System"

In this meeting we decided how we are going to adapt and use the synthetic condensation system designed by Heidenreich et al.

Tetramerization domain design comparison

We compared three different Pumby based construct designs. A standalone tetramerization domain, a combination out of tetramerization- and dimerization domain and a mixed system. Ultimately we decided to go with the simple tetramerization domain – only design since it was more feasible and less prone to error.

Expert meeting (Dr. Franzmann & Prof. Alberti)

On the 16th May Dr. Franzmann and Prof. Alberti reviewed our constructs. They gave us the advice to keep everything on one plasmid to simplify transformation into yeast. Additionally Dr. Franzmann suggested tagging each tetramerization domain with GFP, for better visualization.

Video shoot for “Study in saxony”

This was the day where a video with Saxony’s education ministry was filmed. We talked about how it is to participate in a student research competition. You can find it on our Instagram: @igem.dresden !

Sustainability talk

On May 20, 2025, we discussed lab sustainability. The main focus here was to reduce plastic waste, reusing consumable and to document usage for greener lab practices. The discussed contents became part of our lab routine to commit our part to responsible science.

Expert meeting (MPI CBG)

During the expert meeting at MPI on May 22, 2025 we discussed RNA target selection with the experts. The emphasis was laid on using bioinformatic based prediction tools to avoid off target binding. Additionally they gave us advice on which regions of RNA are most suitable for detection.

BFH european meetup

From May 23 – 25 members of our team attended the BFH European meetup in Frankfurt. This was our first major external event. They presented a poster and a project pitch, engaged feedback with other iGEM teams and gained valuable insights into project presentation and communication. Furthermore this marked our team's official debut into the iGEM community, forming lasting connections.

Backbone finalized

One June first our research team finalized the design of our plasmid backbone. It included multiple restriction cutting sites, terminator/promotor cassettes and detection tags which enabled easier protein identification.

Expert input (Dr. Kar, IPF)

On June 2nd we talked to Dr. Kar from the Institute for Polymer research who especially recommended using established assays for condensate analyses if working with RNA binding Proteins. Microscope-based methods to visualize droplet formation and FRAP for quantitative (e.g. stability, dynamic and aggregation) data were recommended.

Construct orders

After a lot of yeast optimization and synthetic trouble with the complexity of our constructs, we finally ordered them in early June 2025.

B2B presentation training

On June 27, 2025, Fabi and Julius received back-to-back presentation training and advice from Simon Doll. The focus laid on storytelling and pitching skills. This session helped our fellow team members to shape a clear narrative for the following presentation of TRAPS at this year’s Science Slam and following presenting opportunities.

Team breakfast

We had a team breakfast to celebrate our progress and discuss the next steps for our project. Dylan even brought his famous homemade Oleg! mmh tatsy!

B-Cube 2 BIOTEC hour

On this day we joined the B2B Hour hosted in the B-Cube. This is an internal seminar connecting different research groups across the institutions. We presented our project concept and first results to fellow scientists and received a lot of good feedback and advice on our concept. Additionally we used this opportunity as rehearsal for upcoming presentations and pitches.

Long night of science

Our team took part in the Long Night of Science in Dresden. We ran an outreach booth at the CRTD, explained synthetic biology to the public, engaged visitors with interactive activities while raising awareness for both iGEM and our TRAPS project. Most of all we took part in the Science Slam hosted this night and our spokesperson Fabi made 3rd place!

Wiki kick-off

This day marked the official start of our wiki journey. The basic structures and designs were drafted and tasks for writing and coding distributed. Additionally, a lot of ideas were contributed laying the groundwork of our wiki journey!

Connie was born!

Well she existed before, but this was the first time her name was mentioned as an idea for the following name voting in our instagram story.
![Connie](https://static.igem.wiki/teams/5690/siteheader/heart-connie-pointing.webp)

Seniorenakademie

This event was an outreach to elderly citizens. We presented our project at Seniorenakademie, introducing TRAPS and synthetic biology to the audience. We focused on making complex concepts accessible but especially sparking curiosity!

Düsseldorf meetup

Some members of our team traveled to the Düsseldorf iGEM meetup hosted by the HHU. There we presented our project, exchanged thoughts and held conversations with other iGEM teams.

Expert meeting Dr. Mrityunjoy Kar

We met with Dr. Kar to discuss condensate biology. He brought topics such as sub-clusters within condensates closer to us and recommended *in vitro* methods such as Microscale Thermophoresis or Dynamic Light Scattering for the quantification of protein-RNA interactions. This gave us new ideas to research our system beyond light microscopy.

First visual input

On this day we spotted fluorescence induced by our TRAPS system for the first time under a microscope!

Chalk talk

On the 25th July our team member Li Jing held a Chalk Talk at the CRTD, presenting our first lab results. The main discussion was how to distinguish true condensates from unspecific protein aggregates. The brainstorming session with the podium led to plans for new control and more rigorous image analyses.

Promo video shoot

Filming for our iGEM promo video.

Aggregation issues

We faced serious aggregation issues with our Pumby constructs. The Protein clumped together, instead of forming functional condensates. So we shifted the project's focus more on the Cas13 variant, where we also see condensates but know what the potential mistakes in the system are. We decided to order a new Cas13 variant and new gRNAs.

Lab roles split

In a lab meeting on August 5, we reorganized the lab work to be more productive. We assigned specific tasks to different team members. So the task became more routine and completion was faster.

Western blot

This marked the start of protein-level validation for our project. We finished our first Western blot here. Everything went according to plan, unfortunately due to a malfunctioning of the Typhoon imager, the first results were a bit blurry.

Supply boost

We finally collected the leftover lab materials of the previous Dresden iGEM team. And it was more than we expected.

Brainstorm new directions for TRAPS

Our research team held a brainstorm meeting discussing new directions for TRAPS in the future.

Expert feedback (Dr. Jahn)

The discussion with Dr. Jahn gave us new input on how we could advance our system and how we can plan a titration experiment.

Team BBQ

Summer social at Großer Garten, Dresden. 🌭

Expert meeting (Bone Lab Dresden)

Members of our team met with scientists at the Bone Lab, getting feedback on how TRAPS could be applied in research in osteoimmunology or cancer. Comparing TRAPS to existing detection methods like qPCR. TRAPS could become an additionally method to real time or qPCR for a more dynamic analysis.

Expert meeting (Dr. Pekárek)

Dr. Pekárek discussed target strategies for our TRAPS system. Which RNA structres can be targeted and which should be avoided?

Expert meeting (Dr. Jahnel)

We had a discussion about further tests and experiments. A stress response experiment was discussed and planned.

Wiki sprint

Since the end of August most of our time and afford has gone to the wiki. We began to co-work for more productivity. Tasks were divided into the different aspects of the wiki, text writing, figure preparation and coding.

Expert meeting (Dr. Wurm)

We discussed a bunch of potential usecases of TRAPS in an expert meeting with Dr. Alexander Wurm.
Especially interesting could the system be for identifying therapeutic compounds that modulate gene expression in real time.

Pumby westerns

A western blot of our pumby constructs was done. Unfortunately the antibodies do not seem to work.

2025: Prague meetup

From Friday to Saturday members of our team attended the iGEM Meetup in Prague, featuring international teams like Lund or Louvain. We heard many awesome presentations about start-ups formed from iGEM-projects, venture capital and project presentations from participating teams. We also presented a draft of our Paris-presentation. We received great feedback on our system and presentation! The event offered valuable insights, and we got food and beer! Thank you Prague!

Final safety form

Submitted safety forms & check-in for Jamboree.

Potsdam meet up

Our team invited iGEM Potsdam to come to Dresden. Potsdam is the only other east german team and we discussed our iGEM east germany project and our projects. Additionally we gave them a little tour through our lab.

Wiki freeze prep

Times are about to get hot. Everyone works hard on the wiki!

Grand Jamboree (Paris)

The big stage! Team Dresden presents TRAPS journey. 🎉

1. Juni

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# Engineering

![Engineering](https://static.igem.wiki/teams/5690/siteheader/headers-engineering.webp)
![](https://static.igem.wiki/teams/5690/siteheader/lab-connie-pointing.webp)

Glub glub! As I swam through this project with the team, we found challenges, discoveries, and lots of testing...

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All
Design
Build
Test
Learn

* [Our goal](#goal)
* [Design Cycles](#design)
* [Building Cycles](#build)
* [Testing Cycles](#test)
* [Final Learning](#learn)

SynBio is not just about genetic engineering or changing DNA. It is about designing and building new biological parts, devices, and systems or redesigning natural systems to make them useful. It combines ideas from genetic engineering, biochemistry, systems biology, microbiology, and computer science, making it both an engineering and applied science.
Engineering in SynBio follows a few key principles. Abstraction allows a part to be separated from its natural function and used somewhere else. Modularity lets us combine different parts to build bigger systems. Standardization makes sure parts fit together so that we can use the same tools and methods, like BioBrick assembly. Finally, characterization documents on how parts behave, and this information is collected in the Parts Registry, which is useful for future projects. Using these principles, synthetic biologists work through a four-step engineering rollup: design, build, test, and learn. In the next sections, we explain how our team applied this rollup to our project.

## Our Goal

The Goal was clear: we wanted to build an [RNA](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#r) detection system, in which a protein scaffold only condenses in the presence of a specific RNA in order to achieve rapid in vivo sensing abilities. To achieve this, we started by conceptually designing the system, initiating the first phase of the DBTL (Design-Build Test-Learn) engineering rollup.

![Engineering Cycle](https://static.igem.wiki/teams/5690/engineering/webp-50-of-size-cycle-animation-colour-edit.webp)

## Designing

To achieve our goal, two things became clear early on. First, we needed a valency-based synthetic [condensate](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#c) system as a foundation. This system could then be adapted to respond to specific RNAs by exchanging the linkers with RNA. Second, we needed reliable RNA-binding proteins.

During the first expert meetings of this project with Dr. Franzman, Prof. Dr. Alberti and Prof. Dr. Honigmann, a synthetic condensation system engineered by Michael K. Rosen was proposed (Li et al., 2012a).
In our search for adaptable RNA-binding proteins, we found an engineered variant of the Pumilio factor family called ‘Pumby’ (Pumilio-based assembly).
Pumbys exhibit a strong affinity toward RNA and are engineerable to bind RNA sequences up to 18 nucleotides (Adamala et al., 2016).
With these two components, the first iteration of TRAPS was designed.

### Design Cycle 1 – SH3-PRM-TRAPS

 ![Illustration of the valency dependence of the SH3/PRM condensate system. On the upper half SH3 and PRM chains with different length are displayed. Depending on the length of the chains, meaning the amount of domain repeats a condensate can form or not. Five and four repeats allow condensate formation and three repeats do not.](https://static.igem.wiki/teams/5690/engineering/sh3-prm-system-diagram.webp)

Figure 1: The valency dependent SH3-PRM condensate.

The synthetic condensate system designed by Prof. Dr. Rosen consists of two engineered multivalent protein chains, in which each SH3 domain can bind to a PRM. This interaction results in the formation of a network visible as a condensate. The network formation is highly dependent on the valency, meaning, the number of repeats of each binding domain in each chain. For example, if each chain consists of five repeats of the respective binding domain or motive, network formation is highly likely, resulting in a stable condensate. In contrast, if there are only three repeats, the likelihood of network formation is much lower (Figure 1) (Li et al., 2012b).

 ![Animation of the first TRAPS-Pumby design using the SH3/PRM system. The PRM chain consists of only two repeats and the SH3 chain of five. PRM binds to SH3 but does not form a condensate. RNA is introduced to the freely diffusing proteins and connect them to a detectable condensate](https://static.igem.wiki/teams/5690/engineering/webp-50-of-size-sh3-prm-pumby-system.webp)

Figure 2: Design of the first TRAPS-Pumby system based on the SH3-PRM condensate.

We planned to make use of this valency dependence by using the SH3-chain with a high valency consisting of five repeats and reducing the PRM chain to only two repeats.
This valency combination results in a system that does not exhibit condensate formation.
However, by fusing RNA-binding proteins to the C- and N-terminal of the PRM chain, multiple PRM chains can connect to one specific target RNA, effectively increasing the PRM chain valency.
Subsequently, this then results in target RNA-dependent condensate formation.
To achieve this, it is important that we can precisely engineer the RNA-binding proteins, the Pumbys, to our sequence of choice.

Table 1: Overview of engineering Pumby domains for nucleotide specificity by changing the amino acid at the 16th and 20th position

| Ribonucleotide Base | Amino Acid 16 | Amino Acid 20 |
| --- | --- | --- |
| **Adenine (A)** | Cysteine (C) | Glutamine (Q) |
| **Guanine (G)** | Serine (S) | Glutamic Acid (E) |
| **Cytosine (C)** | Serine (S) | Arginine (R) |
| **Uracil (U)** | Asparagine (N) | Glutamine (Q) |

Pumbys consist of concatenating 36 amino acid RNA-binding domains, each binding to one nucleotide.
The nucleotide specificity is determined by the amino acid at position 16 and 20 (Table 1).
To increase specificity while limiting the size of the Pumbys, we decided to design Pumbys consisting of 12 nucleotide-binding regions.

With the Pumbys designed, we built a gene construct encoding for the SH3-chain labelled with a [GFP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#g). For the lower-valency PRM chain, we built two species to increase the chance of network formation. Each species had two Pumbys targeting different sequences on the target RNA. In total four Pumbys were designed to target one RNA at different sites.

Before we ordered the DNA to introduce these proteins into our yeast, we double-checked our design and realized that the initial publication on the SH3-PRM system was *in vitro* work. The likelihood of this system working *in vivo* was clearly very low, since SH3 domains generally bind all proline-rich motifs present in the cell. With no proof that even the simple synthetic system published works in yeast, the uncertainty was too big to continue with this approach. Due to this realization we started looking for a new synthetic condensate system for our idea.

While designing the Pumbys for the first system, we realized that the selection of a correct binding site in the target RNA is vital for the success of TRAPS. There was a multitude of factors to consider when looking for the correct target site. Therefore, we started to design our own software.

## Design Cycles – RNA targeting [Software](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D#t)

### Software Cycle 2.1

In preparation of our first constructs using Pumilio based RBPs we came to realize that common tools for binding sequence design i.e. for sgRNAs - that minimize off target binding in a transcriptome dataset - could not accommodate our requirements of much shorter binding sequences of only 8-12 bp. To counteract the lower specificity of a shorter binding sequence we planned to design multiple RBPs that bind to different sites on the target RNA.

As the core problem of evaluating sequence similarity seemed simple enough, and we predicted more requirements would reveal themself as the project progresses and out of curiosity for the challenge, we decided to build our own software tool. To evaluate a potential binding sequence (query) we count the number of matched base pairs for each possible binding situation in each transcript in the transcriptome and calculated a pseudo binding probability based on the Boltzmann factor using the number of matching base pairs to estimate the energy of the bonds formed.

During the development of a first prototype, we tested different implementations of this moving window sequence comparison, allowing us to arrive at a numba (JIT Python compiler) driven implementation almost 1000x faster than the very first implementation of the algorithm. Furthermore, the batch evaluation of all query sequences, i.e. all substrings of the target RNA, can be sped up by parallel computation distributed over many cores.

In its first iteration the software was designed to compare all subsequence’s of the target RNA (mCherry) of a length n (these are the possible binding site getting evaluated for pumilio RBPs) against all sequences of a yeast transcriptome. While calculating the underlying binding metrics for each potential binding site on the target worked as expected, we were still missing a way to evaluate groups of binding sites simultaneously as we set out to do.

