iGEM 2020 — Team UCopenhagen

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# Team:UCopenhagen/Notebook

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Project

[Description](https://2020.igem.org/Team%3AUCopenhagen/Description)
[Design](https://2020.igem.org/Team%3AUCopenhagen/Design)
[Implementation](https://2020.igem.org/Team%3AUCopenhagen/Implementation)
[Poster](https://2020.igem.org/Team%3AUCopenhagen/Poster)

Science

[Engineering Success](https://2020.igem.org/Team%3AUCopenhagen/Engineering)
[Modeling/Simulation](https://2020.igem.org/Team%3AUCopenhagen/Model)
[Parts Overview](https://2020.igem.org/Team%3AUCopenhagen/Parts)
[Results](https://2020.igem.org/Team%3AUCopenhagen/Results)
[Protocols](https://2020.igem.org/Team%3AUCopenhagen/Experiments)
[Notebook](https://2020.igem.org/Team%3AUCopenhagen/Notebook)
[Safety](https://2020.igem.org/Team%3AUCopenhagen/Safety)

Human Practices

[Integrated Human Practices](https://2020.igem.org/Team%3AUCopenhagen/Human_Practices)
[Entrepreneurship](https://2020.igem.org/Team%3AUCopenhagen/Entrepreneurship)
[Education](https://2020.igem.org/Team%3AUCopenhagen/Education)

Collaborations

[Collaborations](https://2020.igem.org/Team%3AUCopenhagen/Collaborations)
[Partnership](https://2020.igem.org/Team%3AUCopenhagen/Partnership)
[Our Contribution](https://2020.igem.org/Team%3AUCopenhagen/Contribution)

Judging

[Judging Form](https://igem.org/2020_Judging_Form?id=3617)
[Medals](https://2020.igem.org/Team%3AUCopenhagen/Medals)

Team

[Team Members](https://2020.igem.org/Team%3AUCopenhagen/Team)
[Attributions](https://2020.igem.org/Team%3AUCopenhagen/Attributions)
[Sponsors](https://2020.igem.org/Team%3AUCopenhagen/Sponsors)

![](https://static.igem.org/mediawiki/2020/9/99/T--UCopenhagen--notebook-cover.png)

##### Notebook

Due to safety regulations in the lab about contamination and the spread of corona virus, we have recorded all our experiment notes in our lab notebook. This book has lived on our lab bench and has been our dear companion throughout our whole laboratory process. We hope you enjoy it!

## About us

Comprising this team are nine ambitious students from various walks of life, who have come together to produce CIDosis - a non-invasive device that can help people with chronic inflammatory diseases visualize their level of inflammation.

## Helpful Links

[The Team](https://2020.igem.org/Team%3AUCopenhagen/Team)
[Promotional Video](https://video.igem.org/videos/watch/3b21e193-aefc-4d45-a65d-6fa4f7a3b93a)
[Poster](https://2020.igem.org/Team%3AUCopenhagen/Poster)
[Medals](https://2020.igem.org/Team%3AUCopenhagen/Medals)

## Address

University of Copenhagen
Thorvaldsensvej 40, Frederiksberg C
Denmark

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# Team:UCopenhagen/Engineering

[![](https://static.igem.org/mediawiki/2020/4/4d/T--UCopenhagen--Logo_Transparent_BG.png)](https://2020.igem.org/Team%3AUCopenhagen)

Project

[Description](https://2020.igem.org/Team%3AUCopenhagen/Description)
[Design](https://2020.igem.org/Team%3AUCopenhagen/Design)
[Implementation](https://2020.igem.org/Team%3AUCopenhagen/Implementation)
[Poster](https://2020.igem.org/Team%3AUCopenhagen/Poster)

Science

[Engineering Success](https://2020.igem.org/Team%3AUCopenhagen/Engineering)
[Modeling/Simulation](https://2020.igem.org/Team%3AUCopenhagen/Model)
[Parts Overview](https://2020.igem.org/Team%3AUCopenhagen/Parts)
[Results](https://2020.igem.org/Team%3AUCopenhagen/Results)
[Protocols](https://2020.igem.org/Team%3AUCopenhagen/Experiments)
[Notebook](https://2020.igem.org/Team%3AUCopenhagen/Notebook)
[Safety](https://2020.igem.org/Team%3AUCopenhagen/Safety)

Human Practices

[Integrated Human Practices](https://2020.igem.org/Team%3AUCopenhagen/Human_Practices)
[Entrepreneurship](https://2020.igem.org/Team%3AUCopenhagen/Entrepreneurship)
[Education](https://2020.igem.org/Team%3AUCopenhagen/Education)

Collaborations

[Collaborations](https://2020.igem.org/Team%3AUCopenhagen/Collaborations)
[Partnership](https://2020.igem.org/Team%3AUCopenhagen/Partnership)
[Our Contribution](https://2020.igem.org/Team%3AUCopenhagen/Contribution)

Judging

[Judging Form](https://igem.org/2020_Judging_Form?id=3617)
[Medals](https://2020.igem.org/Team%3AUCopenhagen/Medals)

Team

[Team Members](https://2020.igem.org/Team%3AUCopenhagen/Team)
[Attributions](https://2020.igem.org/Team%3AUCopenhagen/Attributions)
[Sponsors](https://2020.igem.org/Team%3AUCopenhagen/Sponsors)

![](https://static.igem.org/mediawiki/2020/b/b5/T--UCopenhagen--ES-cover.png)

## Introduction

This page documents our success with iterative engineering of novel G protein alpha subunits in yeast Saccharomyces cerevisiae. Protein modeling in Rosetta took us most of the way, finally accompanied by wet lab experiments. Our efforts followed the traditional engineering design cycle (see figure 1), with multiple
phases of research followed by testing of our models and continuously refining them. Individual steps are noted chronologically as they happened, thus proceeding from relatively simple to more advanced and specialized methods. Finally, the results challenged our initial assumptions about protein interactions.

![](https://static.igem.org/mediawiki/2020/d/d1/T--UCopenhagen--engineering_cycle.png)

**Fig. 1: Expanded engineering cycle.**
We closely followed the expanded engineering cycle in our engineering process.

As described on our [project design page](https://2020.igem.org/Team%3AUCopenhagen/Design),
our most ambitious design called for modification of the alpha subunit of
endogenous yeast G protein. The challenge here was that we wanted to maintain
its natural functionality while expanding it to serving our purposes.

This meant we needed to design a G-alpha that:

* **A)** has a natural-like affinity for the beta/gamma complex of the
  yeast G protein to stay bound to the beta/gamma complex when no signal
  was present, thus inhibiting leaky activation of the pathway.
* **B)** has a low enough affinity for the beta/gamma complex after
  cleavage by our TEV-protease to enable dissociation from
  the beta/gamma complex.

Our work on G-alpha hinged on finding a mutant that preserved natural
function when there was no signal (i.e. no cytokine), and also dissociated appropriately in the presence of a signal.

This page presents all stages of G-alpha engineering in a chronological
order. The progress followed the engineering cycle (design-build-test-learn)
as we iterated our designs several times. As we utilized primarily dry-lab
experiments, the whole cycle often spans over each stage. Every stage was
informed by all the previous ones.

## Stage One - Design

We based our design choices on knowledge about the interaction interface
of the alpha and beta subunits in bovine G protein, as we found no crystal
structure of yeast G protein. Immediately, we noticed that the yeast G
protein contained an ‘insertion sequence’ (highlighted in figure 2) that
other G proteins did not. This indicated that the sequence was not essential
for the general functionality of the G-alpha subunit. As such, we chose to
search this sequence for a mutagenesis site.

Based on literature review, we identified the residue sequence that the TEV-protease cleaves: **EXLYΦQφ**, where X is any residue, Φ is any large or medium hydrophobic residue, and φ is any small hydrophobic or polar residue. For the sake of interpretability of experiments, however, it was necessary to introduce a single sequence type at all times, as the TEV protease may display different proteolytic rates depending on the recognition site (Dougherty et al., 1989). Following extensive literature search, we opted for a widely used sequence: **ENLYFQG**.

![](https://static.igem.org/mediawiki/2020/1/13/T--UCopenhagen--insertion-seq.png)

**Figure 2: Amino acid sequence of the *S. cerevisiae* G protein alpha subunit.**

The highlighted sequence was initially selected as an optimal region for mutagenesis.

## Stage Two - Design-build

We received a file with the empirically observed binding interface of
bovine G-alpha and G-beta through our supervisors. We could translate
known characteristics of the bovine G protein into what we expected to
see in the yeast G protein because of the similarity of their sequences.
Based on the biophysical properties of different amino acids, we identified
what we believed were the most essential residues for the binding between the
alpha and beta subunits (see figure 3).

![](https://static.igem.org/mediawiki/2020/6/63/T--UCopenhagen--bovine-g.png)

**Figure 3: Crystal structure of the G protein complex.**

The red-colored residues are the ones we identified as the most important for binding of the alpha and beta subunits to each other.

## Stage Three - Test & Learn

Based on suggestions from the Sinisens (Aalto-Helsinki iGEM2020 team), we chose to
use Rosetta for our protein engineering. Rosetta is a powerful tool for
mapping and characterizing protein structures and interactions, and, we
theorized, good for mutagenetic analyses of our G alpha protein.

Using Rosetta, we supplemented our own analysis with an alanine scan,
which provided us with quantitative predictions of the role of selected residues
in the G-alpha in terms of binding to G-beta. Rosetta's alanine scan is
a script that calculates how the Gibb's free energy of two proteins change
upon changing a single amino acid residue to an alanine (see [modeling](https://2020.igem.org/Team%3AUCopenhagen/Model) for more details).

The results of the alanine scan confirmed our intuitions to a good extent,
and offered several valuable insight points that led to the refinement of
our G-alpha designs. As illustrated on figure 4, residue number 227 was
predicted as the single most important residue in the protein-protein interaction
between the alpha and beta subunits. This result, among mutagenetic data
found in literature about what the most functionally important amino acid
residues in G-alpha are , guided us in discerning between which ones we
could change, and which ones were critical for the correct functionality of G-alpha.

![](https://static.igem.org/mediawiki/2020/1/11/T--UCopenhagen--Alanine_Scan.png)

**Figure 4: Change in Gibbs free energy (in kcal/mol) generated by replacing the given residues with alanine.** As Gibbs free energy correlates with a change in affinity, it appears that residue 227 is the most important one for the binding interface of G-alpha and G-beta proteins.

## Stage Four - Redesigning, Building, Testing & Learning

By compiling the information we had received so far, we isolated fitting mutation points in G alpha, where we

1. **1.** Avoid changing residues that lead to loss of function in G alpha (Gladue, D. P. 2008).
2. **2.** Avoid changing residues identified as very important through Rosetta's alanine scan.
3. **3.** Change residues located in the insertion sequence and other non-conserved residues, by comparing the yeast G-alpha to the bovine G-alpha.
4. **4.** Change residues located in loops based on visual analysis in Pymol.

![](https://static.igem.org/mediawiki/2020/0/05/T--UCopenhagen--localization-prediction.png)

**Figure 5: Localization prediction of modified alpha subunits.**

Cello was used to predict the localization of our three GPA1-versions.
For all three versions, an increased cytoplasmic reliability score was observed compared to the wildtype.

With this information in mind, we designed three version of G-alpha, each with
varying degrees of modifications. Using SwissProt protein modeling, we then
confirmed that none of our edits lead to unexpected conformational changes.
In addition, we used Cello to predict protein localization to the plasma membrane,
as this is crucial for the functionality of our biosensor. As shown on figure 5,
our designs yielded even better localization scores than the wildtype.
As shown on the figure, some cytoplasmic localization was also predicted,
but this was desirable as certain enzymes in the cytoplasm are known to modify
G-alpha for it to go to the membrane.

## Stage Five - Design, Build, Test & Learn

Here we faced the reality of laboratory experimentation. Three G-alpha
mutants with many diverse mutations could lead to lots of ambiguity
as the laboratory results could be hard to interpret due to inability
to monitor what mutation had a profound impact. Time constraints in the
wet lab motivated us to limit the number of possible mutation points.
We eliminated point mutations and kept only cleavage sites for TEV
protease. To improve the quality of future experiments, we expanded
the range of G-alpha candidates from three to seven by introducing
all combinations of the three selected mutations. We checked folding
predictions for all seven novel G-alphas which resulted in a significant
overlap (as visible in the figure). None of the proteins seemed likely
to take on a faulty conformation according to the SwissProt analysis.

![](https://static.igem.org/mediawiki/2020/4/46/T--UCopenhagen--mutants-aligned.png)

**Figure 6: Alignment of predicted structures of our G-alpha mutants.** Apart from the intrinsically disordered region on the left, the predicted structures overlap fairly well. This indicates that the conformation of none of the G-alpha mutants was severely impacted by their mutations.

## Stage Six - Test & Learn

In order to predict the behavior of the fragments generated by cleavage
of our mutant G-alpha by the TEV protease, we decided to run a simulation
in Rosetta (see modeling for more details). However, as this was an extremely demanding computational
analysis we could not perform ourselves, we reached out to Assoc. Prof.
Kurt V. Mikkelsen of Theoretical Chemistry at the University of Copenhagen,
who has a supercomputer capable of such demanding calculations. With a help from Andreas Erbs Hillers-Bendtsen, MSc student of chemistry at Copenhagen University, we were able to simulate the change in Gibbs free energy of
the seven mutant G-alphas (see figure 7), and the nine fragments formed
after cleavage (figure 8). Our goal was to identify a G-alpha mutant candidate
with binding affinity similar to the wildtype, but with the lowest possible
binding affinity of its fragments after cleavage. Our simulation lead to
some unexpected findings:

The majority of the fragments seemed to have a higher affinity towards
G-beta than the non-cleaved G-alphas had. Only mutant m124 displayed
a higher affinity than its corresponding fragments.

![](https://static.igem.org/mediawiki/2020/b/ba/T--UCopenhagen--mutants-affinity.png)

**Figure 7: Changes in Gibbs free energy of our G-alpha mutants relative to the wildtype.** Numbers in their labels signify what cleavage sites were included in each mutant. As all changes in energy are negative, it indicates that all mutants have a higher affinity to G-beta protein.

![](https://static.igem.org/mediawiki/2020/f/fe/T--UCopenhagen--fragments-affinity.png)

**Figure 8: Change in Gibbs free energy of fragments produced by cleavage of G-alpha mutants.** The x-axis denotes the length of each fragment. There seems to be a slight trend towards a higher affinity in smaller fragments, most likely caused by more degrees of freedom for a binding region constituting a majority of such fragments.