### Software Cycle 2.2

We came up with more ideas on data we could use to model the binding more accurately: first we decided to switch to a quantitative transcriptome dataset including the expression levels of each transcript allowing us to weight the binding affinities to the transcripts by their concentration. Simultaneously we planned to look into the binding affinity towards the target, where the secondary structure of the target RNA could determine binding site availability. Additional considerations went into the idea of making the tool sequence function aware e.g. to avoid ribosome binding sequences and prefer UTRs. Starting to think about publishing this internal tool we wanted to investigate the common ways tools like these are published, and how the usability of the codebase could be improved.

In this next iteration we changed from a purely qualitative transcriptome dataset to a quantitative one allowing us to weight the similarity to each transcript by the expression level of that transcript, improving the search results by avoiding binding sites on high frequency transcripts, while also reworking most of the visualisations. A major addition was the option to evaluate not just single binding sites, but groups of binding sites to avoid overlapping off target effects in situations where multiple probes are used simultaneously, like in our case with multiple pumilio RBPs and sgRNAs for Cas13. The codebase up to this point being a mix of .py files and jupyternotebooks we decided to put all essential functions into a compact .py file, and additionally offer an example implementation as well as a detailed explanatory jupyter notebook showing each of the core functions. Additionally to the making the tool available for local installation via conda, pip, uv(suggested by iGEM team Potsdam) we started to experiment with a web interface for our tool to be integrated into the wiki.

Talking to more experts and testing example sequences revealed the secondary structure prediction of RNA to be a field very much still in progress, leaving us to decide against a direct integration. Similarly we (for now) did not implement any sequence function based evaluation as this evaluation might depent on the scientific question adressed; focusing purly on off target effects in the transcriptome. However as the list of binding sites to be evaluated by the tool is open for the user to be changed it is easy to apply a pre-selection of potential binding sites, based on prior scoring from other tools, or to restrict the binding site search to certain regions. Looking into the hardware of the iGEM server hosting the wiki we came to realize our project would not be able to run nativly on these servers, for the same performance reasons an implementation running on the clients browser was discarded. Due to iGEMs wiki restrictions hosting our own server to run the backend with the frontend in the wiki is not allowed, leaving us to discard the idea of an online demo tool as part of the wiki.

This second design cycle confronted us with questions and problems addressing the which features are viable, which make sense to include for the target audience, balancing 'smart' i.e. predetermined behaviour against versatility at the cost of complexity and against the time and work it would take to implement. Similar problems came up documentation, having to find a balance between simple and easy to understand documentation for new users while not dropping details and optional parameters inclined users might be interested in.

### Here comes the – Heidenreich System

It became apparent that the initial synthetic condensate wouldn’t work in yeast, so we continued looking for more options. When researching further, we found a fitting synthetic system published by Heidenreich, which had even been tested in the Alberti Lab already (Heidenreich et al., 2020). Additionally, we considered not only using Pumbys as our RNA-binding proteins but also dead [Cas13](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#c). Using [dCas13](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#d) was further evaluated and discussed with Yu (Elsa) Wei from the Hyman lab, who has previously worked extensively with Cas13.

 ![Animation of the synthetic condensate designed by Heidenreich. Tetramerization units form a tetramer and are connected via the E9-Im2 interaction to the dimer. This results in many tetramers connecting to each other resulting in a large network visible as a condensate.](https://static.igem.wiki/teams/5690/engineering/webp-50-size-heidenreich-system-animation.webp)

Figure 3: Synthetic condensate designed by Heidenreich et. al.

The system designed by Dr. Heidenreich consists of two proteins: a tetramerization unit labelled with GFP and fused to a toxin (E9), and a dimerization unit connected to the respective antitoxin (IM2). Due to the multivalence , the system forms a condensate with liquid-like properties based on the [E9-IM2](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#e) link (Figure 1). We then had the idea to replace the dimerization unit with RNA to achieve the linking of multiple GFP-labelled tetramers by the specific RNA.

From this, we designed two new TRAPS systems, both based on the synthetic condensate system, one combined with Pumbys and one with Cas13 as RNA binding proteins.

### Design Cycle 3 – TRAPS-Pumby keeping E9 and IM2

To ensure a high likelihood of the condensate formation, we needed not just two but multiple binding sites of our RNA-binding proteins to our target RNA. With more binding sites, one RNA can connect more than just two tetramers. For the TRAPS-Pumby system, we decided that four binding sites should be sufficient to begin with.

 ![Animation of the first TRAPS-Pumby system using the synthetic condensate designed by Heidenreich. The dimerization domain is replaced by Pumby domains targeting different sites on the target RNA determined by our software. Tetramerization units form a tetramer and are connected via the E9-Im2 interaction to the Pumbys. Once the target RNA is introduced the Pumbys will bind and connect multiple tetramers, forming a large network visible as a condensate.](https://static.igem.wiki/teams/5690/engineering/webp-50-of-size-toxin-antitoxin-pumby-system.webp)

Figure 4: TRAPS-Pumby system with E9 and IM2 connection.

We initially planned on keeping the E9-IM2 link to maintain the liquid-like nature of the condensate. Therefore, we kept the tetramer as it was and created three versions of an IM2-Pumby fusion protein, each targeting different sites on our target mRNA. These three proteins should bind to the available E9-binding sites at the tetramer via IM2. If the target RNA is present in the cell tetramers will be connected via the Pumbys (Figure 4).

It was time to get serious and think of a cloning strategy. For this we met with two cloning experts from the MPI-CBG in Dresden, Dr. Aliona Bogdanova and Andrey Pozniakovsky. In this meeting we decided to use conventional cloning and ordered all the genes with SacI and MluI restriction sites at the ends for insertion into the plasmids available in the Alberti lab.

But most importantly one more thing became apparent; the E9/IM2 ratio should be close to 1:1 in order to avoid many freely diffusing Pumbys that would not contribute to the network when bound to the target RNA. With three designed proteins containing IM2 (the three different IM2-Pumbys) and only one containing E9 (the tetramerization unit), this ratio is almost impossible to achieve.
With this realization we went back to the drawing board designing a new TRAPS-Pumby system.

### Design Cycle 4.1 – TRAPS-Pumby

We decided to leave out the E9-IM2 interaction altogether, since there was no viable option to keep the interaction while still having multiple different Pumbys targeting different sites on the target RNA. We still wanted to have three Pumbys and decided to replace the E9 toxin entirely with Pumbys. This resulted in the tetramerization domain being covalently connected to its respective Pumby. Three versions of these tetramerization–Pumby fusion proteins were created.

 ![Animation of the second and final TRAPS-Pumby design. Pumby domains are directly connected to the tetramerization units replacing the E9-toxin. Tetramers will assemble. Additionally, a Pumby is fused to the dimer and dimers will assemble. All proteins are freely diffusing in the cytosol. Once the target RNA is introduced it will be bound by the Pumbys connecting multiple tetramers, forming a large network visible as a condensate.](https://static.igem.wiki/teams/5690/engineering/webp-50-of-size-pumby-with-dimers.webp)

Figure 5: Final Pumby design.

If we had stopped here, there would have been a chance that one tetramer contained all three versions and could occupy one target RNA completely, meaning all possible binding sites of the RNA would be bound by one tetramer, which in turn would not contribute to network formation. Even if this outcome is statistically unlikely, we still wanted to ensure that at least two tetramers would be connected by one RNA. To achieve this, we planned to introduce a fourth pumby connected to the original dimerization unit of the system. With this, the dimer will have two identical binding sites to the RNA, so in theory each dimer is connected to two target RNAs, while the tetramers can bind to the residual binding sites on the RNA, thereby forming a network (Figure 5).

These four proteins, the three tetramerization units connected to different Pumbys and the one dimerization unit connected to a pumby, composed our TRAPS-Pumby System.

### Design Cycle 4.2 - TRAPS-Cas13 Design

 ![Animation of the TRAPS-Cas13 design. Cas13 domains are fused to the Im2-antitoxin. Tetramerization units form a tetramer and are connected via the E9-Im2 interaction to the Cas13 domain. The binding site of the Cas13 unit are defined by the introduction of four gRNAs targeting different sites on the target RNA. Once the target RNA is introduced it will be bound by the Cas13 connecting multiple tetramers, forming a large network visible as a condensate.](https://static.igem.wiki/teams/5690/engineering/webp-50-of-size-trapscas13-protein-model.webp)

Figure 6: Figure 6: TRAPS-Cas13.

The [TRAPS](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#t)-Cas13 design is simpler, since the binding site of Cas13 on the RNA is not defined by its amino acid sequence, as in the case of [Pumbys](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#p), but by the [gRNA](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#g) bound to Cas13. The [tetramerization unit](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#t) can remain as it is, resulting in tetramer with four E9 binding sites, and the dimerization unit is replaced by Cas13. We initially used a miniaturized Cas13 version, Cas13X.1(Xu et al., 2021). Cas13 fused to [IM2](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#i) will bind to the four available E9 binding sites, maintaining the liquid like E9-IM2 connection (Figure 6).

If now a set of gRNAs is introduced, the gRNAs will be distributed to the Cas13 proteins, thereby defining their binding site on the target RNA. We again decided to initially have four binding sites on the target RNA, so four gRNAs were designed, with each one binding to a Cas13 domain. To introduce the gRNAs we used an approach with a self-cleaving gRNA cassette that can be genomically integrated, producing a self cleaving RNA that results in a functional gRNA (Utomo et al., 2021).

The introduction of two proteins, the tetramerization domain as it is and the IM2-Cas13 fusion protein, results in freely diffusing tetramers with multiple Cas13 units noncovalently bound. The introduction of gRNAs now defines the target RNA for the Cas13 binding, resulting in network formation and condensation.

We have two systems that both can form a large network once a targeted RNA is present, resulting in visible local up-concentration of GFP signal. With TRAPS-Pumby, the RNA target is defined by the amino acid sequence of the Pumbys, whereas the TRAPS-Cas13 system defines the target by the introduction of gRNAs. In the Pumby system, four proteins need to be designed and integrated, whereas in the TRAPS-Cas13 system only two proteins are needed. The E9-IM2 bond that ensured the liquid-like behaviour of our synthetic condensate is still present in the TRAPS-Cas13 system but was lost in the TRAPS-Pumby system.

At this point, it became apparent that Cas13 is most likely the better RNA-binding protein for our application, being substantially more adaptable to new targets and significantly less complicated. Additionally, the resulting network once an RNA is bound, might also behave more like a liquid due to the E9-IM2 link. The only upside of the TRAPS-Pumby system is the very high affinity for the Pumbys towards their target RNA sequences.

## Building

As a proof of concept we used the mRNA of the fluorescent protein [mCherry](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#m) as our first target RNA in [S. cerevisiae](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#s). This has multiple advantages. Since it is an artificially introduced gene that is not naturally expressed in yeast we can control the expression with a conditional promoter. For this we used a [galactose-dependent promoter](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#g), which only activates when we switch from glucose medium to galactose medium for the growing cells (Hovland et al., 1989). This allows us to precisely observe whether condensates form when we activate the promoter. Additionally, we can directly assess the impact of our system on translation of the target [RNA](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#r) by looking at the mCherry fluorescence.

### Build Cycle 1 – Cloning Strategy: Gateway Cloning

We planned on using [Gateway cloning](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#g), since it is a well-established method in our lab. For this, we planned to add attB sites to the genes that we order and introduce them into donor vectors (Hartley, 2000).

When we became more concrete with the design of the gene cassettes, we realized that we would probably need to replace promoters and exchange genes quite often to improve our system step by step. We realized that for such a fast, iterative process, Gateway cloning is not optimal and decided instead to use conventional cloning to introduce our TRAPS genes. However, to integrate the conditional mCherry, we used the Gateway cloning approach, since a yeast-optimized mCherry entry clone was already available.

### Build Cycle 2 – Cloning Strategy: Conventional Cloning

For our TRAPS proteins, we decided on using conventional cloning and selection with [auxotrophic](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#a) markers (Burke et al., 2000). To avoid using too many auxotrophic markers, we integrated two proteins per plasmid, resulting in only three auxotrophic markers in total per system.

In the TRAPS-Cas13 system, a tryptophan-selectable plasmid (Figure 7A) was used for mCherry transformation, an uracil-selectable plasmid (Figure 7B) was used for the integration of both our proteins and a leucine-selectable plasmid (Figure 7C) was used to integrate the gRNA coding cassette. In the TRAPS-Pumby system again tryptophan (Figure 7A) was used for mCherry, uracil (Figure 7B) for two of the tetramerization-Pumby units and leucin (Figure 7C) for the last tetramerization-Pumby unit and the dimer-Pumby unit.

To ligate two protein cassettes, a SphI restriction site was placed at one end of one cassette and to the opposite end of the associated cassette. For the introduction into the vectors, we used SacI and a MluI restriction sites to remove the toxic ccdB cassette and insert our cassette. Additionally, the enzymes cut directly in front of or behind M13 primer sites, allowing us to sequence the insert after ligation.

![Illustration of the plasmids used for the transformation of yeast with our proteins. A: pAG304GAL-ymCherry is an expression vector containing a yeast optimised mCherry. It is a genomically integrating vector with a Tryptophan coding cassette. B: pAG416GPD-ccdb destination vector is the vector used for ligation with the designed gene coding cassettes of the TRAPS-Cas13 system and two of the four proteins of the TRAPS-Pumby system. Restriction and ligation will happen at the SacI and MluI restriction sites. It is a centromeric vector with an Uracil coding cassette. C: pAG305GPD-ccdb destination vector is the vector used for ligation with the designed gRNA coding cassettes TRAPS-Cas13 system and two of the four proteins of the TRAPS-Pumby system. Restriction and ligation will happen at the SacI and MluI restriction sites. It is a genomically integrating vector with an Leucin coding cassette.](https://static.igem.wiki/teams/5690/engineering/figure-7-used-plasmids-for-the-transformation-of-traps.webp)

Figure 7: Used plasmids for the transformation of TRAPS.

### Build Cycle 3 - Construct Building

For each protein, the respective construct was built. All constructs are controlled by a GAP (BBa\_K4292002) promoter and a ADH1 (BBa\_K1486025) terminator derived from the iGEM registry. As previously mentioned, the ratios of the proteins relative to each other are important. In the TRAPS-Pumby system the expression rate of all three different tetramerization units should be 1:1:1 and in the TRAPS-Cas13 system the ratio of both proteins should also be 1:1 for efficient network formation.

Since protein expression has many variables, expression rates are hard to predict. Therefore, we added an antibody tag to all proteins to check expression levels with a [western blot](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#w) after integration. Proteins with similar size, specifically the three tetramerization-unit-Pumby variants, were tagged with different antibodies. Once we know the expression rates, we can increase or reduce it by exchanging the promoter of individual cassettes, depending on the needs. For this purpose, each promoter was flanked by two unique restriction sites within the plasmid, enabling simple exchange.

### Build Cycle 4 - Considering potential failure

Before we finalized and ordered our fragments, we considered all likely failure points of our system and tried to implement strategies that would allow us to directly demonstrate failure.

The first possible failure would be if our large fusion proteins were not expressed, or misexpressed. This was already testable by the [western blot](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#w). Since both Cas13 (444 aa) and Pumbys (432 aa), which are either directly or indirectly connected to the tetramer, are substantially larger than the tetramerization domain (31 aa), we hypothesized that the domain might not form a tetramer anymore due to steric clashes of the large bound proteins. We planned a strategy of testing this in the TRAPS-Pumby system. For one of the three different tetramerization-pumby units, no [GFP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#g) was fused. This unit is the only one of the three units that has a [myc tag](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#m). With this setup, we could perform a [pulldown assay](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#p) against the myc-tag, resulting in this specific tetramerization unit remaining bound in the gel. If the other two units labelled with GFP were unable to bind and form a tetramer with the mentioned unit, there would be no GFP fluorescence remaining in the gel. On the other hand, if they form a tetramer, there should be strong fluorescence in the gel even though only the unlabelled tetramerization unit was directly bound.