Initially, we were startled by these findings, however after thorough
discussions we concluded that the overall G-alpha conformation probably
restricted some degrees of freedom of the high-affinity regions.
These regions facilitated most interaction between G-alpha and G-beta,
as we learned in one of the earlier stages. Our cleavage sites were
located between these high-affinity regions, therefore, cleavage
effectively freed the high-affinity regions from the previous restrictive
conformation and allowed them to bind G-beta more tightly. Obviously,
these findings turned our initial conclusions about the protein-protein
interaction on its head. We also realized that affinities do not
necessarily provide the full answer to successful signal transduction.
In accordance with this, further analysis showed that conformational changes
of G-beta and their causes could not answer our questions.

Since mutant m124 was the only mutant whose corresponding fragments had
a lower affinity to G-beta compared to the mutant itself, we decided
it was the most promising candidate for our biosensor based on the data
from the simulation.

## Stage Seven - Test & Learn

Based on the conclusion from the previous Rosetta simulation, we designed
and ran another simulation to obtain more refined details about the
m124 fragments (see [modeling](https://2020.igem.org/Team%3AUCopenhagen/Model) for more details). The results showed that all generated fragments had
a significantly higher affinity to the G-beta compared to m124. Although
exploration of the PDB files revealed that spatial assignment of the
fragments showed clashes (figure 9) so that the affinity of each fragment
could be limited by the presence of the other fragments, ultimately
leading to diminished presence at G-beta. Nevertheless, the results of
our simulations could not inform us about conformational changes of
G-beta which protected us from formulating clear conclusions.

![](https://static.igem.org/mediawiki/2020/9/99/T--UCopenhagen--m124.png)

**Figure 9: Change in Gibbs free energy of fragments produced by the cleavage of G-alpha mutant m124.**
The labels on the x-axis signify the position of each fragment in the original protein.

![](https://static.igem.org/mediawiki/2020/5/5d/T--UCopenhagen--m124-clash.png)

**Figure 10: An example of clashing positions of the fragments.** This indicates a situation that cannot happen in the real-world conditions, thus reducing our trust in the prediction.

## Wet Lab

As part of our wet lab, we created four different mutant constructs of GPA1,
named GPA1m1, GPA1m2, GPA1m3, and GPA1m4. Mutants 1, 2 and 4 correspond to
the m1, m2, and m4 denotations used in our dry lab modeling. The Wt
GPA1 was replaced with the different GPA1 mutants in an adenosine biosensor
strain of *Saccharomyces Cerevisiae*, previously created by our supervisor Sotirios
Kampranis' lab. In addition, an inducible TEV protease was inserted into
the same locus of the *S. Cerevisiae* chromosome as the rest of
the biosensor constructs.

As a result, we were able to test out both the maintenance of the original
functions of GPA1 in a GPCR receptor system, and to verify that the GPA1
mutants were cleaved by the induced TEV protease, as this would result
in higher concentrations of the reporter.
INSERT FIGURE OF A2A.

The experiments showed that the amount of reporter was increased
drastically in all biosensors when the TEV protease was induced by
growing the cells in galactose. This included the biosensor with wt
GPA1 which made it hard to conclude whether some of the increased
signal was caused by cleavage of the GPA1 mutant or that the increase
in signaling came from other parts of the system.

The experiments further showed a loss-of-function in the sense that
none of the biosensors with mutated GPA1 proteins were able to respond
to different levels of adenosine whereas we were able to reproduce
the A2A adenosine biosensor created in the Sotirios Kampranis lab when
we used the wt GPA1 and grew the cells in glucose media.

## Future Vision

#### Dry lab

The next round of simulations could be based on molecular dynamics
for us to see whether the results of Rosetta could be trusted. Apart
from that, the focus could shift from affinities to conformations,
and particularly G-beta could be more explored.

#### Wet lab

In the wet lab, the next step would be to understand why the signaling
also increases in the biosensor with wt GPA1 when the cells are grown
in galactose and raffinose media. First, we would create a strain of
the biosensor without the inducible TEV protease to see whether the
spike in signaling is caused by the induction of the TEV protease or
something else related to the change of the media. If the TEV protease
does not seem to be the cause we would investigate the promoters that
we use, some of these are glycolytic promoters like pPGK1 and the
different media might cause slightly different expression levels
even though the promoters are normally classified as constituent.
To further investigate the loss-of-function of the mutants, we would
like to create strains of the biosensor with the mutated GPA1 proteins
but without the inducible TEV promoter. This would enable us to see
whether leaky expression of the TEV protease in the glucose fed cells
might have caused the loss-of-function.

1. Douglas P. Gladue, James B. Konopka, Scanning mutagenesis of regions in the Gα protein Gpa1 that are predicted to interact with yeast mating pheromone receptors, FEMS Yeast Research, Volume 8, Issue 1, February 2008, Pages 71–80, https://doi.org/10.1111/j.1567-1364.2007.00311.x[↩](#mutant-ref)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UCopenhagen/Experiments

[![](https://static.igem.org/mediawiki/2020/4/4d/T--UCopenhagen--Logo_Transparent_BG.png)](https://2020.igem.org/Team%3AUCopenhagen)

Project

[Description](https://2020.igem.org/Team%3AUCopenhagen/Description)
[Design](https://2020.igem.org/Team%3AUCopenhagen/Design)
[Implementation](https://2020.igem.org/Team%3AUCopenhagen/Implementation)
[Poster](https://2020.igem.org/Team%3AUCopenhagen/Poster)

Science

[Engineering Success](https://2020.igem.org/Team%3AUCopenhagen/Engineering)
[Modeling/Simulation](https://2020.igem.org/Team%3AUCopenhagen/Model)
[Parts Overview](https://2020.igem.org/Team%3AUCopenhagen/Parts)
[Results](https://2020.igem.org/Team%3AUCopenhagen/Results)
[Protocols](https://2020.igem.org/Team%3AUCopenhagen/Experiments)
[Notebook](https://2020.igem.org/Team%3AUCopenhagen/Notebook)
[Safety](https://2020.igem.org/Team%3AUCopenhagen/Safety)

Human Practices

[Integrated Human Practices](https://2020.igem.org/Team%3AUCopenhagen/Human_Practices)
[Entrepreneurship](https://2020.igem.org/Team%3AUCopenhagen/Entrepreneurship)
[Education](https://2020.igem.org/Team%3AUCopenhagen/Education)

Collaborations

[Collaborations](https://2020.igem.org/Team%3AUCopenhagen/Collaborations)
[Partnership](https://2020.igem.org/Team%3AUCopenhagen/Partnership)
[Our Contribution](https://2020.igem.org/Team%3AUCopenhagen/Contribution)

Judging

[Judging Form](https://igem.org/2020_Judging_Form?id=3617)
[Medals](https://2020.igem.org/Team%3AUCopenhagen/Medals)

Team

[Team Members](https://2020.igem.org/Team%3AUCopenhagen/Team)
[Attributions](https://2020.igem.org/Team%3AUCopenhagen/Attributions)
[Sponsors](https://2020.igem.org/Team%3AUCopenhagen/Sponsors)

## PROTOCOLS

PCR Cleanup

**Reagents required:**
• PCR clean up kit (E.N.Z.A)
• PCR reaction product

**Procedure:**
1.) Transfer 30-50 μL of the PCR product to a new 1.5 mL microcentrifuge tube.
2.) Add 4-5 times volume of the CP buffer.
3.) Briefly vortex and centrifuge the samples.
4.) Insert the Hibind DNA mini columns into the 2 mL collection tubes.
5.) Add the samples from step 3 to the Hibind DNA mini column.
6.) Centrifuge at 13000 g for 60 seconds at room temperature. Discard the filtrate and re use the collection tubes.
7.) Add 700 μL of DNA wash buffer diluted with ethanol to the tubes.
8.) Centrifuge at maximum speed for 60 seconds. Discard the filtrate and re use the collection tubes.
9.) Repeat step 7 for a second DNA wash buffer step.
10.) Centrifuge the empty Hibind DNA columns at maximum speed for 2 minutes to dry the column. This step is critical to remove the trace ethanol.
11.) Transfer the Hibind DNA column to a new 1.5 mL microcentrifuge tube.
12.) Add 50 μL of the elution buffer directly into the Hibind DNA column.
13.) Keep at room temperature for 2 minutes and centrifuge at maximum speed for 60 seconds.
14.) Store the DNA at -20℃.

Plasmid DNA Extraction

**Reagents required:**
• E.N.Z.A Plasmid DNA mini prep kit
• Culture

**Procedure:**
1.) Vortex the liquid cultures and use 1-5 mL for the plasmid extraction.
2.) Centrifuge at 10000 g for 1 min at room temperature. Discard the pellet.
3.) Add 250 μL of solution I, vortex the samples and transfer the suspension to a new microcentrifuge tube.
4.) Add 250 μL of solution II, invert the samples and wait for 2-3 minutes.
5.) Add 350 μL Solution III and invert the tube several times.
6.) Centrifuge the samples at maximum speed for 10 minutes.
7.) Place the Hibind DNA columns onto the 2 mL collection tubes.
8.) Transfer the supernatant from step 6 into the Hibind DNA columns.
9.) Centrifuge at maximum speed for 1 minute and discarded the filtrate.
10.) Add 500 μL of HBC buffer and centrifuge it at maximum speed for 1 minute and discard the supernatant.
11.) Add 700 μL DNA Wash Buffer and centrifuge it at maximum speed for 30 seconds and discard the supernatant.
12.) Repeat step 11 for a second DNA wash step.
13.) Centrifuge the empty Hibind DNA column for 2 min at maximum speed to dry the column.This will remove trace amounts of ethanol.
14.) Transfer the Hibind DNA column into a new microcentrifuge tube.
15.) Add 50 μL of Elution Buffer, let it sit at room temperature for 2 minutes and centrifuge at maximum speed for 1 minute.
16.) Store DNA at -20℃.

NanoDrop DNA quantification

**Reagents required:**
• DNA samples
• Milli-Q water
• Nanodrop device
• Pipette

**Procedure:**
1.) Clean the measurement surface with a piece of tissue and ethanol.
2.) Use 1 μL of Milli-Q water as a blank.
3.) Clean the measurement surface with a piece of tissue.
4.) Use 1 μL of sample to measure the concentration.
NOTE: It is best to measure the same sample in triplo and use the average value. If you have multiple samples, clean the measurement surface in between measurements.
5.) When done measuring, wipe the measuring surface with a tissue and Milli-Q water.

USER Cassette linearization reaction

**Reagents required:**
• Purified PCR product
• Restriction enzyme NotI HF
• Milli-Q water
• 10X cut smart buffer

**Procedure:**
1.) Prepare sample in a 0.5 mL microcentrifuge tube as follows.

| Component | Volume (μL) |
| --- | --- |
| 10X cut smart buffer | 4 |
| Purified PCR product | 30 |
| Restriction enzyme Dpn1 | 1 |
| Milli-Q | 5 |

2.) Incubate for 1.5 hours at 37℃.
3.) Heat inactivate the enzyme by heating it at 80℃ for 20 minutes.

Yeast Colony PCR

**Reagents required:**
• NaOH solution
• MangoMix master mix solution
• F1-primer
• F2-primer
• R1-primer
• Template: Colonies from overnight plates

**Procedure:**

**Yeast cell lysis:**
1.) Calculate the number of colonies and mark required number of PCR tubes.
2.) Add 20 μL of 20mM NaoH to all the tubes.
3.) Pick selected colonies using a pipette tip and inoculate into the tubes containing NaoH.
4.) Place the tubes in the PCR machine and run it for 10 minutes at 99℃.

**PCR reaction:**
1.) Use a commercial mastermix called MangoMix. This mastermix solution contains taq polymerase and dNTPs.
2.) Take new PCR tubes and add 5 μL of Mango master mix, 0.5 μL of each primers and 3.5 μL of water to a total volume of 9 μL.
3.) Add 1 μL of the template to each of the respective tubes.
4.) Place the tubes in the PCR machine and run a programme with following steps:

30-35 cycles for steps marked in red
96 degree celsius: 30 seconds

96 degree celsius: 30 seconds
55 degree celsius: 25 seconds
72 degree celsius: 2 minutes and 30 seconds

72 degree celsius: 5 minutes
12 degree celsius: Infinity

Bacterial Colony PCR

**Reagents required:**
• 10X X7 PCR buffer
• dNTP
• F-primers (10 μM)
• R-primers (10 μM)
• Template
• X7-Polymerase
• mQ Water

**Procedure:**
1.) Selected isolated colonies from the plate and using a sterile cocktail stick, remove a small quantity (such that it is visible on the end of the stick) of cells from the plate. This will serve as the template in the PCR mix.
2.) Label the PCR tubes with a number of g-blocks to be amplified.
3.) Calculate the number of samples and prepare a master mix (all the PCR reagents except the template).
4.) Transfer the master mix to the labelled PCR tubes and add the necessary templates to all the tubes.
5.) Set the PCR machine - Calculate the annealing temperature and extension time (20-30s/Kb)
6.) Place all the PCR tubes in the machine and run the PCR with the pre-set programme.

Competent Bacterial Cells

**Reagents required:**
• LB agar plates
• LB liquid media
• E.coli (WT)
• Eppendorfs
• TSS buffer

**Procedure:**
1.) From the overnight culture, add 1 mL of E.coli to 99 mL of fresh LB media and grow to OD600 to 0.4-0.5.
2.) Place the eppendorfs on ice and make sure that TSS buffer is chilled in the fridge (4℃).
3.) Split the culture into different falcon tubes and incubate them on ice for 10 minutes.
4.) Centrifuge the falcon tubes for 10 minutes at 3000 RPM and 4℃ ( The centrifuge needs to be cooled down to 4℃ before use).
5.) Discard the supernatant and resuspend the pellet in TSS buffer.
6.) Make aliquots into cooled eppendorf tubes and freeze them in -80℃.

Standard PCR

**Reagents required:**
• 10X X7 PCR buffer
• dNTP
• F-primers (10 μM)
• R-primers (10 μM)
• Template
• X7-Polymerase
• mQ Water

**Procedure:**
1.) Label the PCR tubes with number of g-blocks to be amplified.
2.) Calculate the number of samples and prepare a master mix (all the PCR reagents except the template).
3.) Transfer the master mix to the labelled PCR tubes and add the necessary templates to all the tubes.
4.) Set the PCR machine - Calculate the annealing temperature and extension time (20-30s/Kb).
5.) Place all the PCR tubes in the machine and run the PCR with the pre-set programme.

Dilution of G-blocks and primers

**Reagents required:**
• Primers
• g-blocks
• mQ water

**Procedure:**
1.) Dilute the primers and g-blocks in the tubes they came in, by making the solutions as following:
2.) Add 100 μL of Milli-Q water to the g-block fragments (1000ng) to make it 10 ng/μL .
3.) For the primers, make up to 100 µM concentrations.