A further possible failure point is the successful binding of our RNA-binding proteins to the respective RNA binding site. Here again, a pulldown assay could demonstrate binding. If we perform a pulldown against one of the antibody tags connected to our RNA-binding proteins, we should be able to extract the RNA that remains in the gel. One common problem in protein purification is contamination with RNA, so we already know that anyway RNA often remains in the gel anyway. However if our proteins bind efficiently the target RNA, it should be enriched compared to the usual RNA impurities.

### Cas13-Constructs

![Illustration of the TRAPS-Cas13 units and coding cassettes. On the top the illustration of the tetramerization unit, with individual domains parts and restriction sites labelled on the coding cassette depiction. On the bottom the illustration of the Im2-Cas13 unit, with individual domains, parts and restriction sites labelled on the coding cassette depiction. ](https://static.igem.wiki/teams/5690/engineering/figure-8-traps-cas13-protein-coding-cassettes.webp)

Figure 8: TRAPS-Cas13 protein coding cassettes, with the Tetramerization Unit and the Im2-Cas13 Unit, designed to be cloned into the pAG416-ccdb plasmid.

![Illustration of the TRAPS-Cas13 gRNA coding cassettes. Four different sequences for the gRNA are placed into the general self-cleaving gRNA coding cassette with individual parts and restriction sites labelled.](https://static.igem.wiki/teams/5690/engineering/figure-9-traps-cas13-grna-coding-cassettes.webp)

Figure 9: TRAPS-Cas13 gRNA coding cassette designed to be cloned into the pAG305GAP-ccsdb plasmid.

### Pumby-Constructs

![Illustration of two of the TRAPS-Pumby units and coding cassettes. On the top the illustration of the one tetramerization-Pumby unit, with individual domains parts and restriction sites labelled on the coding cassette depiction. On the bottom the illustration of a second tetramerization-Pumby unit, with individual domains, parts and restriction sites labelled on the coding cassette depiction.](https://static.igem.wiki/teams/5690/engineering/figure-10-traps-pumby-protein-coding-cassettes-1.webp)

Figure 10: TRAPS-Pumby protein coding cassettes, with two Tetramerization-Pumby Units, designed to be cloned into the pAG416GAL-ccdb plasmid.

![Illustration of the other two TRAPS-Pumby units and coding cassettes. On the top the illustration of the third tetramerization-Pumby unit, with individual domains parts and restriction sites labelled on the coding cassette depiction. On the bottom the illustration of a dimerization-Pumby unit, with individual domains, parts and restriction sites labelled on the coding cassette depiction.](https://static.igem.wiki/teams/5690/engineering/figure-11-traps-pumby-protein-coding-cassettes-2.webp)

Figure 11: TRAPS-Pumby protein coding cassettes, with one Tetramerization-Pumby Unit and the Dimerization-Pumby Unit, designed to be cloned into the pAG305GAL-ccdb plasmid.

## Testing

Once the protein-coding cassettes were designed and the cloning strategy was clear, we started the testing by first introducing the mCherry gene into the yeast cells.

### Testing Cycle 1 – mCherry integration, expression and microscopy

The mCherry DNA was genomically integrated with a galactose-dependent promoter in the tryptophan locus using Gateway cloning.

Upon changing the medium from glucose to galactose, thereby activating the galactose promoter, a strong increase in fluorescence is observed. Almost no fluorescence is visible in the cytosol of the uninduced cells, whereas strong fluorescence can be seen in the induced cells (Figure 12 A, B, C, D), which is also reflected in the analysis of the cumulative fluorescence intensity of the cells (Figure 12 E). Interestingly, we observed fluorescence in the vacuoles of the uninduced cells (Figure 12 B).

![](https://static.igem.wiki/teams/5690/engineering/figure-12-mcherry-expression-test.webp)

Figure 12: mCherry expression test. A, B: Images of uninduced cells continuously grown in glucose medium. C, D: Gal-induced cells grown in galactose medium. E: Analysis of the fluorescence intensity of both groups.

The conditional expression of mCherry under the galactose-dependent promoter was successful, and the resulting yeast strain was used for further tests of the TRAPS System targeting the mCherry RNA. The fluorescence of the vacuoles, even in the uninduced sample, was initially confusing. However, upon further research, we learned that adenine-deficient yeast strains, such as ours, accumulate an intermediate of the adenine biosynthesis pathway that has auto fluorescent properties and is typically stored in the vacuole (Banta et al., 1988). Thus, the vacuole fluorescence we observed is independent of mCherry expression and not relevant for our further testing.

After we created our mCherry-expressing strain, we introduced the TRAPS-Cas13 proteins and the gRNA targeting the mCherry mRNA

### Testing Cycle 2 – TRAPS-Cas13 integration, expression and microscopy

Once the genes for our TRAPS-Cas13 proteins arrived, the two cassettes were ligated and introduced to our centromeric plasmid with the uracil auxotrophic marker. This plasmid was then transformed into the conditionally mCherry-expressing strain using our optimized transformation protocol. The same was performed with the gRNA coding cassette.

After successfully transforming the yeast, we started testing the functionality of TRAPS. We imaged one uninduced group, in which no mCherry RNA should be present, and one galactose-induced group expressing mCherry (Figure 13 B, E). When looking at our GFP-labelled TRAPS proteins, we saw local up-concentrations (Figure 13 C, F, arrows) indicating that our proteins have formed “droplets” in the cell. This is the desired outcome for our TRAPS system, but only when the RNA is present. Unfortunately, we found the droplet formation was independent of mCherry expression, therefore independent of our target RNA.

![](https://static.igem.wiki/teams/5690/engineering/figure-13-testing-first-iteration-of-traps-cas13.webp)

Figure 13: Testing first iteration of TRAPS-Cas13. A-C: Images of uninduced cells continuously grown in glucose medium. D-E: Gal-induced cells grown in galactose medium.

At this point, we already speculated that we were not observing condensate formation but rather [aggregation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#a) of our proteins independent of RNA, but this had to be proven. First, we needed to confirm that the formation of these droplets is RNA-independent since the conditional promoter can be leaky (Peng et al., 2015), resulting in RNA expression and condensate formation even without galactose induction. Then, we aimed to determine which of the two proteins caused the aggregation by splitting apart the two cassettes and transforming them individually.

Having observed the first microscopy results, we wanted to test whether our proteins are expressed at the correct size and, if so, in what ratio. As mentioned previously, an optimal ratio is 1:1. To test this, we performed a [western blot](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#w).

### Testing Cycle 3 – Western Blot

Western blotting was conducted to confirm that our protein target construct is expressed and to assess its molecular weight, thereby also confirming the success of the transformation process. Furthermore western blotting allows the detection of potential protein degradation and enables the comparative analysis of construct expression levels in cell lines with gRNA and without gRNA expression, providing insight into expression efficiencies. To further investigate whether the expressed proteins are soluble or insoluble, western blotting was performed on the Total cell lysate (T), which includes both soluble and insoluble fractions, as well as on the Supernatant (S), which contains primarily soluble proteins.

The protein constructs were designed to be detected through immunoblotting. Both constructs were tagged with a myc tag. As the GFP-E9-Tetra construct is approximately 48 kDa and the Cas13-Im2 construct approximately 65 kDa a differentiation between the two constructs in the immunoblot should be possible. Furthermore the GFP-E9-Tetra construct is GFP tagged so the samples were split into heated and non-heated samples before performing a SDS-Page so that the GFP will still be functional in its fluorescence in the non-heated sample.

After two failed western blot attempts and extensive trouble shooting a successful western blot was performed. Both protein constructs are expressed at the expected molecular weight of as can be seen in figure 14A with a band at ~ 48 kDa corresponding to the GFP-E9-Tetra construct and a band at ~65 kDa which corresponds with the Cas13-Im2 unit. An additional GFP-fluorescent band (~27 kDa) was detected, likely representing a degradation product as 27 kDa is also the molecular weight of GFP (10.1385/0-89603-481-X:305). The experiments also revealed a more effective expression in the cell lines without gRNA expression in direct comparison to the cell lines with gRNA expression as can be seen in figure 14C. As can be seen in figure 14C the results from the Supernatant and Total fraction of the cell lysate confirmed that our proteins are primarily insoluble, as there is more protein material visible in the T fraction in direct comparison to the S fraction in all samples by a factor of 2.1 to 11.7. Another finding is that the GFP-E9-Tetra construct is expressed at higher labels than the Cas13-Im2 construct across all heated samples as can be seen in figure 14C.

### Testing Cycle 4 – Testing the Aggregation

To check whether our proteins aggregate independent of RNA, we transformed our plasmid carrying TRAPS into WT yeast, where no mCherry RNA is present. Additionally, we separated the two cassettes and integrated and transformed them individually to test whether our labelled tetramer also aggregates without the IM2-Cas13 unit bound to it.

As we expected, the droplets formed completely independent of RNA, since we also observed them during the microscopy of the WT cells that were transformed with our TRAPS proteins (Figure 15 A-C).

![](https://static.igem.wiki/teams/5690/engineering/figure-15-testing-for-aggregation.webp)

Figure 15: Testing for aggregation. A-B: Images of TRAPS-Cas13 system introduced into WT W303 yeast cells, not having the mCherry gene. D-E: Images of yeast cells only having the tetramerization unit of the TRAPS-Cas13 system.

The individual expression of the GFP-labelled tetramerization unit resulted in distributed GFP signal within the cytosol (Figure 15 D-F), further confirming our suspicion that the aggregation is caused by misfolding of the Cas13 domain.

### FRAP

Another possible explanation for our observation is that Cas13 lacks specificity in its binding to the gRNAs. This would mean that Cas13 not only binds the designed gRNAs and subsequently the target RNA, but that binds other RNAs in the cell instead. These unspecifically bound RNAs could connect the TRAPS tetramers, resulting in condensate formation independent of our target RNA. In that case, the system should still behave liquid-like, since the bond between the tetramers is RNA-mediated, even though it is not mediated by the target RNA. This contrasts with the expectation of aggregates, which behave more solid like (Taylor et al., 2019).

To test this, we performed a [FRAP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27glossary%27%29%20%7D%7D#f) (Fluorescence Recovery After Photobleaching) experiment to examine at the fluidity properties of our droplets. If there is substantial intramolecular movement within the droplet, as expected for liquid-like behaviour, the fluorescent recovery of a bleached part of the droplet should be faster, since unbleached molecules migrate into the bleached area. On the other hand, if there is little intramolecular movements in the droplet, as expected in solid-like aggregates, the recovery should be slow, since unbleached molecules struggle to migrate to the bleached area.

Intracellular FRAP experiments of condensates or aggregates is quite difficult, since they are usually small in size. Typically, FRAP is performed in vitro on large size aggregates of purified Proteins. Nevertheless, we performed the experiment bleaching a part of the droplet and observed almost no recovery (Figure 16). A similar experiment was performed on the original condensate system published by Heidenreich (Heidenreich et al., 2020, Figure 3b). An appropriate adaptation of the result was depicted here as the expected recovery (Figure 16).

![](https://static.igem.wiki/teams/5690/engineering/figure-16-frap.webp)

Figure 16: FRAP of TRAPS-Cas13: A-I: Images of fluorescently bleached droplets in our cells before bleaching, and 2 seconds or 10 minutes after bleaching. J: Graph plotting the fluorescence recovery over time in seconds.

As expected for aggregates, there is almost no recovery after photobleaching, and the recovery is not comparable to the observed recovery in the initial system. This provides final conformation that we are observing aggregates and not condensates.

We can therefore conclude with good confidence that the Cas13 domains do not fold properly, leading to direct protein-protein interactions subsequent aggregation of the entire system.

After confirming that the TRAPS-system does not correctly respond to our target RNA because aggregation of the Cas13 unit, resulting in a signal without the involvement of our target RNA, we explored further Cas13 options.

### Testing Cycle 5 – TRAPS-Cas13 with a RfxCas13d unit

We continued searching for a suitable Cas13 version to fuse to our IM2 unit. We found a catalytically inactive RfxCas13d variant that was reported to effectively bind to RNA in yeast (Zhang et al., 2022) A new gene fragment was constructed by replacing the Cas13 unit, then integrated into our plasmid, transformed and tested.

![](https://static.igem.wiki/teams/5690/engineering/figure-17-testing-second-iteration-of-traps-cas13.webp)

Figure 17: Testing the second iteration of TRAPS-Cas13. A-C: Images of uninduced cells continuously grown in glucose medium. D-E: Gal-induced cells grown in galactose medium.

Again, we observed the previously described aggregates independent of RNA presence (Figure 17). Extensive research was needed to overcome the aggregation problem.

### Testing Cycle 6 – TRAPS-Cas13 with redesigned RfxCas13d unit

The observed aggregation was disappointing, since the RfxCas13d version of Cas13 is the only catalytically inactive variant reported to work in yeast. We therefore considered that Cas13 might not be compatible our system, which would mean the end of our approach, at least in yeast. However, we did not give up and tried to determine whether there was a plausible and resolvable cause for this aggregation.

Upon a close look at the publication about the RfxCas13d, we realized two things. First, the version that they used was not yeast-optimised, as the gene for the protein was directly taken from a provided plasmid of previous work on human cell lines with this Cas13 application (Han et al., 2020). Yeast optimisation could potentially increase the expression levels, although it would likely not resolve the aggregation issue. Second, we found in the supplementary data of the initial publication on human cells that the efficacy of the RfxCas13d depends on the position of protein fusion: N-terminal fusion leads to aggregation and C-terminal fusion results in a functional Cas13 domain (Han et al., 2020, Figure S5).

In our construct the IM2 unit is fused N-terminally to the RfxCas13d domain, which is therefore the likely cause of aggregation. A new yeast-optimised version of our protein with a C-terminal IM2 fusion has been ordered and is expected to arrive on the 07.10. This new version will be tested and, hopefully, will lead to a functional system.

With this we found a very reasonable explanation of the continued aggregation and moved forward by addressing the problem with a new gene. However, we had reached the end of the development of TRAPS-Cas13 up to this point.

### Testing Cycle 7 -TRAPS-Pumby integration, expression and microscopy

The TRAPS-Pumby genes were ligated, integrated into the respective vectors, and transformed into the yeast strain carrying the mCherry gene. After successfully transforming the yeast, we again examined one galactose-induced group and one uninduced group.

Microscopy revealed a slightly different result compared to the first TRAPS-Cas13 test. We again observed droplets independent of galactose induction, and therefore independent of our target RNA (Figure 18 C, F), but there were some interesting additional observations. Compared to the fluorescence in the TRAPS-Cas13 microscopy, we observed a substantial amount of seemingly unaggregated cytosolic GFP signal. While droplets were present, distributed GFP signal was also visible in the cytosol.

Another interesting observation was that mCherry fluorescence appeared reduced in cells that showed high GFP intensity and numerous droplets (Figure 18 E, F, arrows).

![](https://static.igem.wiki/teams/5690/engineering/figure-18-testin-traps-pumby.webp)

Figure 18: Testing the second iteration of TRAPS-Cas13. A-C: Images of uninduced cells continuously grown in glucose medium. D-E: Gal-induced cells grown in galactose medium.

We also observed aggregation independent of RNA, but since unaggregated GFP was still present, we can conclude that at least some of our proteins are not aggregating. Of the four proteins that we introduced, only two are labeled with GFP (two of the tetramerization units) and two are unlabeled (the third tetramerization unit and the dimer). It is possible that only one of the two labeled proteins aggregates while the other does not.