DNA transformation in E. Coli

**Reagents required:**
• Competent cells
• Vector containing DNA

**Procedure:**
1.) Add 1% volume of the vector to competent cells.
2.) Mix by gently flicking the tube. Vortex will prevent transformation.
3.) Incubate the cells on ice for 30-60 minutes.
4.) Water bath at 42℃ for 45 seconds.
5.) Again incubate on ice for 3-5 minutes.
6.) Add 950 μL of LB and place them in shaking incubator at 37℃ for 1 hour.
7.) Centrifuge at 13000 RPM for 1 minute.
8.) Discard the supernatant slowly, leaving behind 50 μL-100 μL in the tube.
9.) Resuspend the pellet using a pipette and spread on to an appropriate selection plate and incubate overnight at 37℃.

Agarose Gel Electrophoresis

**Reagents required:**
• 1% agarose
• Gel red
• Electrophoretic chamber
• Samples to be loaded
• Molecular weight marker (DNA ladder)

**Procedure:**
1.) For a 1% (w/v) gel in TAE, weigh agarose powder for a 1% (w/v) gel.
2.) Add weighed agarose in TAE buffer (1X) and warm the solution (in a microwave) until it is completely dissolved. Let the solution cool down to hand warm temperature.
3.) Pour gel in gel tray and mix well with Gel Red. For a small gel (~ 40 mL solution) add 1 μL of Gel red; for a large gel (~80 mL) add 2 µL of Gel red. Add a comb to create wells for the samples. Allow the agarose to solidify.
4.) Transfer the gel to the electrophoresis cell, remove the combs and cover the gel in TAE buffer (1X).
5.) Prepare the electrophoresis samples by adding Nucleic Acid Loading Buffer conform the manufacturer’s instructions.
6.) Load the molecular weight marker (DNA ladder) in the first well according to manufacturer’s instructions (generally 3-5 µL) and load 5-10 µL of the dyed samples in the other wells.
7.) Connect the cables of the gel tray following the colour code and run at 80-110V for 40-60 min.

Preparation of agar plates

**Reagents required:**
• LB agar
• Milli-Q water
• Petri dishes

**Procedure:**
1.) Dissolve X grams of LB-agar in X mL of Milli-Q.
2.) Autoclave the LB-agar solution.
3.) Cool the agar down to room temperature.
4.) Add the correct amount of the correct antibiotic. (We used 2 μL of antibiotic per mL of LB agar)
5.) Under sterile conditions, pour the LB-agar in empty Petri dishes (20-25 mL per plate).
6.) Leave the Petri dishes in the sterile bench with the lid partially off for approximately 5 minutes so the agar can solidify.

Restriction digestion assay

**Reagents required:**
• 10X cut smart buffer
• Restriction enzymes
• DNA sample

**Procedure:**
1.) Decide on which restriction enzyme(s) to cut with. Find out what buffer the enzyme(s) work(s) in. In general, SmartCut buffer can be used.
2.) Prepare the sample as follows:

| Component | Volume (μL) |
| --- | --- |
| DNA | (~1 µg is required) |
| Buffer (CutSmart) | 2 |
| Restriction enzyme(s) (5u/µg) | 1 µL each |
| Milli-Q | Up to 20-25 µL |

2.) Incubate for one hour at 37 °C.
3.) Inactivate the restriction enzyme(s) by heating to 65 °C for 10 minutes.
NOTE: This last step can be skipped if the sample is evaluated on gel immediately after.

Raffinose media

**Reagents required:**
• Raffinose
• Yeast nitrogen base
• Synthetic drop out media
• Amino acids
• NaOH

**Procedure:**
1.) Add all the necessary reagents in necessary quantities.
2.) The quantities used to prepare 1L of the media are given below:
Raffinose - 20g
Yeast nitrogenous base - 6.7g
Synthetic drop out media - 1.4g
Amino acids:
0.04g Trp, 0.04g Leu, 0.02 His
3.) Make up the volume to 1 L.
4.) Adjust the pH(5.8-6.2) using 10% NaOH.

USER Ligation

This experiment is carried out to ligate the gene of interest with its promoter and vector backbone.
Note: The Gene of interests used in this experiment have already been amplified with primers containing USER sequence.
**Reagents required:**
• USER enzyme
• Cut smart buffer
• Promoter
• Gene of interest
• Vector backbone
• Milli-Q water

**Procedure:**
1.) Create a master mix using Milli-Q water, USER enzyme and cutsmart buffer as shown below.

2.) For each ligation, add the gene fragments, promoters and vector backbones separately.
3.) Add 7 μL of the master mix to each tube. Finally, place the samples in the PCR and set the following conditions:
37 °C for 30 min
25 °C for 15 min
20 °C for 10 min
12 °C indefinitely

| Compound | Volume (μL) |
| --- | --- |
| USER enzyme | 1 |
| CutSmart buffer | 1 |
| Promoter | 1 |
| Gene | 1 |
| Vector backbone | 1 |
| Vector backbone | 1 |
| Milli-Q | 5 |
| Total | 10 |

2.) Incubate for 1.5 hours at 37℃.
3.) Heat inactivate the enzyme by heating it at 80℃ for 20 minutes.

Yeast transformation

**Reagents required:**

**Reagents required:**
• O/N yeast culture
• YPD media
• Cuvettes
• Salmon sperm
• Lithium acetate
• Vector backbones
• Milli-Q

**Procedure:**
1.) Mix 0.9 mL YPD media and 0.1 mL of overnight culture in a cuvette and measure OD at 600nm.

2.) Dilute the culture to an OD(600nm) = 0.25 based on the OD value measured.
3.) Incubate the culture for another 4h to reach an OD(600nm) of approximately 1.
4.) Boil the ssDNA(Salmon sperm) for 10 mins at 99°C in the heat block and keep it on ice.
5.) Spin down the culture in the falcon tube at 3000RCF for 10 mins at room temperature.
6.) Pour the supernatant into a waste tube and resuspend in 1mL of LiAc.
7.) Transfer the culture into 1.5 mL of Eppendorf tubes and spin down at 7000 RCF for 1 min at room temperature.
8.) Remove the supernatant carefully with a pipette and resuspend the pellet in a 100 μL of 0.1M LiAc.
9.) Add 30 μL of salmon sperm and mix everything gently.
10.) Add 1 mL of PLI and mix well.
11.) Transfer the DNA samples into 1.5 mL eppendorf tubes and 200 μL of the yeast culture.
12.) Heat shock the samples for 30 mins at 42 °C in a heat block.
13.) After transformation, spin down the cells at 7000RCF for 1 min at room temperature and discard the supernatant.
14.) Resuspend the pellet in 1 mL of water.
15.) Spin down the cells at 8000RCF for 1 min at room temperature.
16.) Remove 900 μL of the water and resuspend the cells in the remaining water.
17.) Finally, add the transformed cells onto the necessary plates and spread it uniformly.
18.) Incubate the plates for 3 nights at 30°C.