A more likely explanation for this partial aggregation is that, since we expressed four very similar proteins all under the highly expressive GAP promoter, we may have reached a concentration threshold where aggregation begins. Because we expressed four only slightly different Pumbys targeting different sites on the target RNA, this high Pumby concentration may have caused some of them to aggregate.

The interesting observation that mCherry fluorescence appeared reduced in cells with high GFP fluorescence may indicate that free, unaggregated Pumbys were binding our target mCherry mRNA and reducing its translation.
All these assumptions still need to be tested. To determine whether only one of the tetramers aggregates, we would need to separate the protein cassettes and transform them individually. To test whether the aggregation is caused by high protein concentration, we could replace the GAP promoter with a less expressive promoter.

These results (Figure 18) were obtained at almost the same time that we confirmed aggregation in the TRAPS-Cas13 system, and we realized that it would be impossible within the available timeframe to continue testing and validating both TRAPS systems. We therefore actively decided, after the first iteration of the TRAPS-Pumby system, to pause its development and fully focus our efforts on the TRAPS-Cas13 system, since it appeared more promising due to its rapid adaptability to new targets, reduced complexity, and retention of the E9–IM2 interaction.

## Learning

TRAPS, as a new RNA detection system, holds strong potential not only for sensing RNA but also for sequestering it into a protein-scaffold condensate. This feature opens the door for a wide range of additional applications, since the RNA becomes localized within the scaffold. This became clear to us during numerous discussions with experts and scientists throughout the development of TRAPS. We realized that we had not just designed an RNA detection method but also created an RNA sequestration platform that could be applied in several different ways. If you want to know more, read the sections Future Prospects and Integrated Human Practices.

The aggregation observed in many of our experiments was caused by design errors in the Cas13 or Pumby coding cassettes, or more generally, by the choice of the particular domain versions we used, which only became apparent during testing. We are confident that this aggregation issue is solvable, and we expect it to be resolved in the coming months, allowing us to continue testing the system in multiple directions.

As you have read, we researched, adapted, and learned throughout every step of this project. On the scientific level, each of us has acquired new knowledge and skills. But by the end of this project, and this large rollup, we realized it was not just about the science. For all of us, it was the first time carrying out such a large project with full self-responsibility. We experienced everything that comes with it: from fundraising and bureaucracy to PR and outreach events. This experience has made us more prepared than ever to advance in our scientific careers.

Most importantly, we grew not only as a team, moving from initial brainstorming to a concrete idea and testing a new tool in the toolbox of biology, but also as individuals, evolving from loose connections into real friendships. We didn’t just meet in the lab, but also outside of it, celebrating together and sharing good times.

The biggest lesson we take from this project is that science is not just about science, but also about enjoying the process and the fellow scientists around you!

## References

* Adamala, K. P., Martin-Alarcon, D. A., & Boyden, E. S. (2016). Programmable RNA-binding protein composed of repeats of a single modular unit. *Proceedings of the National Academy of Sciences, 113*(19). <https://doi.org/10.1073/pnas.1519368113>
* Alberti, S., & Hyman, A. A. (2021). Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing. *Nature Reviews Molecular Cell Biology, 22*(3), 196–213. <https://doi.org/10.1038/s41580-020-00326-6>
* Banta, L. M., Robinson, J. S., Klionsky, D. J., & Emr, S. D. (1988). Organelle assembly in yeast: Characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. *The Journal of Cell Biology, 107*(4), 1369–1383. <https://doi.org/10.1083/jcb.107.4.1369>
* Burke, D. J., Dawson, D. S., & Stearns, T. (with Cold Spring Harbor laboratory). (2000). *Methods in yeast genetics: A Cold Spring Harbor laboratory course manual (2000 ed)*. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
* Han, S., Zhao, B. S., Myers, S. A., Carr, S. A., He, C., & Ting, A. Y. (2020). RNA–protein interaction mapping via MS2- or Cas13-based APEX targeting. *Proceedings of the National Academy of Sciences, 117*(36), 22068–22079. <https://doi.org/10.1073/pnas.2006617117>
* Hartley, J. L. (2000). DNA Cloning Using In Vitro Site-Specific Recombination. *Genome Research, 10*(11), 1788–1795. <https://doi.org/10.1101/gr.143000>
* Heidenreich, M., Georgeson, J. M., Locatelli, E., Rovigatti, L., Nandi, S. K., Steinberg, A., Nadav, Y., Shimoni, E., Safran, S. A., Doye, J. P. K., & Levy, E. D. (2020). Designer protein assemblies with tunable phase diagrams in living cells. *Nature Chemical Biology, 16*(9), 939–945. <https://doi.org/10.1038/s41589-020-0576-z>
* Hovland, P., Flick, J., Johnston, M., & Sclafani, R. A. (1989). Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose. *Gene, 83*(1), 57–64. [https://doi.org/10.1016/0378-1119(89)90403-4](https://doi.org/10.1016/0378-1119%2889%2990403-4)
* Li, P., Banjade, S., Cheng, H.-C., Kim, S., Chen, B., Guo, L., Llaguno, M., Hollingsworth, J. V., King, D. S., Banani, S. F., Russo, P. S., Jiang, Q.-X., Nixon, B. T., & Rosen, M. K. (2012a). Phase transitions in the assembly of multivalent signalling proteins. *Nature, 483*(7389), 336–340. <https://doi.org/10.1038/nature10879>
* Li, P., Banjade, S., Cheng, H.-C., Kim, S., Chen, B., Guo, L., Llaguno, M., Hollingsworth, J. V., King, D. S., Banani, S. F., Russo, P. S., Jiang, Q.-X., Nixon, B. T., & Rosen, M. K. (2012b). Phase transitions in the assembly of multivalent signalling proteins. *Nature, 483*(7389), 336–340. <https://doi.org/10.1038/nature10879>
* Peng, B., Williams, T. C., Henry, M., Nielsen, L. K., & Vickers, C. E. (2015). Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: A comparison of yeast promoter activities. *Microbial Cell Factories, 14*(1), 91. <https://doi.org/10.1186/s12934-015-0278-5>
* Taylor, N. O., Wei, M.-T., Stone, H. A., & Brangwynne, C. P. (2019). Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching. *Biophysical Journal, 117*(7), 1285–1300. <https://doi.org/10.1016/j.bpj.2019.08.030>
* Utomo, J. C., Hodgins, C. L., & Ro, D.-K. (2021). Multiplex Genome Editing in Yeast by CRISPR/Cas9 – A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways.*Frontiers in Plant Science, 12,* 719148.<https://doi.org/10.3389/fpls.2021.719148>
* Xu, C., Zhou, Y., Xiao, Q., He, B., Geng, G., Wang, Z., Cao, B., Dong, X., Bai, W., Wang, Y., Wang, X., Zhou, D., Yuan, T., Huo, X., Lai, J., & Yang, H. (2021). Programmable RNA editing with compact CRISPR–Cas13 systems from uncultivated microbes. *Nature Methods, 18*(5), 499–506. <https://doi.org/10.1038/s41592-021-01124-4>
* Zhang, Y., Ge, H., & Marchisio, M. A. (2022). A Mutated Nme1Cas9 Is a Functional Alternative RNase to Both LwaCas13a and RfxCas13d in the Yeast S. cerevisiae. *Frontiers in Bioengineering and Biotechnology, 10*, 922949. <https://doi.org/10.3389/fbioe.2022.922949>

[![Connie](https://static.igem.wiki/teams/5690/siteheader/connie-pointing.webp)

Follow me to check out our SEA-STAR Software on the here!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D)

![Connie](https://static.igem.wiki/teams/5690/graphics/exclamation-mark-pointing-connie.webp)

Glub glub! The fun began when the team launched two parallel engineering cycles, one for TRAPS-Cas13 and one for TRAPS-Pumby, even though Cas13 seemed more promising

![Connie](https://static.igem.wiki/teams/5690/graphics/question-hmm-connie.webp)

Even before testing, the lab suspected that Cas13 was causing aggregation—since the tetramerization unit was unchanged from Heidenreich’s original protein and Cas13 is unstable in yeast—but as always, we aim to know, not assume.

[![Connie](https://static.igem.wiki/teams/5690/graphics/compact-happy-connie.webp)

Woohoo! You’ve reached the end of the engineering journey so far. A fully optimized Cas13 is on the way, and if it finally lets TRAPS sense RNA as hoped, a wave of experiments and applications will follow. Curious about what’s next? Dive into Future Prospects to find out!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27prospects%27%29%20%7D%7D)

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# Experiments

![experiments](https://static.igem.wiki/teams/5690/siteheader/headers-experiments-new.webp)
![](https://static.igem.wiki/teams/5690/siteheader/lab-connie-pointing.webp)

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Welcome to the experiments! In this tab you can explore what we did in the lab.
You will find detailed protocols that we used and optimized, all the materials that were necessary and a step-by-step explanation how we carry out our experiments.

* [Methods](#methods)
  + [Plasmid Construction](#plasmid-construction)
  + [Transformation](#transformations)
  + [Microscopy](#microscopy)
* [Protocols](#protocols)
* [Materials](#materials)

## Methods

### Plasmid Construction [![Go to Results](https://static.igem.wiki/teams/5690/siteheader/lab-connie-pointing.webp) Results](/tu-dresden/results "Results")

For plasmid construction, restriction digestion cloning was employed in all cases except for the integration of mCherry into pAG-304, which was generated using Gateway cloning.
For this construct, the original plasmid pENTRY-ymCherry served as the template. During the LR reaction, the mCherry insert from pENTRY-ymCherry was recombined into the destination vector pAG-304.
All other DNA fragments were inserted into their respective destination vectors via restriction digestion. The oligonucleotide fragments used in our project were all designed by us using existing BioBricks and other parts found in papers.
The oligos were then ordered and synthesised by IDT, GenScript, or Twist Bioscience.
The cloning workflow involved digestion of both fragments and vectors with the appropriate restriction enzymes ([Protocol 1](#restriction)), followed by gel electrophoresis to prove successful cutting ([Protocol 2](#pcr)).
For a few restriction enzymes (NOT1, BSU36I, BstX1, XHO1) the restriction digestion initially failed, new respective enzymes were validated by control digestions and gel electrophoresis to confirm their activity. Here 1% agarose gels and 90V/60min running conditions were used. As a marker 1kb marker GeneRuler (NEB) was used. All our gels were analyzed under UV light.
Following successful digestion, the fragments and vectors were combined and incubated at 37 °C for 1 h prior to ligation.
For a the gRNA inserts for both variants of our Cas13 a Ligation was necessary to combine multiple gRNA oligonucleotides.
During our Project we constructed the following Constructs that way:

| Fragment | Vector | Purpose | Cutting Enzyme | Auxotrophy Marker |
| --- | --- | --- | --- | --- |
| GFP-E9-Tetra Im2-MiniCas13.X1 | pAG-416 | This is the first version of our completed TRAPS System with Cas13 | Sac1, Mlu1 | URA3 |
| GFP-E9-Tetra Im2-RfxCas13d | pAG-416 | This is the second version of our completed TRAPS System with Cas13 | Sac1, Mlu1 | URA3 |
| gRNAsMiniCas13.X1 | pAG-305 | This Construct contains the gRNAs for the MiniCas13.X1 version of our TRAPS system | Mlu1, Sac1 | LEU |
| gRNAsRfxCas13d | pAG-305 | This Construct contains the gRNAs for the RfxCas13d version of our TRAPS system | Mlu1, Sac1 | LEU || GALymCherry | pAG-304 | mChery RNA is the first target for our RNA detection system and is under a conditional promotor to make working with it easy | Gateway Cloning | TRP |
| Pumby1 | pAG-416 | Pumby is a RNA binding protein that binds to a specific 8nt sequence. We use it to bind our target RNA In this Construct half of the TRAPS system with Pumbys is present | Sac1, Mlu1 | URA3 |
| Pumby2 | pAG-305 | In this Construct half of the TRAPS system with Pumbys is present. Together with the pAG416-Pumby1 Construct they form the completed TRAPS system with Pumbys instead of Cas13. | Mlu1, Sac1 | LEU |
| GFP-E9-Tetra | pAG-416 | This Construct is part of the TRAPS system and used to test the interaction of the Tetramerization domains | Sac1, Sph2 | URA3 |
| Im2-MiniCas13.X1 | pAG-416 | This Construct is part of the TRAPS system and used to test if the Im2-MiniCas13.X1 form aggregates. | Mlu1, Sph2 | URA3 |

Resulting in the following strains:

| Strain | Genotype | Selection |
| --- | --- | --- |
| *E. coli* DH5α | fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 | - |
| *E.coli* DH5α | pAG304-GALymCherry | Ampicillin |
| *E.coli* DH5α | pAG416-GFP-E9-Tetra-Im2-MiniCas13.X1 | Ampicillin |
| *E.coli* DH5α | pAG305-gRNAsMiniCas13.X1 | Ampicillin |
| *E.coli* DH5α | pAG416-GFP-E9-Tetra-Im2-RfxCas13d | Ampicillin |
| *E.coli* DH5α | pAG305-gRNAsRfxCas13d | Ampicillin |
| *E.coli* DH5α | pAG416-Pumby1 | Ampicillin |
| *E.coli* DH5α | pAG305-Pumby2 | Ampicillin |
| *E.coli* DH5α | pAG416-GFP-E9-Tetra | Ampicillin |
| *E.coli* DH5α | pAG416-Im2-MiniCas13.X1 | Ampicillin |
| *E.coli* DH5α | pAG416-Im2-RfxCas13d | Ampicillin |
| *S. cerevisiae* W303 | MATa/MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+] | - |
| *S. cerevisiae* W303 | pAG304-GALymCherry | TRP |
| *S. cerevisiae* W303 | pAG416-GFP-E9-Tetra-Im2-MiniCas13.X1 + pAG305-gRNAsMiniCas13.X1 + pAG304-GALymCherry | URA3, LEU, TRP |
| *S. cerevisiae* W303 | pAG416-GFP-E9-Tetra-Im2-RfxCas13d + pAG305-gRNAsRfxCas13d + pAG304-GALymCherry | URA3, LEU, TRP |
| *S. cerevisiae* W303 | pAG416-Pumby1 + pAG305-Pumby2 + pAG304-GALymCherry | URA3, LEU, TRP |
| *S. cerevisiae* W303 | pAG416-GFP-E9-Tetra + pAG304-GALymCherry | URA3, TRP |
| *S. cerevisiae* W303 | pAG416-Im2-MiniCas13.X1 + pAG304-GALymCherry | URA3, TRP |

### Transformations [![Go to Results](https://static.igem.wiki/teams/5690/siteheader/lab-connie-pointing.webp) Results](/tu-dresden/results "Results")

All plasmids were initially transformed into *E. coli* DH5α cells for propagation using the chemical transformation method (see Protocol *E. coli* Transformation) and grown on selective plates with ampicillin as a selective marker.
Single colonies were grown in overnight cultures (4mL 100µg/ml ampicillin) and plasmids were then isolated from those cultures of the transformed strains using the Monarch Spin Plasmid Miniprep Kit from NEB. The DNA concentration was afterwards measured with a NanoPhotometer and to confirm the correct size a test restriction of the plasmid was performed and subjected to an agarose gel-electrophoresis. An empty vector sample was used as negative control and loaded as well to the agarose gel.
The purified plasmids intended for further use were subsequently prepared and sent to Microsynth for sanger sequencing.
After confirming the desired sequences, the plasmids were introduced into our *S. cerevisiae* strain W303 ([see Protocol Yeast Transformation](#yeast)). The final strains were kept as glycerol (25 % (v/v)) stocks at -80°C.
Over the course of the project, we optimized the yeast transformation procedure. Initially, transformations were carried out without the use of sorbitol using a standard yeast transformation protocol, which resulted in low success rates.
Further investigation led us to adopt a sorbitol-containing protocol, which we refined throughout the project, ultimately achieving high transformation efficiencies.
The final protocol is detailed in the Protocols section.
The vectors pAG304 and pAG305 are genomically integrating vectors and were therefor genomically integrated into the Tryptophan (pAG304) and Leucin (pAG305) locus of the W303 yeast cells. Therefore, the Plasmid was first linearized with the appropriate restriction enzyme and then used in the transformation reaction.
Successful transformation was verified by plating the cells on selective media lacking the appropriate auxotrophic markers corresponding to the plasmids used (see Table 1 for details on auxotrophic markers).