Interleukine Induction Assay (200 µL, 96-well)

**Reagents required:**
• Cell cultures of the strains to be measured
• Growth media
• Dilution series of receptor ligands
• YeastBuster
• Luciferase substrate
• Sterile 96-well plate
• black 96-well plate

**Procedure:**
Incubation - keep sterile!
1.) Grow cells to desired OD600 in falcon tubes with YPD
2.) Optional: Spin down cells for 1 min. at 5000 RCF and discard supernatant
3.) Resuspend OR dilute cells in desired growth medium to desired concentration
4.) Transfer cells to sterile 96-well plate
5.) Add desired volume of biomarker dilution series to the wells (remember to note down what is in each well)
6.) Optional: Wrap plate in parafilm to reduce evaporation.
7.) Incubate in shaker at 30°C for desired incubation time

**Luciferase assay**
8.) Transfer ¾ of desired measurement volume to black 96-well plate
9.) Add ¼ of desired measurement volume YeastBuster with 1% luciferase substrate to each well
10.) Immediately measure luminescence from top with plate reader.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UCopenhagen/Results

[![](https://static.igem.org/mediawiki/2020/4/4d/T--UCopenhagen--Logo_Transparent_BG.png)](https://2020.igem.org/Team%3AUCopenhagen)

Project

[Description](https://2020.igem.org/Team%3AUCopenhagen/Description)
[Design](https://2020.igem.org/Team%3AUCopenhagen/Design)
[Implementation](https://2020.igem.org/Team%3AUCopenhagen/Implementation)
[Poster](https://2020.igem.org/Team%3AUCopenhagen/Poster)

Science

[Engineering Success](https://2020.igem.org/Team%3AUCopenhagen/Engineering)
[Modeling/Simulation](https://2020.igem.org/Team%3AUCopenhagen/Model)
[Parts Overview](https://2020.igem.org/Team%3AUCopenhagen/Parts)
[Results](https://2020.igem.org/Team%3AUCopenhagen/Results)
[Protocols](https://2020.igem.org/Team%3AUCopenhagen/Experiments)
[Notebook](https://2020.igem.org/Team%3AUCopenhagen/Notebook)
[Safety](https://2020.igem.org/Team%3AUCopenhagen/Safety)

Human Practices

[Integrated Human Practices](https://2020.igem.org/Team%3AUCopenhagen/Human_Practices)
[Entrepreneurship](https://2020.igem.org/Team%3AUCopenhagen/Entrepreneurship)
[Education](https://2020.igem.org/Team%3AUCopenhagen/Education)

Collaborations

[Collaborations](https://2020.igem.org/Team%3AUCopenhagen/Collaborations)
[Partnership](https://2020.igem.org/Team%3AUCopenhagen/Partnership)
[Our Contribution](https://2020.igem.org/Team%3AUCopenhagen/Contribution)

Judging

[Judging Form](https://igem.org/2020_Judging_Form?id=3617)
[Medals](https://2020.igem.org/Team%3AUCopenhagen/Medals)

Team

[Team Members](https://2020.igem.org/Team%3AUCopenhagen/Team)
[Attributions](https://2020.igem.org/Team%3AUCopenhagen/Attributions)
[Sponsors](https://2020.igem.org/Team%3AUCopenhagen/Sponsors)

![](https://static.igem.org/mediawiki/2020/4/45/T--UCopenhagen--results2.png)

## Introduction

The ambition of CIDosis is to design and create an inflammation-detecting patch, containing a *Saccharomyces cerevisiae*-based interleukin biosensor able to track concentrations of selected interleukins in sweat. For this, we decided to create three biosensor designs with progressing complexity based on interleukin receptor association.
The general CIDosis biosensor design ([project design page)](https://2020.igem.org/Team%3AUCopenhagen/Design) is highly modular as exemplified here with four different biosensors (IL-1, IL-10, IL-6 design1 IL-6 design 2) each consisting of a different extracellular receptor domain pair. Each biosensor can be constructed with either of three different intracellular signal transduction modules.

The modular nature of the biosensor enables multiple setups for different purposes. From the implementation point of view, it also creates multiple points for optimization and verification. In order to verify that all of our different modules work we divided designs into smaller constituents and created experiments around them in order to test their functionality. This was done in parallel with creating and assaying our complete whole cell biosensors.

To lay out what experiments we had to do, we first had to think of the pitfalls of each of the modules, and what could go wrong in different scenarios. Therefore, we chose the following questions to guide our choice of experiments:

![](https://static.igem.org/mediawiki/2020/8/82/T--UCopenhagen--design_system_loop.gif)

**Figure 1: General biosensor design.**

* ✧ Are the extracellular domains functioning as intended? Are they dissociated in the absence of the interleukins and will they associate in the presence of the interleukins?
* ✧ Do the receptors and accessory proteins have the intended subcellular localization?
* ✧ Is the membrane bound transcription factor of the intermediate design able to be cleaved by a TEV protease?
* ✧ Is the TEV protease able to cleave the modified Gα (Gpa1p) proteins and trigger the yeast pheromone pathway?
* ✧ Do the modified Gα (Gpa1p) proteins retain their ability to transduce signalling from a GPCR?

To answer these questions we followed the scientific workflow shown in figure 2.

![](https://static.igem.org/mediawiki/2020/1/15/T--UCopenhagen--ugly_process.png)

**Figure 2: Scientific workflow** - Obtaining synthetic genes and promoters → PCR amplification → USER plasmid ligation → transformation into *Escherichia coli* → colony PCR → sequencing → yeast transformation → yeast colony PCR → Biosensor (and other) assays.

## Cloning

First we had to create the plasmids that we wanted to use in our experiments. All cloning steps were performed with USER cloning. For an explanation of the concepts behind USER cloning, watch our introductive video on the [education page.](https://2020.igem.org/Team%3AUCopenhagen/Education) For further details, see also the article by Geu-Flores et al. 2007.

Most of the gene fragments used were ordered as synthetic DNA from IDT and Twist Bioscience after designing and codon optimizing them for expression in *S. cerevisiae* using Benchling. Other fragments like the promoters we used and some of the genes, e.g. our reporter, were already available in our supervisor Sorterios Kampranis' lab

As a first step, we started by PCR amplifying the promoters and ORFs needed for biosensor construction. To confirm that the gene fragments were correctly amplified, the PCR products were analyzed using gel electrophoresis on an agarose gel.

![](https://static.igem.org/mediawiki/2020/5/5f/T--UCopenhagen--results-12.8_PCRgel3v4.png)

**Figure 3: Representative image of a gel electrophoresis of positive PCR fragment amplifications.** The bands that indicate the length of our desired PCR products are marked with a star (\*).

The PCR products with the correct size were isolated by gel extraction.

Then, the fragments were digested with the USER enzyme to create custom sticky ends. These fragments were ligated by incubation with the relevant linearized vector. Subsequently, competent *E. coli* cells were transformed with the products of the USER ligation and grown on agar plates with carbenicillin. This allowed for selection for cells that had been transformed with plasmids from our USER ligation since all our plasmid backbones contained a gene encoding β-lactamase (AmpR).

![](https://static.igem.org/mediawiki/parts/d/dc/T--UCopenhagen--results-plates.jpg)

**Figure 4: Representative picture of plates with transformed bacteria and yeast.** Background: Our transformed *E.coli* was grown on agar plates with carbinicilin to select for colonies containing our plasmids. Foreground: Transformed *S. cerevisiae* was grown on glucose plates without uracil to select for the colonies with the URA3 gene in the X3A vector inserted. The vectors used for yeast transformation are described in the next chapter.

To identify colonies with plasmids with the desired inserts, we picked between 4-16 colonies for colony PCR. We used primers that annealed to the vector backbone on either side of the USER cassette (figure 5a). If nothing was inserted, analyzing the PCR product with gel electrophoresis would yield a band at 300 bp. A band at the sum of the length of our gene and promoter + 300 bp from flanking regions would mean that the construct had been successfully inserted in the backbone vector (figure 5b). By using primers that anneal to the backbone we automatically had a process control since no bands would mean that something had gone wrong with the colony PCR reaction.