### Microscopy [![Go to Results](https://static.igem.wiki/teams/5690/siteheader/lab-connie-pointing.webp) Results](/tu-dresden/results "Results")

Before microscopy we prepared the cells ([see protocol Microscopy](#microscopy)). We either microscoped the samples from a 384 Well Plate, a sliding dish or fixed the cells for long time measurements and FRAP.
All microscopies were performed with a Nikon ECLIPSE Ti2 microscope, equipped with a photometrics prime 95B camera (Teledyne Technologies Incorporated, CA, USA), at a temperature of 25 °C. For imaging the yeast cells, a 1000x magnification was used.
Imaging always included Bright Field and Fluorophore images. Imaging of the fluorophores was enabled by exciting RFP and GFP.
All our created strains were microscoped always induced cells expressing mCherry RNA compared to uninduced cells as a control. ([see Protocol Inducing of the GAL1 Promoter](#gal1)). All measurements were done on at least three times on at least 4 different spots per sample.
For fluorescence microscopy image analysis, we used a Python pipeline (see Software). We first employed Cellpose 2.0 and their cyto3 model in the brightfield channel to obtain the segmentation labels of all the yeast cells. Then, we extracted the fluorescence intensities within these segmentation labels from the fluorescence channels. For quantification, we calculated the total cell fluorescence by multiplying the corrected mean fluorescence intensity of a cell with the total amount of pixels in the cell region. The corrected mean is calculated as mean intensity of the region minus the mean fluorescence intensity of all the non-cell pixels.

### Protein Analysis [![Go to Results](https://static.igem.wiki/teams/5690/siteheader/lab-connie-pointing.webp) Results](/tu-dresden/results "Results")

Yeast cell culture of cell lines W303-mCherry-Cas13X.1, W303-mCherry-Cas13X.1-gRNAs, W303-RfxCas13.d, W303-mCherry-305-Pumby1 and W303-mCherry-416-Pumby were grown in SD -TRP -URA Medium, while W303 wild type (WT) culture was grown in YPD medium. All cultures were grown at 30°C with shaking at 180 rpm until they reached an OD₆₀₀ of 0.5 to ensure the cells had reached the exponential phase. The cultures were harvested at a volume equivalent to 10 OD₆₀₀₀ units, calculated from OD₆₀₀ measurements of a 1 ml sample. The 10 OD₆₀₀ samples were then spun down at 2,000 rcf for 4 min. The cells of every sample were washed in 1 ml of ice cold water and centrifuged at 3,000 rpm for 2 min at 4°C. The cell pellets were resuspended in 800 µl lysis buffer and lysed by bead beating with 500 µl silica beads shaken at 30 oscillations/s for 20 min at 4°C. Afterwards the lysate was centrifuged for 1 min at 500 rpm at 4 °C to reduce foam. 300 µl of lysate were taken and centrifuged at 4°C for 30 sec at 3,000 rcf to get the crude extract. 61 µl were taken from the crude extract and stored as the total fraction (T). 200 µl of the crude extract were further centrifuged at 4°C for 5 min at 20,000 rcf. 121 µl were taken as the Supernatant (S).
The protein concentration of the T and S samples were measured with Detergent Compatible (DC) Assay. Bovine Serum Albumin (BSA) was used as the standard protein. A standard curve was generated from a dilution series of BSA covering the concentrations 2.58-0.00 mg/ml in a consecutive 7 step 1:2 dilution. In a 96-well plate 1 µl of the T and S sample as well as each dilution step of the BSA standard was diluted in 4 µl water and mixed with 25 µl SA-mix (50:1 ratio of A to S). After adding 200 µl reagent B to each sample, they were incubated at room temperature in the dark for 15 min. Absorbance was measured at 750 nm using the TECAN Spark® microplate reader.
The T and S samples of the different constructs were split in half and one half was heated at 95°C for 5 minutes (Heated), the other half was not heated (Non-Heated). An SDS-PAGE of the Cas13- and Pumby-constructs was performed with a 12 % SDS-Gel each. An equal amount of protein of each sample and prestained protein marker VI (10-245 kDa) was loaded on the respective Cas13 and Pumby SDS gels in duplicates, so after the blotting the membranes could be cut in half and each half would include a duplicate of the samples and a ladder. The gels were run at a constant voltage of 200 V for 40 min. A scan of the gels was performed using the Cy2 525BP20 and IRshort 720BP20 emission filters of the Amersham Typhoon to detect GFP and the ladder. Proteins were transferred to nitrocellulose membranes at 200 mA for two hours, followed by blocking with 5% (w/v) in PBS-T for 1h. The membranes were washed four times for 5 min in PBS-T. An antibody staining with the primary antibodies fitting the different tags of our target constructs was performed overnight at 4°C. For the Cas13-constructs an α-myc-tag antibody was used. For the Pumby constructs an α-FLAG-tag antibody and an α-HA-tag antibody were used. Each respective primary antibody were mouse host and added to the membranes in a 1:5000 dilution in 2% skim milk (w/v) PBS-T and incubated overnight at 4°C. The membranes were washed four times for 5 min in PBS-T. Both the Cas13 and the Pumby membranes were then cut at the duplicate line so each half contained the target samples and a ladder. The secondary antibody Alexa-Flurophor 488 anti-mouse diluted 1:10,000 in 5% skim milk was added and incubated for 1h at room temperature. After conducting a first fluorescent measurement of the membrane, the immunostaining protocol was repeated with a second primary antibody against the loading control PGK1 (mouse, 1:5000 dilution in 2% skim milk (w/v) PBS-T) followed by the incubation with secondary antibody Alexa-Flurophor 488 anti-mouse (1:10,000 in 5% skim milk).
The membranes were washed four times for 5 minutes in PBS-T. They were then scanned using the Cy2 525BP20 and IRshort 720BP20 emission filters of the Amersham Typhoon.
Densitometric analysis was performed using the Gel Analysis tool in Fiji (doi:10.1038/nmeth.2019). Band intensities were quantified by selecting rectangular regions of interest (ROIs) around each lane and subtracting the background signal. The intensity of the target protein construct bands was normalized to the corresponding loading control (PGK1).

## Protocols

### Restriction Digestion

#### Abstract

Restriction Digestion is used to create defined DNA fragments by cutting plasmids
and other fragments with restriction endonucleases. Here we use a protocol adapted
for HF-Endonucleases.

---

#### Material

* Plasmid DNA or DNA fragment
* Restriction enzyme(s) (e.g., HF-Endonucleases)
* rCutSmart Buffer (10X)
* Nuclease-free water
* Microcentrifuge tubes
* Pipettes and tips
* Thermoblock or water bath (37 °C)

---

#### Protocol

Composition of restriction digestion reaction:

| Component | Volume/Amount |
| --- | --- |
| DNA | 1 µg |
| Restriction enzyme | 1 µl |
| rCutSmart Buffer 10X | 5 µl |
| Nuclease free water | up to 50 µl |

Mix and incubate at 37 °C for 1 h.

### PCR

#### Abstract

PCR (Polymerase Chain Reaction) is a method to amplify DNA fragments.
Here we use Q5 High-Fidelity DNA Polymerase for high accuracy.

---

#### Material

* Template DNA
* Forward and reverse primers (10 µM)
* dNTP mix (10 mM each)
* Q5 High-Fidelity DNA Polymerase
* Q5 Reaction Buffer (5X)
* Nuclease-free water
* Microcentrifuge tubes
* Pipettes and tips
* Thermocycler

---

#### Protocol

Composition of PCR reaction:

| Component | Volume/Amount |
| --- | --- |
| Template DNA | 1 µl |
| Forward Primer (10 µM) | 0.5 µl |
| Reverse Primer (10 µM) | 0.5 µl |
| dNTPs (10 mM each) | 1 µl |
| Q5 High-Fidelity DNA Polymerase | 0.5 µl |
| Q5 Reaction Buffer (5X) | 5 µl |
| Nuclease free water | up to 25 µl |

Mix and run the following program in a thermocycler:
1. Initial denaturation: 98°C for 30s
2. 25-35 cycles of:
a. Denaturation: 98°C for 10s
b. Annealing: X°C for 20s (X = Tm of primers - 3°C)
c. Extension: 72°C for Y min (Y = 30s per kb of target length)
3. Final extension: 72°C for 2min
4. Hold at 4°C

### Ligation

#### Abstract

Ligation is used to join DNA fragments together. Here we use T4 DNA Ligase to ligate
fragments with compatible ends.

---

#### Material

* Linearized vector DNA
* Insert DNA fragment
* T4 DNA Ligase
* T4 DNA Ligase Buffer (10X)
* Nuclease-free water
* Microcentrifuge tubes
* Pipettes and tips
* Thermoblock or water bath (37 °C)

---

#### Protocol

Composition of ligation reaction:

| Component | Volume/Amount |
| --- | --- |
| Vector | 50 ng |
| Insert (3:1 molar ratio) | X ng |
| T4 DNA Ligase | 1 µl |
| T4 DNA Ligase Buffer 10X | 2 µl |
| Nuclease free water | up to 20 µl |

Mix and incubate at 37 °C for 1.5 h.

### Agarose Gel electrophoresis

#### Abstract

Agarose Gel electrophoresis is a simple method for analysing DNA Fragments.
It is used to separate different DNA fragments with different lengths from each other.
Here we show Gel electrophoresis with agarose gels stained with Ethidium Bromide.

---

#### Material

* Agarose
* 1x TAE buffer
* Ethidium Bromide (or alternative DNA stain)
* Gel casting tray and comb
* DNA samples
* Purple gel loading dye (6x)
* 1kb extend DNA Ladder (NEB)
* Electrophoresis chamber and power supply
* Gel imager with UV light
* Pipettes and tips

---

#### Protocol

1. Prepare 1% agarose gel:
• Weigh 1g agarose and add it to 100ml 1x TAE buffer.
• Heat the solution in a microwave until the agarose is completely dissolved.
• Cool the solution to about 60°C and add 5µl of Ethidium Bromide.
• Pour the solution into a gel casting tray with a comb and let it solidify for at least 30 minutes.
• After solidification pour 1x TAE Buffer into the tray.
2. Prepare samples:
• For sample Preparation add purple gel loading dye 6x at a final concentration of 1x to each sample.
• Load each sample in a separate well and alongside 5µl 1kb extend DNA Ladder (NEB).
3. Run the gel:
• Let the gel run at 90V for 60-90min depending on the loaded samples and agarose concentration (1-2%).
• Image the gel in a gel imager under UV light

### *E. coli*Transformation

#### Abstract

*E. coli* Transformation is a method to introduce foreign DNA into *E. coli* cells.
Here we use chemically competent cells and a heat shock protocol.

---

#### Material

* Chemically competent *E. coli* (e.g., DH5α)
* Plasmid DNA
* ddH2O
* 5x KCM buffer
* LB medium
* Microcentrifuge tubes
* Pipettes and tips
* Incubator (37 °C)
* Shaker
* Water bath (42 °C)
* Agar plates with appropriate antibiotic

---

#### Protocol

Mix:

| Component | Volume/Amount |
| --- | --- |
| dH2O | 7 µl |
| 5x KCM | 2 µl |
| DNA | 1 µl |

•Set the mixture on ice for 10min
•Add 10µl chemical competent *E. coli* (DH5α)
•Set it on ice for another 20min
•Heat shock the cells at 42°C for 1min, 300 RPM
•Add 900µl LB for regeneration
•Incubate the cells for 45min at 37°C
•Spin the cells down and resuspend them in ddH2O before plating them

### Yeast Transformation

#### Abstract

Yeast transformation is a widely used method to introduce plasmid DNA into Saccharomyces cerevisiae. This protocol is based on the lithium acetate/PEG method, which relies on chemical permeabilization of the yeast cell wall.

---

#### Material

* *S. cerevisiae* cells
* Linearized plasmid DNA
* Salmon sperm DNA (ssDNA)
* Sorbitol/LiAc solution (1M/1M)
* PEG
* DMSO
* YPD medium
* ddH2O
* Selective agar plates
* Microcentrifuge tubes
* Pipettes and tips
* Incubator (30 °C)
* Shaker
* Centrifuge

---

#### Protocol

1. Preparation of yeast culture:
• Grow 5 ml overnight pre-culture to saturation
• Dilute to OD₆₀₀ = 0.1 in 50 ml of fresh medium in 250 ml flask
• Incubate at 30 °C for 3–4 h to reach OD₆₀₀ ≈ 0.5-0.7
2. Spin cells at 2000x g for 3 min
3. Wash once with 25 ml of Sorbitol/LiAc (1M/1M) solution,
4. Spin cells at 2000x g for 3min and discard supernatant.
5. Resuspend cells in the rest drops of liquid (usually it is 400-500 microliters)

6. Assemble transformation reaction:

| Component | Volume/Amount |
| --- | --- |
| Linearized plasmid | ~25 µl (1 µg) |
| ssDNA (salmon sperm DNA) | 15 µl |
| Yeast cells (resuspended in rest drops Sorbitol/LiAc) | 100 µl |
| PEG | 280 µl |

7. Assemble transformation reaction for a negative control with everything excluding the linearized plasmid
8. Mix well and incubate at room temperature for 45-60min
9. Add 40µl DMSO to the mixture
10. Incubate at 42°C for 15min, 300RPM
11. Add 1ml of YPD and mix well 11. Add 1ml of YPD and mix well
12. Allowing the cells to recover for 1-2h at 30°C with shaking at 200rpm is optional.
13. Spin at 4000rpm for 5min and discard the
14. Resuspend the cell pellet in 200µl ddh3O
15. Plate on the appropriate selective media.
16. Incubate plate at 30°C for 2-3 days until colonies appear.

### Inducing of the GAL1 Promoter

#### Abstract

Conditional promoters enable precise regulation of gene expression in response to defined stimuli. In our case the GAL1 promotor is used, which is a very strong promoter that is activated through galactose

---

#### Material

* Yeast strains with GAL1 promoter constructs
* SC medium with glucose
* SC medium with 2% raffinose
* SC medium with 2% galactose (or raffinose + galactose)
* Sterile water
* Microcentrifuge tubes
* Pipettes and tips
* Incubator (30 °C)
* Shaker
* Spectrophotometer (for OD600 measurement)

---

#### Protocol

Pick a single colony and bring it into the appropriate media containing Glucose.

1. Incubate overnight at 30 °C with shaking at 200 rpm.

2. Removal of Glucose:

 • Pellet 1–5 mL of the overnight culture by centrifugation at 3,000 × g for 5 minutes.

 • Wash the cell pellet twice with sterile water or SC medium lacking a carbon source to eliminate residual glucose.

3. Pre-induction in Raffinose:

 • Resuspend the washed cells in SC medium supplemented with 2% raffinose.

 • Adjust the cell density to OD₆₀₀ = 0.2–0.4.

 • Incubate at 30 °C with shaking (200 rpm) for 4–6 hours, or until cells reach mid-log phase (OD₆₀₀ ~0.6–0.8).

 • This step allows for derepression of GAL promoters without activation.

4. Induction with Galactose:

 • Add galactose to the raffinose culture to a final concentration of 2% (w/v), or alternatively, pellet and resuspend the cells in fresh SC medium containing 2% raffinose and 2% galactose.

 • Continue incubation at 30 °C with shaking.

 • Induction times may vary

### Microscopy

#### Abstract

Microscopy is used to visualize protein expression and localization in yeast cells. This protocol describes the preparation of yeast samples and the use of fluorescence microscopy to analyze induced and control cultures.

---

#### Material

* Yeast cultures (induced and control)
* Microscope slides and cover slips
* Fluorescence microscope with appropriate filters
* Well plates (optional, for multiple samples)
* Pipettes and tips

---

#### Protocol

Preparation of Yeast cells:
•Set the OD₆₀₀ of your yeast culture to ~0.3
•Take 20µl of the induced culture and put it on a microscope slide and add a cover slip.
•Optional: If you want to analyze multiple samples at the same time add 20µl of each sample to a well of a appropriate well plate.
Image the cells with a fluorescence microscope using the appropriate filters for your fluorophore.
Analyze the images using image analysis software.

### FRAP

#### Abstract

Fluorescence Recovery After Photobleaching (FRAP) is a technique used to study the dynamics of fluorescently labeled molecules within living cells. This protocol outlines the steps for performing FRAP experiments on yeast cells expressing fluorescent proteins. For FRAP it is important to have immobilized cells. Therefore, we show a immobilization protocol using Concanavalin A.

---

#### Material

* Yeast cultures expressing fluorescent proteins
* Fluorescence microscope with FRAP capabilities
* Microscope slides and cover slips
* NaOH
* Concanavalin A solution
* Imaging software
* Pipettes and tips

---

#### Protocol

**1. Preparation of Yeast cells:**
•Set the OD₆₀₀ of your yeast culture to ~0.3
**2. Immobilize the yeast cells to the cover slip:**
•Wash dish with 1M NaOH.
•Add Concanavalin A solution to the dish and coat for 20min while gently shaking.
•2.2ml 1x PBS
•125µl 0.2M K2HPO4
•2.5µl 1M CaCl2
•5µl 10% NaN3
•250µl 5mg/ml Concanavalin A
•Rinse dish 3 times with ddh3O
•Do not let your dish dry after the coating as this will give you a high background
•Add your yeast suspension to the dish and let it sit for 10min
**3. Image the cells:**
Image the cells with a fluorescence microscope using the appropriate filters for your fluorophore.
Analyze the images using image analysis software.

### Yeast Lysate Preparation & Protein Quantification

#### Abstract

This protocol describes the preparation of yeast lysates for protein analysis and subsequent quantification using the DC Protein Assay (Bio-Rad). The procedure includes cell growth, lysis by bead beating, and quantification against a BSA standard curve.

---

#### Protocol

1. **Grow yeast culture:**
   Inoculate an overnight culture. Measure OD600 (dilute 1:10 if necessary). Dilute to OD600 ≈ 0.2 and incubate until OD600 ≈ 0.5–0.7 (typically ~4 h in SD -TRP -URA medium).
2. **Harvest cells:**
   Collect enough culture for a total OD of 10 (e.g., for OD 0.52: 10/0.52 = 19.2 ml). Spin down at 2000g, 4 min.
3. **Wash cells:**
   Discard supernatant, resuspend pellet in 4°C autoclaved water, transfer to 1.5 ml tube, spin at 3000 rpm, 2 min, 4°C.
4. **Prepare for lysis:**
   Prepare a 1.5 ml tube with 500 µl silica beads. Remove supernatant from washed cells.
5. **Cell lysis:**
   Resuspend pellet in 800 µl complete lysis buffer (with DTT and protease inhibitor), transfer to bead tube. Bead-beat at 30 oscillations/s for 20 min at 4°C.
6. **Clarify lysate:**
   Spin at 500 rpm, 1 min, 4°C to reduce foam. Transfer 200 µl lysate to new tube (avoid beads/foam). Spin at 3000g, 30 sec, 4°C to obtain crude extract.
7. **Prepare samples:**
   For "Total" (T): Take 61 µl crude extract into new tube (1 µl for quantification).
   For "Supernatant" (S): Take 120 µl crude extract, spin at 20,000g, 5 min, 4°C, then take 61 µl supernatant (1 µl for quantification).
8. **Add sample buffer:**
   Add 20 µl sample buffer (with 10% β-mercaptoethanol) to T and S after taking 1 µl for quantification. Freeze samples for later SDS-PAGE.
9. **Protein quantification (DC Protein Assay, Bio-Rad):**
   * Prepare BSA standard curve: 2.58 mg/ml, 1.29, 0.64, 0.32, 0.16, 0.08, 0.04, 0.00 mg/ml (serial 1:2 dilutions in lysis buffer).
   * Prepare S and A mixture (50:1 ratio of A to S).
   * Dilute 1 µl sample in 4 µl water.
   * Load samples and standards into a 96-well plate.
   * Add 25 µl SA mixture to each well.
   * Add 200 µl B reagent to each well, mix gently (avoid bubbles).
   * Incubate 15 min at room temperature in the dark (color changes from orange to blue).
   * Measure absorbance at 750 nm (e.g., with Tecan plate reader).
   * Determine protein concentration using the BSA standard curve.
10. **Notes:**
    * Quantification and freezing/loading the gel should be done ASAP to avoid protein degradation.

### SDS-PAGE

#### Abstract

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a technique used to separate proteins based on their molecular weight. This protocol describes the steps for preparing and running SDS-PAGE gels to analyze protein expression and purity.

---

#### Protocol

**1. Gel Casting**

If using pre-cast gels, skip this section.
**Note:** In our experiments, self-cast gels with 10–12.5% acrylamide were used (Mini-PROTEAN Tetra Cell system).

**Separating Gel (10 ml, 12.5% acrylamide, 1 mm thickness preferred):**

| Component | Volume |
| --- | --- |
| H₂O | 3.7 ml |
| 1.5 M Tris-HCl pH 8.8 | 3.0 ml |
| 10% SDS | 178 µl |
| 30% Acrylamide | 5.0 ml |
| 10% APS | 79 µl\* |
| TEMED | 8 µl\* |

**\* Important:** Add APS and TEMED last, mix quickly, and pour immediately.
Allow separating gel to polymerize (check solidification with leftover mix in Falcon tube).

**Stacking Gel (4%, 1 gel):**

| Component | Volume |
| --- | --- |
| H₂O | 1.8 ml |
| 1 M Tris-HCl pH 6.5 | 0.8 ml |
| 10% SDS | 96 µl |
| 30% Acrylamide | 0.5 ml |
| 10% APS | 32 µl\* |
| TEMED | 4 µl\* |

**\* Important:** Add APS and TEMED last, mix quickly, insert comb, and allow to polymerize.
Cast gels can be stored in SDS running buffer at 4 °C or used directly.

**2. Sample Preparation**

•Load 10µg protein per lane (calculated from quantification).
•Load 4µl prestained protein ladder per gel.
•Load samples directly without additional denaturation steps.

**3. Sample Loading**

•Insert gels into the running chamber. Push the glass plates to the rim to avoid leaks.
•If running only one gel, insert a dummy plate in the second slot.
•Fill inner chamber with fresh SDS running buffer. Once confirmed tight, fill the outer chamber halfway with used buffer.
•After removing the comb, rinse wells with running buffer.
•Load marker and samples as indicated above.

**4. Running the Gel**

•Run the gel at a Constant voltage: **200 V, 40 min**
•Run until dye front reaches the end of the gel (slightly running out).
•After run: recycle buffer into used buffer flask.

**5. Post-Run**

•Open cassette carefully with spatula under running water.
•Transfer gel into container with water.
**Scanning (Typhoon):**
•Select appropriate channels for scanning.
•Perform prescan: if signal appears pink = oversaturated → reduce voltage.
•Otherwise, lower voltage slightly before final scan.
•Save scan file!
•Clean glass plates carefully, ensure no lint remains. If organic solvents were used, do not place gel directly on glass → use foil support instead.

### Western Blot

#### Abstract

Western blotting is a technique used to detect specific proteins in a sample after separation by SDS-PAGE and transfer to a membrane.

---

#### Protocol

1. **Run SDS-PAGE:**
   Perform SDS-PAGE as described in the "SDS-Page Protocol".
2. **Prepare transfer sandwich:**
   Prepare a container with transfer buffer.
   Assemble the sandwich in the following order (from bottom to top):
   * Black side of clamping cassette (down)
   * Sponge
   * Whatman paper
   * Gel
   * Nitrocellulose membrane (roll flat with roller!)
   * Whatman paper
   * Sponge**Note:** Ensure the gel is oriented so that the ladder will be on the left side after transfer.
3. **Close cassette:**
   Place the white side of the clamping cassette on top and close it.
4. **Transfer proteins:**
   Place the sandwich in the container with transfer buffer.
   Run the transfer for 2 hours at 200 mA.
   **Note:** For proteins >300 kDa, extend transfer time beyond 2 hours.
5. **Blocking:**
   Incubate the membrane for 1 hour in blocking buffer.
6. **Primary antibody incubation:**
   Dilute the primary antibody in blocking buffer.
   Incubate the membrane overnight at 4 °C with gentle shaking.
7. **Wash membrane:**
   Wash the membrane 3 times for 10 minutes each with TBST.
8. **Secondary antibody incubation:**
   Dilute the secondary antibody in blocking buffer.
   Incubate the membrane for 1 hour at room temperature with gentle shaking.
9. **Wash membrane:**
   Wash the membrane 3 times for 10 minutes each with TBST.
10. **Imaging:**
    Image the membrane using the appropriate imaging technique.

## Materials

Chemical compounds used in this study were purchased from Merck (Darmstadt, Germany), Roche Diagnostics (Basel, Switzerland), Carl Roth (Karlsruhe, Germany) and Qiagen (Venlo, Netherlands). Restriction enzymes were purchased from New England Biolabs (NEB, Ipswitch, USA). Water used for buffers, solutions and media was filtered using Milli-Q Ultrapure water systems (Merck)

### Chemicals and Reagents

* 10% APS
* 10% SDS
* 10x TAE Buffer
* 1.5 M Tris-HCl pH 8.8
* 1 kb DNA ladder Generuler (NEB)
* 1 kb extend DNA Ladder (NEB)
* 1M Tris-HCl pH 6.5
* 30% Acrylamide
* 5x KCM buffer
* Agarose
* Agar plates with antibiotic selection
* Alexa-Fluor 488 anti-mouse secondary antibody (1:10,000)
* Antibiotics: Ampicillin, Kanamycin
* Blocking Buffer: 5% Milk powder in PBST
* Bovine Serum Albumin (BSA)
* Complete lysis buffer (with DTT and protease inhibitor)
* β-Mercaptoethanol
* Coating solution:
  + 1x PBS
  + 0.2M K2HPO4
  + 1M CaCl2
  + 10% NaN3
  + 5mg/ml Concanavalin A
* Competent *E. coli* cells (DH5α)
* ddh3O
* DMSO
* dNTPs
* DTT
* EDT
* Ethanol
* Ethidium bromide
* Forward and reverse primers (10 µM)
* Galactose, Glucose, Raffinose
* Gel casting tray and comb
* Gel loading dye, Purple (NEB)
* HEPES (pH 7.6)
* High-Fidelity Buffer
* Ice cold water
* Insert DNA fragment
* KCM
* LB medium
* LiAc
* Ligation Buffer
* Linearized plasmid DNA
* Linearized vector DNA
* Lysis buffer
* Magnesium acetate
* NaOH
* Nitrocellulose membranes
* Nuclease-free water
* PBS
* PBS-T (PBS with Tween-20)
* PEG 3350
* Plasmid DNA
* Potassium acetate
* Purple gel loading dye (6x)
* Q5 High-Fidelity DNA Polymerase
* Q5 Reaction Buffer (5x)
* Primary antibodies:
  + 9E10 α-myc mouse
  + α-FLAG M2 mouse
  + α-HA.11 mouse
  + α-PGK1 mouse
* Protein Marker VI (10 - 245 kDa)
* Protease inhibitors:
  + 0.1M PMSF
  + 0.5M Benzamidine
  + Aprotinin
  + Chymostatin
  + E-64
  + Leupeptin
  + Pepstatin
* rCutSmart Buffer (NEB)
* Restriction enzymes (HF): BstXI, EcoRI, MfeI, MluI, SacI, SphI, XhoI (NEB)
* Running buffer (1x):
  + 0.025 M Tris base
  + 0.192 M Glycine
  + 0.1% SDS
* *S. cerevisiae* cells (strain W303)
* Salmon sperm DNA (ssDNA)
* Sample buffer (with 10% β-mercaptoethanol)
* SC medium with glucose/raffinose/galactose
* Selective agar plates
* Skim milk (2% and 5% w/v in PBS-T)
* Sorbitol
* Sorbitol/LiAc solution (1M/1M)
* Sterile water
* T4 DNA Ligase (NEB)
* T4 DNA Ligase Buffer (10X)
* Taq polymerase (Thermofisher)
* TBST
* TEMED
* Template DNA
* Transfer Buffer:
  + 0.025 M Tris base
  + 0.192 M Glycine
  + 20% Methanol
* Triton-X
* Whatman paper
* Yeast cultures (induced and control)
* Yeast synthetic dropout media (-LEU, -TRP, -URA)
* YPD medium

### Kits

* DC Protein Assay Kit (Bio-Rad)
* Gel Extraction Kit (Qiagen, NEB)
* Monarch Spin Plasmid Miniprep Kit (NEB)
* PCR Clean Up Kit (Qiagen, NEB)

### Equipment & Instruments

#### Laboratory Equipment

* 96-well plate
* Amersham Typhoon laser scanner
* Bead beating device (30 oscillations/s)
* Cover slips
* Culture tubes
* Electrophoresis chamber and power supply
* Eppendorf centrifuge 5425 R
* Erlenmeyer Flask
* Falcon Tubes (15 mL, 50 mL)
* Gel documentation system (Bio-Rad Gel Doc XR+)
* Gloves (nitrile)
* Heidolph Rotamax 120
* Incubator (30 °C and 37 °C)
* Microcentrifuge tubes (1.5 mL, 2 mL)
* Microscope slides
* Microwave
* Mini-PROTEAN Tetra Cell system (Bio-Rad)
* NanoPhotometer® NP80
* Nikon ECLIPSE Ti2 microscope
* Parafilm
* Petri dishes
* Pipettes and tips (sterile)
* PCR tubes (0.2 mL)
* Shaker
* Silica beads (0.1 mm, 500 µL)
* Spectrophotometer (for OD₆₀₀ measurement)
* TECAN Spark® microplate reader
* Thermoblock
* Thermocycler (Bio-Rad T100)
* Thermo Scientific Multifuge X Pro series centrifuge
* UV transilluminator
* Vortex mixer
* VWR Perfect Blue Power Supply Universal
* Water bath (42 °C)
* Well plates (384-well and general)

## References

Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P., & Cardona, A. (2012a). Fiji: An open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. https://doi.org/10.1038/nmeth.2019

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# Results

![Results](https://static.igem.wiki/teams/5690/siteheader/headers-results.webp)
![](https://static.igem.wiki/teams/5690/siteheader/lab-connie-pointing.webp)

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* [DNA Constructs](#dna-constructs)
* [Microscopy Images](#microscopy-images)
* [Protein Analysis](#protein-analysis)

Here you can find an overview of our experimental results.
We present the construction and validation of our DNA constructs, show microscopy imaging of yeast colonies expressing our TRAPS and Pumby systems, and finally have protein expression and analysis results.
We highlight how the constructs were verified, how the TRAPS systems behaves *in vivo*, and how protein expression was quantified across different conditions.