![](https://static.igem.org/mediawiki/parts/2/24/T--UCopenhagen--results-scgal1tevmap.png)

![](https://static.igem.org/mediawiki/parts/7/76/T--UCopenhagen--results-bestgelever.png)

**Figure 5b: Gel electrophoresis of colony PCR products.** Green arrow: The band indicated that the colony had been transformed with the full construct from figure 5a. Red arrow: Colony with an empty plasmid. Yellow arrow: The plasmid did not have our full construct inserted. It might be caused by a deletion or a single missing fragment.

**Figure 5a: A map of one of our vector constructs.** The vector consists of the Ass2c backbone with a gene encoding TEV protease and an inducible promoter, scGAL1, inserted. Primers used in the colony PCR are shown. The distance between the forward and backward primers is about 1700bp. Bands of approximately this size would indicate that the plasmid had our full construct inserted.

Colonies corresponding to positive colony PCRs were grown overnight in liquid media with antibiotic selection and plasmid purified with a miniprep kit. The resulting plasmids were sent for sequencing to confirm that our construct had been inserted without mutations. In total, we managed to clone 53 constructs. The following table shows what plasmids we created or planned to create:

After confirming the right sequence, the constructs could be used for creating different yeast strains.

Constructs

SP= Wsc1 ER import signal peptide, TMD= Wsc1 transmembrane domain.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Construct name** | **Vector** | **Promoter** | **Domains in gene** | **Status** |
| Sgp130(D1-D6)-Cub | X3A | pCCW12 | SP-sgp130(D1-D6)-TMD-Cub-LexA-VP16 | Done |
| Sgp130(D1-D6)-Cub | X3A | pCCW12 | SP-sgp130(D1-D6)-TMD-Cub-LexA-VP16-sfGFP | Done |
| Sgp130(D1-D6)-Nub | Ass2C | pTDH3 | SP-Sgp130(D1-D6)-TMD-Nub | Done |
| Sgp130(D1-D6)-Nub | Ass2C | pTDH3 | SP-Sgp130(D1-D6)-TMD-Nub-sfGFP | No - two colonies had mutations did not sequence third |
| sIL-6R (secreted) | Ass2C | pPGK1 | Alpha factor-pre-pro-sequence -sIL-6R | Done |
| sIL-6R (secreted) | Ass2B | pPGK1 | Alpha factor-pre-pro-sequence -sIL-6R | Done |
| sIL-6R (secreted) | Ass2C | pPGK1 | Alpha factor-pre-pro-sequence -sIL-6R | Done |
| Sgp-130(D1-D3)-Cub | X3A | pCCW12 | SP-Sgp130(D1-D3)-TMD-Cub-LexA-VP16 | Done |
| Sgp-130(D1-D3)-Cub | X3A | pCCW12 | SP-Sgp130(D1-D3)-TMD-Cub-LexA-VP16-sfGFP | Done |
| sIL-6R-Nub | Ass2C | pTDH3 | SP-sIL-6R-TMD-Nub | Done |
| sIL-6R-Nub | Ass2C | pTDH3 | SP-sIL-6R-TMD-Nub-sfGFP | Done |
| sIL-1R2-Cub | X3A | pCCW12 | SP-sIL-1R2-TMD-Cub-LexA-VP16 | Done |
| sIL-1R2-Cub | X3A | pCCW12 | SP-sIL-1R2-TMD-Cub-LexA-VP16-sfGFP | Done |
| sIL-1RAcP-Nub | Ass2C | pTDH3 | SP-sIL-1RAcP-TMD-Nub | Done |
| sIL-1RAcP-Nub | Ass2C | pTDH3 | SP-sIL-1RAcp-TMD-Nub-sfGFP | Redo USER ligation |
| sIL-10R1-Cub | X3A | pCCW12 | SP-sIL-10R1-TMD-Cub-LexA-VP16 | Done |
| sIL-10R1-Cub | X3A | pCCW12 | SP-sIL-10R1-TMD-Cub-LexA-VP16-sfGFP | Done |
| sIL-10R2-Nub | Ass2C | pTDH3 | SP-sIL-10R2-TMD-Nub | Done |
| sIL-10R2-Nub | Ass2C | pTDH3 | SP-sIL-10R2-TMD-Nub-sfGFP | Done |
| MJDOD | X3C | LexAop | Already in lab | Already in lab |
| Sgp-130(D1-D6)-CTEV | X3A | pCCW12 | SP-Sgp-130(D1-D6)-TMD-CTEV | Done |
| Sgp-130(D1-D6)-NTEV | Ass2C | pTDH3 | SP-Sgp-130(D1-D6)-TMD-NTEV | Done |
| Sgp-130(D1-D6)-CTEV | X3A | pCCW12 | SP- Sgp-130(D1-D6)-TMD-CTEV-sfGFP | Done |
| Sgp-130(D1-D6)-NTEV | Ass2C | pTDH3 | SP- Sgp-130(D1-D6)-TMD-NTEV-sfGFP | Done |
| Sgp130-(D1-D3)-CTEV | X3A | pCCW12 | SP-Sgp130-(D1-D3)-TMD-CTEV | Done |
| sIL-6R-NTEV | Ass2C | pTDH3 | SP-sIL-6R-TMD-NTEV | Done |
| Sgp130(D1-D3)-CTEV | X3A | pCCW12 | SP-sgp130-TMD-CTEV-sfGFP | USER-ligation produced stop codon. |
| sIL-6R-NTEV | Ass2C | pTDH3 | SP-sIL-6R-TMD-NTEV-sfGFP | Done |
| sIL-1R2-CTEV | X3A | pCCW12 | SP-sIL-1R2-TMD-CTEV | Done |
| sIL-1RAcp-NTEV | Ass2C | pTDH3 | SP-sIL-1RAcp-TMD-NTEV | Done |
| sIL-1R2-CTEV | X3A | pCCW12 | SP-sIL-1R2-TMD-CTEV-sfGFP | USER-ligation produced stop codon. New primer needed |
| sIL-1RAcP-NTEV | Ass2C | pTDH3 | SP-sIL-1RAcP-TMD-NTEV-sfGFP | Done |
| sIL-10R1-CTEV | X3A | pCCW12 | SP-sIL-10R1-TMD-CTEV | Done |
| sIL-10R2-NTEV | Ass2C | pTDH3 | SP-sIL-10R2-TMD-NTEV | Done |
| sIL-10R1-CTEV | X3A | pCCW12 | SP-sIL-10R1-TMD-CTEV-sfGFP | USER-ligation produced stop codon. |
| sIL-10R2-NTEV | Ass2C | pTDH3 | SP-sIL-10R2-TMD-NTEV-sfGFP | Done |
| E3-CTEV (BBa\_K1159102) | X3A |  | SP-E3-TMD-CTEV(BBa\_K1159102) | Do colony PCR |
| ZE-CTEV (BBa\_K1159102) |  |  | SP-ZE-TMD-CTEV (BBa\_K1159102) | Do colony PCR |
| K3-NTEV |  |  | SP-K3-TMD-NTEV | Do colony PCR |
| ZR-NTEV |  |  | SP-ZR-TMD-NTEV | Do colony PCR |
| E3-CTEV (truncated C-terminal) |  |  | SP-E3-TMD-CTEV(truncated C-terminal) | Do colony PCR |
| ZE-CTEV (truncated C-terminal) |  |  | SP-ZE-TMD-CTEV (truncated C-terminal) | Do colony PCR |
| E3-CTEV (wt) |  |  | SP-E3-TMD-CTEV(wt) | Do colony PCR |
| ZE-CTEV (wt) |  |  | SP-ZE-TMD-CTEV (wt) | Do colony PCR |
| 40 |  |  |  | cancelled |
| 41 |  |  |  | cancelled |
| 42 |  |  |  | cancelled |
| 43 |  |  |  | cancelled |
| 44 | Ass2A | pPGK1 | SP-TMD-ENLYFQG-LexA-VP16 | Done |
| 45 |  |  |  | cancelled |
| 46 | Ass2a | ppgk1 | SP-TMD-ENLYFQG-LexA-VP16-sfGFP | Done |
| 47 |  |  |  | cancelled |
| 48 |  |  |  | new plan for G-alpha |
| 49 |  |  |  | new plan for G-alpha |
| 50 |  |  |  | new plan for G-alpha |
| 51 | Ass2A | pPGK1 |  | already in lab |
| 52 | Ass2C | scGAL1 | TEV protease | done |
| 53 | Ass2B | pRET | Ste12 | cancelled |
| 54 | Ass2B | pRET | LexA-Ste12 | already in lab |
| 55 | Ass2B | pFIG | LexA-nanoluciferase | already in lab |
| 56 | X3A | pCCW12 | A2A (R199A) | already in lab |
| GPA1 mutations: |  |  |  |  |
| GPA1mut1 | Ass2A | pPGK1 | GPA1 with cut site inserted at site one | done |
| GPA1mut2 | Ass2A | pPGK1 | GPA1 with cut site inserted at site two | done |
| GPA1mut3 | Ass2A | pPGK1 | GPA1 with cut site inserted at site three | done |
| GPA1mut4 | Ass2A | pPGK1 | GPA1 with cut site inserted at site four | done |
| GPA1mut5 | Ass2A | pPGK1 | GPA1 with cut site inserted at site five | done |