## DNA Constructs

![Figure 1](https://static.igem.wiki/teams/5690/results/bild6.webp)

### Figure 1: Cutting of pAG304GAL-ccdB and pAG304GPD-ccdB

**Figure 1.** Agarose gel electrophoresis of restriction digestion products. (1 % agarose in 1× TAE buffer, stained with EtBr). Vectors pAG304GAL-ccdb and pAG304GPD-ccdB cut with different restriction enzymes. Gene Ruler 1kb Ladder is used as a size marker. Control is the uncut vector.

Figure 1 shows Agarose gel electrophoresis which the presence of the empty pAG-304GAL-ccdB and pAG304GPD-ccdB vector at the size of 6.7 kb and the successful cutting with the enzymes Bsu361 and Xho1. Cutting with BstX1 and Not1 show unwanted cutting patterns. 1kb Ladder is used as a size marker. Control is the uncut vector.
The successful cutting with Bsu361 and Xho1 indicates that these enzymes can be used for further cloning steps, while the unwanted cutting patterns with BstX1 and Not1 suggest that these enzymes are not suitable for our purposes.
By using the enzymes Bsu361 and Xho1 for further cloning steps, we can ensure that our constructs are assembled correctly and efficiently.
The presence of the empty vector at the correct size also confirms that our plasmid preparation was successful and that we have a good starting point for cloning a conditional mCherry under a GAL-Promoter for our first RNA target. The pAG304 vector is a genomically integrating vector to ensure the yeast strain does not lose the plasmid.

![Figure 2](https://static.igem.wiki/teams/5690/results/bild5.webp)

### Figure 2: Cutting of pAG304-mCherry

**Figure 2.** Agarose gel electrophoresis of restriction digestion products. (1 % agarose in 1× TAE buffer, stained with EtBr). Vector pAG304-mCherry cut with the restriction enzymes Mfe1 and Bsu361. 1kb Ladder is used as a size marker. Control is the uncut vector.

Figure 2 shows an agarose gel electrophoresis of plasmid DNA obtained from miniprep of successfully transformed E. coli colonies showing a band corresponding to the expected size of the construct pAG304-ymCherry (6,4kb). The ymCherry was previously successfully cloned into the vector.
The plasmid carries the mCherry gene cloned under control of a conditional GAL promoter.
Plasmids were subsequently sent for sequencing, which confirmed the correct sequence of the construct.
The yeast strain containing this construct was used for further integrating our TRAPS system to target the mCherry RNA.

![Figure 3](https://static.igem.wiki/teams/5690/results/bild1.webp)

### Figure 3: Cutting of pAG305

**Figure 3.** Agarose gel electrophoresis of isolated pAG305. (1 % agarose in 1× TAE buffer, stained with EtBr). Vector pAG305 after cutting with SacI and MluI.
1kb Ladder is used as a size marker.

Figure 3 confirm the successful isolation of the pAG305 vector without the ccdb cassette, which is essential for our cloning and transformation strategy. pAG305 is a genomically integrating vector that allows for stable integration of our genes of interest into the yeast genome. The presence of a clear band at the expected size of 5,5 kb indicates that the vector was correctly cut. Bands visible at ~1,6kb and ~1kb confirm the cut out fragment (combined size of ~2,6kb corresponding to the size of the cut out including the ccdb cassette).

![Cas13 V2 Results](https://static.igem.wiki/teams/5690/results/bild2.webp)

### Figure 4: Cutting of pAG416

**Figure 4.** Agarose gel electrophoresis of isolated pAG416. (1 % agarose in 1× TAE buffer, stained with EtBr). Vector pAG416 after being cut with SacI and MluI. 1kb Ladder is used as a size marker.

Figure 4 confirms the successful isolation of the pAG416 vector.
The pAG416 vector is a high-copy plasmid that allows for strong expression of our genes of interest in yeast cells. We will further use it to integrate our TRAPS system to target the mCherry RNA. The presence of a clear band at the expected size of ~5kb indicates a successful digestion. Bands are also visible at ~1kb and ~1.6kb confirming the correct size of the cut out fragment (combined size of ~2.6kb).

![]()
×

![Cas13 Wild Type](https://static.igem.wiki/teams/5690/results/bild3.webp)

### Figure 5: Isolation of Cas13-TRAPS Inserts

**Figure 5.** Agarose gel electrophoresis of Inserts and destination vector. (1 % agarose in 1× TAE buffer, stained with EtBr). Empty Vector pAG-416 cut with SacI and MluI, Insert Im2-MiniCas13.X1 and Insert GFP-E9-Tetra. 1kb Ladder is used as a size marker.

Figure 5 shows the successful isolation of our inserts Im2-MiniCas13.X1 and GFP-E9-Tetra after restriction digestion and gel extraction. The presence of clear bands at the expected sizes of ~3.2 kb for Im2-MiniCas13.X1 and ~1.2 kb for GFP-E9-Tetra indicates that our cloning strategy worked. The empty vector pAG-416 linearized with Mlu1 shows a band at the expected size of ~5 kb (+bands at the expected combined size (~2.6kb) of the cut out fragments), confirming that the vector is ready for ligation with our inserts.
These inserts were then ligated together into the pAG-416 vector to create our TRAPS system.

![Cas13 V1 Results](https://static.igem.wiki/teams/5690/results/grna-gel.webp)

### Figure 6: Isolation of individual gRNAs

**Figure 6.** Agarose gel electrophoresis of gRNA inserts isolated. Gel was run after gRNA inserts were cut with gRNA1: SacI and EcorI, gRNA2: EcoRI and SphI, gRNA3: SphI and NotI and gRNA4: NotI and MluI to create compatible ends for ligation. (1 % agarose in 1× TAE buffer, stained with EtBr). 1kb Ladder is used as a size marker.

Figure 6 shows the successful cutting and isolation of our gRNA inserts at the size of ~250b. These inserts were then isolated via gel extraction and ligated into the pAG-305 vector to create our gRNA collection for our TRAPS system.

![Cas13 V1 Results](https://static.igem.wiki/teams/5690/results/bild4.webp)

### Figure 7: Isolation of Pumby-TRAPS plasmids

**Figure 7.** Agarose gel electrophoresis of Pumby-Plasmids after ligation of the inserts into the vector and restriction digestion of the vector for linearization with MluI. (1 % agarose in 1× TAE buffer, stained with EtBr). pAG416 Vector with pumby1 insert and pAG305 Vector with pumby 2 insert1kb Ladder is used as a size marker.

Figure 7 shows the successful isolation of our Pumby inserts cloned into the respective vectors after restriction digestion. The presence of clear bands at the expected sizes of ~8kb for pAG416-Pumby1 and ~10kb for Pumby2 indicates that our cloning strategy worked. In the pAG416-pumby1 samples is another band visible at ~0.5kb. This is because the vector was cut with Sac1 which cuts twice in the vector. The combined size of the drop out and the main band corresponds to the expected size of the vector of ~8.5kb thereby confirming that the vectors are ready for transformation into our yeast strain.
The pAG305 is thereby a genomically integrating vector to ensure the yeast strain does not lose the plasmid. The pAG416 is a high-copy plasmid that allows for strong expression of our genes of interest in yeast cells.

![Pumby Results](https://static.igem.wiki/teams/5690/results/gel-new2.webp)

### Figure 8: Second version of Cas13-TRAPS Inserts

**Figure 8.** Agarose gel electrophoresis of the second version of Cas13-TRAPS Inserts cut with MluI/SacI and SphI. (1 % agarose in 1× TAE buffer, stained with EtBr). Insert Im2-RfxCas13d cut with Mlu1 and Sph1, and Insert GFP-E9-Tetra cut with Sph1 and Sac1. 1kb Ladder is used as a size marker.

Figure 8 shows the successful isolation of our second version of Cas13-TRAPS insert Im2-RfxCas13d and the previously used insert GFP-E9-Tetra after restriction digestion. The presence of clear bands at the expected sizes of ~4 kb for Im2-RfxCas13d (marked with red arrow) and ~2 kb for GFP-E9-Tetra show the successful isolation of our inserts.
These inserts were then ligated together into the pAG-416 vector to create our updated TRAPS system with the RfxCas13d variant. Besides the band of the empty vector, there are more bands visible at different sizes. This could be due to incomplete cutting of the vector or binding activity of the enzymes or of the inserts themselves.
After this gel electrophoresis a gel extraction of the main band was performed to ensure that only the correct band is used for the ligation. The ligation and transformation of *E. coli* showed successfully transformed colonies which were further verified via plasmid mini prep and sequencing.

## Microscopy Images

![Figure 9](https://static.igem.wiki/teams/5690/results/mcherry-expression-bsui-2.webp)

### Figure 9: mCherry expressing yeast colonies (BsuI)

**Figure 9.** Microscopy of gal-induced an uninduced yeast colonies after transformation with the genomically integrating plasmid linearized with BsuI. Total of four different colony microscopies are displayed.

![Figure 10](https://static.igem.wiki/teams/5690/results/bsui.avif)

### Figure 10: Total cell fluorescence distributions (BsuI)

**Figure 10.** Histograms showing the distribution of total cell fluorescence in uninduced and gal-induced yeast cells transformed with the BsuI linearized plasmid.

![Figure 10](https://static.igem.wiki/teams/5690/results/mcherry-expression-mfei-2.webp)

### Figure 11: mCherry expressing yeast colonies (MfeI)

**Figure 11.** Microscopy of gal-induced an uninduced yeast colonies after transformation with the genomically integrating plasmid linearized with BsuI. Total of four different colony microscopies are displayed.

![Figure 12](https://static.igem.wiki/teams/5690/results/mcherry-expression-mfei-2.webp)

### Figure 12: mCherry expressing yeast colonies (MfeI)

**Figure 12.** Microscopy of gal-induced an uninduced yeast colonies after transformation with the genomically integrating plasmid linearized with BsuI. Total of four different colony microscopies are displayed.

As described above, we linearized the mCherry-containing plasmid and transformed the linearized plasmid into the W303 S. cerevisiae strain.
After transformation, a set of the resulting colonies was imaged and a candidate for further experimentation was selected.
Linearization with both BsuI and MfeI restriction enzymes resulted in successful transformation for most of the colonies, as indicated by the homogeneous fluorescence when galactose medium was used for cell growth (Figure 9,11).
In addition, we plotted histograms of total cell fluorescence in uninduced and gal-induced yeast cells (Figure 10, 12) using the image analysis code (see Software), further showing ymCherry expression upon gal induction.
For further experiments with our TRAPS constructs, a colony showing strong fluorescence from the transformation linearized with MfeI was selected.

![Figure 13](https://static.igem.wiki/teams/5690/results/figure10-microscopy-of-traps-cas13-v1.webp)

### Figure 13: Microscopy of TRAPS-Cas13 (dCas13X.1)

**Figure 13.** Microscopy of the first TRAPS-Cas13 iteration containing miniaturized dCas13X.1. A-C: TRAPS-Cas13 with gRNA targeting mCherry grown in glucose medium. D-F: TRAPS-Cas13 with gRNA targeting mCherry grown in galactose medium. G-I: TRAPS-Cas13 without gRNA targeting mCherry grown in glucose medium. J-L: TRAPS-Cas13 without gRNA targeting mCherry grown in galactose medium. M-O: Control with yeast cells containing the GFP without proteins fused. P-R: Control of WT yeast

The first version of the TRAPS-Cas13 proteins with the miniaturized dCas13x.1 variant was transformed and introduced into the yeast strain containing the galactose-dependent mCherry. Green droplets within the cells were visible with and without galactose induction, as well as with and without gRNAs (Figure 13C, F, I, L), indicating that droplet formation is independent of RNA’s presence. Droplet formation was not observed in the yeast strain where only GFP, without any fusion, was transformed.

![Figure 14](https://static.igem.wiki/teams/5690/results/figure-11-traps-cas13-in-wt.webp)

### Figure 14: Microscopy of TRAPS-Cas13 in WT yeast

**Figure 14.** Microscopy of TRAPS-Cas13 transformed into WT yeast and controls. A-C: TRAPS-Cas13 transformed into WT yeast. D-F: Control with yeast cells containing the GFP without proteins fused. G-I: Control of WT yeast.

To see if the droplet formation is completely independent of the presence of the target RNA, we transformed our constructs into W303 WT yeast, which do not contain the gene for mCherry. Even when transformed into yeast lacking the mCherry gene, droplet formation still occurred (Figure 14C), showing that no mCherry RNA is needed for this, further strengthening the hypothesis that our proteins aggregate

![Figure 15](https://static.igem.wiki/teams/5690/results/figure-12-only-tetramer-in-wt.webp)

### Figure 15: Microscopy of TRAPS-Cas13 tetramerization domain

**Figure 15.** Microscopy of the TRAPS-Cas13 tetramerization domain transformed into yeast and controls. A-C: TRAPS-Cas13 tetramerization unit transformed into WT yeast. D-F: Control with yeast cells containing the GFP without proteins fused. G-I: Control of WT yeast.

To determine which of the two proteins introduced into the cell aggregates, we separated the cassettes, added the respective missing restriction sites, and transformed the tetramerization unit individually. When only the tetramerization unit was transformed, we observed distributed cytosolic GFP fluorescence (Figure 15C) without droplets, indicating that the IM2-Cas13 unit is the driver of aggregation.

![Figure 16](https://static.igem.wiki/teams/5690/results/figure-13-microscopy-of-traps-cas13-v1.webp)

### Figure 16: Second version of TRAPS-Cas13 with RfxCas13d

**Figure 16.** Microscopy of the second version of TRAPS-Cas13 containing the RfxCas13d variant. A-C: TRAPS-Cas13 with gRNA targeting mCherry grown in glucose medium. D-F: TRAPS-Cas13 with gRNA targeting mCherry grown in galactose medium. G-I: TRAPS-Cas13 without gRNA targeting mCherry grown in glucose medium. J-L: TRAPS-Cas13 without gRNA targeting mCherry grown in galactose medium. M-O: Control with yeast cells containing the GFP without proteins fused. P-R: Control of WT yeast.

After determining that the Cas13 unit is the driver of aggregation, we integrated a second version of the IM2-Cas13 protein using an RfxCas13d variant. We again observed droplet formation independent of the presence of our target RNA (Figure 16C, F, I, L). Similar aggregates formed as in the first TRAPS-Cas13 iteration. We realized that N-terminal fusion of this specific Cas13 version leads to aggregation (Han et al., 2020) and therefore designed a third iteration of the TRAPS-Cas13 system, which is currently in progress but has not yet been tested.

![Figure 17](https://static.igem.wiki/teams/5690/results/figure-14-traps-pumby.webp)

### Figure 17: Microscopy of TRAPS-Pumby System

**Figure 17.** Microscopy of the TRAPS-Pumby System.1. A-C: TRAPS-Pumby targeting mCherry grown in glucose medium. D-F: TRAPS-Pumby targeting mCherry grown in galactose medium. M-O: Control with yeast cells containing the GFP without proteins fused. P-R: Control of WT yeast.

We also tested the TRAPS-Pumby system. Similar to the TRAPS-Cas13 system, droplets were observed independent of the target RNA (Figure 17C, F). The cause of this droplet formation is likely, again, aggregation of the Pumby domains or general instability of the fusion proteins, although this was not experimentally proven.

## Protein Analysis

![Figure 18](https://static.igem.wiki/teams/5690/results/sds-page-gfp-e9-tetra.webp)

### Figure 18: SDS-Page gel with GFP-signal from GFP-E9-Tetra protein constructs in yeast.

**Figure 18.** Lysates were prepared from the yeast cell lines W303 WT (WT), W303 mCherry Cas13 gRNA(constructs +gRNA) and W303 mCherry Cas13 (constructs -gRNA). 12% SDS Page gel after running 40 min constant 200 V. Molecular weight protein marker VI (10 - 245) prestained was used. First four lanes show cell lysate from W303 mCherry Cas13 gRNA: firstly the Total (T) cell lysate, first a not-heated, then heated sample. Secondly Supernatant (S) fraction, first not heated, then heated sample. Next four samples show cell lysate from W303 mCherry Cas13: T, fist not-heated, then heated sample, S non-heated, then heated sample. Lastly WT yeast cell lysate as control. GFP-Fluorescence signal at ~45 kDa can be seen in non-heated samples of W303 mCherry Cas13 gRNA and W303 mCherry Cas13.

Lysates were prepared from the yeast cell lines W303 WT (WT), W303 mCherry Cas13 gRNA(constructs +gRNA), where gRNA is expressed alongside the constructs of interest and W303 mCherry Cas13 (constructs -gRNA), where no gRNA is expressed. Equal amounts of total protein were loaded for all lanes. Molecular weight protein marker VI (10 - 245) prestained was used. The first four lanes correspond to lysates from W303 mCherry Cas13 gRNA. Total cell lysate (T) was loaded first, with a non-heated (N) and then a heated (H) sample, followed by the supernatant fraction (S), also as a non-heated and heated sample. The next four lanes show lysates from W303 mCherry Cas13, in which the constructs are expressed without gRNA, again displaying total lysate and supernatant fractions in non-heated and heated conditions. The final lane contains lysate from WT yeast as negative control. A 12 % SDS Page gel was run for 40 min at constant 200 V. Afterwards gel was imaged in Amersham Typhoon. Distinct fluorescence bands can be detected in non-heated samples of W303 mCherry Cas13 gRNA and W303 mCherry Cas13 at the size of ~ 45 kDa (Figure 18). Next to the expected lanes a weak signal can be seen at ~27 kDa in TN and SN of mCherry Cas13.
Afterwards the proteins from the gel were transferred onto a nitrocellulose membrane

![Figure 19](https://static.igem.wiki/teams/5690/results/oldcas-pgk1-blot.webp)

### Figure 19: Expression of Cas-Im2 and GFP-E9-Tetra protein constructs in yeast.

**Figure 19.** Lysates were prepared from the yeast cell lines W303 WT (WT), W303 mCherry Cas13 gRNA(constructs +gRNA) and W303 mCherry Cas13 (constructs -gRNA). Molecular weight protein marker VI (10 - 245) prestained was used. A Nitrocellulose membrane was probed with primary anti-Myc-Antibody (1:5000 in 2% skim milk, mouse) against Myc-tagged constructs Im2-Cas13 and GFP-E9-Tetra and secondary antibody Rabbit Anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk, rabbit, fluorescence detection) was used. B Nitrocellulose membrane was probed with primary anti-PGK1-Antibody (1:5000 in 2% skim milk, mouse) for detection of housekeeping gene PGK1 (loading control) and secondary antibody Rabbit Anti-Mouse Alexa Fluor 790 (1:10000 in 5% skim milk, rabbit, fluorescence detection) was used.

The image presented was generated by probing the nitrocellulose membrane with anti-Myc-Antibody (1:5000 in 2% skim milk, mouse) for the detection of the constructs Im2-Cas13 and GFP-E9-Tetra and anti-PGK1-Antibody (1:5000 in 2% skim milk, mouse) for detection of housekeeping gene PGK1. The latter served as loading control to normalize the relative expression of the protein constructs between lanes. As secondary antibody Rabbit Anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk, rabbit) was used for fluorescence detection of proteins of interest and Rabbit Anti-Mouse Alexa Fluor 790 (1:10000 in 5% skim milk, rabbit) for detection of PGK1. Distinct bands corresponding to GFP-E9-Tetra (~48 kDa) and Cas13-Im2 (~65 kDa) were observed in the TN and TH samples of gRNA-expressing cell lines (Figure 19A). In the absence of gRNA, both constructs were detectable across all fractions, including TN, TH, SN, and SH. Fluorescence of the loading control PGK1 was detected in all lanes (Figure 19B).

![Figure 20](https://static.igem.wiki/teams/5690/results/bild8.webp)

### Figure 20: Quantification of protein expression.

**Figure 20.** Densitometric analysis was performed using the Gel Analysis tool in Fiji (doi:10.1038/nmeth.2019) on the image in Figure 2. Quantification revealed higher band intensity in cell lines without gRNA compared to cell lines with gRNA expression, higher band intensity in T compared to band intensity in S, higher band intensity of GFP-E9-Tetra in heated samples compared to Cas13-Im2 band intensity.

Densitometric analysis was performed using the Gel Analysis tool in Fiji (doi:10.1038/nmeth.2019). To correct for differences in sample loading, measured band intensity values were normalized to the corresponding loading control PGK1. Since band intensities in gRNA-expressing cell line W303 mCherry Cas13 gRNA did not exceed background levels for both SH and SN fractions, only TH and TN samples were used in analysis for gRNA expressing cell line. In the cell line W303 mCherry Cas13, without gRNA expression all samples (TH, TN, SH, SN) produced a measurable band signal.

| Sample | Condition | Cas13-Im2 signal | GFP-E9-Tetra signal | PGK1 signal |
| --- | --- | --- | --- | --- |
| **W303 mCherry Cas13 gRNA** | TN | 1.466,426 | 1.176,820 | 10.511,095 |
| TH | 3.452,004 | 6.299,075 | 17.148,238 |
| SN | - | - | 11.299,288 |
| SH | - | - | 14.743,359 |
| **W303 mCherry Cas13** | TN | 4.868,660 | 7.365,782 | 16.956,995 |
| TH | 5.447,125 | 16.718,652 | 17.547,459 |
| SN | 2.206,891 | 1.868,477 | 16.412,752 |
| SH | 1.156,527 | 1.523,305 | 18.744,652 |
| **WT** | TH | - | - | 19.287,116 |

**Table 1. Comparison cell lines with gRNA vs. without gRNA:**

Corresponding samples in both cell lines were compared and analysis revealed a constantly higher signal in in cells without gRNA compared to cell lysate with gRNA. A 2-fold higher TN GFP-E9-Tetra band intensity a 3.8 fold higher TN Cas13-Im2 band intensity, a 1.5 fold higher TH GFP-E9-Tetra and a 2,6 fold higher TH Cas13-Im2 band intensity in cell lines without gRNA expression in comparison to cell lines with gRNA expression was measured.

 **2. Comparison of Total (T) vs. Supernatant (S) fraction:**

Corresponding samples in both cell lines were compared, as no band intensity higher than the background signal was measured in cell lines with gRNA expression for both SH and SN, only cell lines without gRNA expression were compared. For GFP-E9-Tetra samples a 3.8 fold higher band intensity was measured in TN than in SN and a 11.7 fold higher band intensity was measured in TH than in SH. For Cas13-Im2 a 2.1times higher band intensity was measured in TN than in SN and a 5 fold higher band intensity was measured in TH than in SH.

**3. Comparison of Cas13-Im2 vs GFP-E9-Tetra expression:**

The band intensity for the relative expression of the two constructs in each sample was analysed and signal intensity was higher for the GFP-E9-Tetra construct in comparison to the Cas13-Im2 construct in cell lines with gRNA for TH by a factor of 1.8, and for cell lines without gRNA in TN by a factor of 1.5, for TH by a factor of 3.1, for SH by a factor of 1.3. In the other samples the signal intensity was higher in Cas13-Im2 than GFP-E9-Tetra in by 1.2 in TN (with gRNA) and in SN (without gRNA).

![Figure 21](https://static.igem.wiki/teams/5690/results/sds-page-gfp-e9-tetra-2.webp)

### Figure 21: SDS-Page gel with GFP-E9-Tetra protein constructs in yeast

**Figure 21.** Lysates were prepared from the yeast cell lines W303 WT (WT), W303 mCherry Cas13. 12% SDS Page gel after running 40 min constant 200 V. Molecular weight protein marker VI (10 - 245) prestained was used. Firstly WT yeast cell lysate as control. Next four lanes show cell lysate from W303 mCherry Cas13: firstly the Total (T) cell lysate, first a heated, then non-heated sample. Secondly Supernatant (S) fraction, with first heated and then non-heated sample. GFP-Fluorescence signal at ~65 kDa can be seen in non-heated samples of W303 mCherry.

Lysates were prepared from the yeast cell lines W303 WT, W303 mCherry Cas13. Equal amounts of total protein were loaded for all lanes. Molecular weight protein marker VI (10 - 245) prestained was used. The first lane contains lysate from WT cells, serving as a negative control. Next four lanes show lysates from W303 mCherry Cas13: first, the total (T) fraction, with non-heated (N) and heated (H) samples applied, followed by the soluble (S) fraction, also with non-heated and heated samples. A 12 % SDS Page gel was run for 40 min at constant 200 V. Afterwards gel was imaged in Amersham Typhoon. Distinct GFP fluorescence bands can be detected in non-heated samples of T and S at approximated molecular weight of 65 kDa and no other bands were observed (Figure 21). Afterwards the proteins from the gel were transferred onto a nitrocellulose membrane.

![Figure 22](https://static.igem.wiki/teams/5690/results/newcas-pgk1-blot.webp)

### Figure 22: Western Blot of new Cas13 constructs.

**Figure 22.** Molecular weight protein marker VI (10 - 245) prestained was used. A) Nitrocellulose membrane was probed with primary anti-Myc-Antibody (1:5000 in 2% skim milk, mouse) against Myc-tagged constructs Im2-Cas13 and GFP-E9-Tetra and in B) anti-PGK1-Antibody (1:5000 in 2% skim milk, mouse) for detection of housekeeping gene PGK1 (loading control). Secondary antibody Rabbit Anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk, rabbit, fluorescence detection) was used. Lanes: WT control, followed by total (T) and soluble (S) fractions, with heated (H) and non-heated (N) samples as indicated.

Western blot analysis was performed to assess the expression of Myc-tagged protein constructs in yeast cell lysates. Proteins were transferred to a nitrocellulose membrane and probed with anti-Myc antibody (1:5000 in 2% skim milk, mouse) to detect the constructs and anti-PGK1 antibody (1:5000 in 2% skim milk, mouse) for the housekeeping protein pGK1, which served as a loading control. Detection was performed using Rabbit anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk) for fluorescence imaging. Molecular weight reference was provided by Protein Marker VI (10–245 kDa, prestained). Distinct fluorescence bands can be detected in TH and TN samples at approximated size of 65 kDa (Figure 22, top, middle two lanes). No second set of bands and no fluorescent signal were observed in the solube samples (Figure 22, top, outer right lanes). Fluorescence of the loading control pGK1 was detected in all lanes (Figure 22, bottom).

![Figure 23](https://static.igem.wiki/teams/5690/results/pum-gel.webp)

### Figure 23: SDS-Page gel with GFP-PUM2-Tetra and GFP-PUM3-Tetra protein constructs in yeast.

**Figure 23.** Lysates were prepared from the yeast cell lines W303-mCherry-416-Pumb2y. 12% SDS Page gel after running 40 min constant 200 V. Molecular weight Protein Marker VI (10–245 kDa, prestained) is shown in first lane. Following lane shows SN followed by TN. Two distinct fluorescent bands at ~65 kDa and ~67 kDa and lighter signal at ~37kDa, ~39 kDa and ~25 kDa.

Lysates were derived from yeast cell lines W303-mCherry-416-Pumb2y, expressing the constructs of interest. Equal amounts of protein were loaded in all lanes. Molecular weight protein marker VI (10 - 245) prestained was used. The first lane after molecular weight marker contains non-heated Supernatant fraction from W303-mCherry-416-Pumb2y lysate, afterwards the non-heated Total fraction of W303-mCherry-416-Pumb2y lysate is applied. A 12 % SDS Page gel was run for 40 min at constant 200 V. Afterwards gel was imaged in Amersham Typhoon. Distinct GFP fluorescence bands can be detected in samples of T and S at approximated size of ~65 kDa and ~67 kDa. A lighter and less distinct signal can also be spotted in both T and S at ~37kDa, ~39 kDa and ~25 kDa. Afterwards the proteins from the gel were transferred onto a nitrocellulose membrane.

![Figure 24](https://static.igem.wiki/teams/5690/results/pumby-pgk1-blot.webp)

### Figure 24: Failed western blot of Pumby constructs from yeast.

**Figure 24.** Nitrocellulose membrane was probed with primary anti-Myc-Antibody (1:5000 in 2% skim milk, mouse) against Myc-tagged constructs PUM1-Dim and PUM4-Tetra, anti-HA-Antibody (1:5000 in 2% skim milk, mouse) against HA-tagged GFP-PUM2-Tetra, anti-Flag-Antibody (1:5000 in 2% skim milk, mouse) against Flag-tagged GFP-PUM3-Tetra and anti-PGK1-Antibody (1:5000 in 2% skim milk, mouse) for detection of housekeeping gene PGK1 (loading control). Secondary antibody Rabbit Anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk, rabbit, fluorescence detection) was used. Molecular weight protein marker VI (10 - 245) prestained was used. First two membranes were incubated with anti-Myc primary antibody. Second two membranes were incubated with anti-Flag primary antibody. Last two membranes were incubated with anti-HA-primary antibody. No signal from immunostaining is visible.

Western blot analysis was performed to assess the expression of Myc-tagged protein constructs PUM1-Dim and PUM4-Tetra, Flag-tagged protein construct GFP-PUM3-Tetra and HA-tagged protein construct GFP-PUM2-Tetra in yeast cell lysates. Proteins were transferred to a nitrocellulose membrane and probed with anti-Myc antibody (1:5000 in 2% skim milk, mouse) to detect Myc-tagged constructs PUM1-Dim and PUM4-Tetra, with anti-HA-Antibody (1:5000 in 2% skim milk, mouse) to detect HA-tagged GFP-PUM2-Tetra and anti-Flag-Antibody (1:5000 in 2% skim milk, mouse) to detect Flag-tagged GFP-PUM3-Tetraand. Anti-PGK1 antibody (1:5000 in 2% skim milk, mouse) was used for detection of the housekeeping protein pGK1, which served as a loading control. Detection was performed using secondary antibody Rabbit anti-Mouse Alexa Fluor 488 (1:10000 in 5% skim milk) for fluorescence imaging. Molecular weight reference was provided by Protein Marker VI (10–245 kDa, prestained). Membranes were cut into pieces, so that each membrane could be incubated into a different primary antibody. First two membranes were incubated in anti-Myc-primary antibody: the first Myc-incubated membrane contains non-heated samples, second membrane contains heated samples, each first Total (T) fraction then Supernatant (S). Second two membranes were incubated in anti-HA-antibody: first HA-incubated membrane shows non-heated samples, second membrane shows heated samples, each first T then S. Third two membranes were incubated in anti-Flag-antibody: first Flag-incubated membrane shows non-heated samples, second membrane shows heated samples, each first T then S. There was no immunostaining signal (Figure 24A) and fluorescence of the loading control pGK1 was detected in all lanes (Figure 24B).

## References

Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P., & Cardona, A. (2012a). Fiji: An open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. https://doi.org/10.1038/nmeth.2019

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