Table 1: Constructs

## Yeast strains

All the yeast strains (table 2) were constructed by genomic integration of the different modules into chromosome X site 3 using the homologous recombination based 5-plasmid assembly system (unpublished) available at the host lab (figure 5).

![](https://static.igem.org/mediawiki/2020/9/9a/T--UCopenhagen--five_ass.png)

**Figure 6: The 5 assembly system.** Convenient assembly of different biosensors through the shuffling of parts in the five modules dedicated to different functions is allowed by the modular structure of the 5 assembly system (Unpublished). Extra flexibility is provided by the Aux module by accommodating additional parts, such as for constructing multi-part reporters. One assembles the biosensor by co-transforming five linearized module plasmids that contain a promoter and a coding sequence, with both being flanked by homologous recombination regions. To ensure consistent and controllable expression of the module genes of interest, the five modules are then stably integrated into the genome of *Saccharomyces cerevisiae* (Unpublished).

Correct insertion of all five vectors can was confirmed with a single colony PCR with three primers: a forward and a reverse primer anneal up- and down-stream of the homologous recombination site on the yeast chromosome respectively and a second forward primer anneals to the inserted construct (explained in figure 7).

![](https://static.igem.org/mediawiki/2020/1/1e/T--UCopenhagen--inserted.png)

![](https://static.igem.org/mediawiki/parts/1/1e/T--UCopenhagen--results-ycolPCR.jpg)

**Figure 7: Yeast colony PCR** A) A band at about 1500 bp would mean no insertion into the locus, while a band at 1000 bp implies that all five vectors have been inserted successfully. B) Arrow a: Positive band at 1000 bp. Arrow b: Negative band at 1500 bp.

In total, 21 different strains were created. Beneath are tables showing all the strains we created or intended to create in order to do the different experiments:

Biosensors

Our biosensors

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| **Assembly nr.** | **Description** | **X3A** | **Ass2A** | **Ass2B** | **Ass2C** | **X3C** | **Status** |
| CD1 | IL-10 biosensor using IL-10RI and IL-10II and the split ubiquitin | Il-10RI-Cub (15) | empty | empty | Il-10RII-Nub (17) | LexAop-nanoluc | Confirmed |
| CD2 | IL-6 biosensor using the IL-6R and truncated sgp130 and split ubiquitin design | sgp130(D1-D3)-Cub (7) | empty | empty | sIL-6R-Nub (9) | LexAop-nanoluc | Confirmed |
| CD3 | IL-1 biosensor using IL-1R2 and IL-1RAcP and the split ubiquitin design | sIL-1R2-Cub (11) | empty | empty | sIL-1RAcP-Nub (13) | LexAop-nanoluc | Confirmed |
| CD4 | IL-6 biosensor using the IL-6R and truncated sgp130 and the split-TEV and TMD-LexA-VP16 design | sgp130(D1-D3)-CTEV (24) | TMD-LexA-VP16 (44) | empty | sIL-6R-NTEV (25) | LexAop-nanoluc | no |
| CD5 | IL-1 biosensor using IL-1R2 and IL-1RAcP and the split-TEV and TMD-LexA-VP16 design | sIL-1R2-CTEV (28) | TMD-LexA-VP16 (44) | empty | sIL-1RAcP-NTEV (29) | LexAop-nanoluc | Confirmed |
| CD6 | IL-10 biosensor using IL-10RI and IL-10II and the split-TEV and TMD-LexA-VP16 design | sIL-10RI-CTEV (32) | TMD-LexA-VP16 (44) | empty | sIL-10RII-NTEV (33) | LexAop-nanoluc | no |
| CD7 | IL-6 biosensor using the IL-6R and truncated sgp130 and the split-TEV protease and Gα cleavage  design | sgp130(D1-D3)-CTEV (24) | GPA1-mut2 | LexA-Ste12 | sIL-6R-NTEV (25) | LexAop-nanoluc | no |
| CD8 | IL-1 biosensor using IL-1R2 and IL-1RAcP and the split-TEV protease and Gα cleavage  design | sIL-1R2-CTEV (28) | GPA1-mut2 | LexA-Ste12 | sIL-1RAcP-NTEV (29) | LexAop-nanoluc | no |
| CD9 | IL-10 biosensor using IL-10RI and IL-10II and the split-TEV protease and Gα cleavage design | sIL-10RI-CTEV (32) | GPA1-mut2 | LexA-Ste12 | sIL-10RII-NTEV (33) | LexAop-nanoluc | no |
| CD16 | IL-6 biosensor using sgp130 and secreted sIL-6R and split ubiquitin design | sgp130(D1-D6)-cub (1) | empty | sIL-6R (5) (ass2B) | sgp130(D1-D6)-Nub (3) | LexAop-nanoluc | Confirmed |
| CD17 | IL-6 biosensor using sgp130 and secreted sIL-6R and the split-TEV and TMD-LexA-VP16 design | sgp130(D1-D6)-cTEV (20) | TMD-TF (44) | sIL-6R (5) (ass2B) | sgp130(D1-D6)-nTEV (21) | LexAop-nanoluc | no |
| CD18 | IL-6 biosensor using sgp130 and secreted sIL-6R and the split-TEV protease and Gα cleavage  design | sgp130(D1-D6)-cTEV (20) and sgp130(D1-D6)-nTEV (21) (57) | GPA1 mut | LexA-Ste12 | sIL-6R (5) | LexAop-nanoluc | no |

Localization assays

These strains were designed in order to do subcellular localization assays on the different components of our biosensors and some of the accessory proteins needed for the designs to work.

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| **Assembly nr.** | **Description** | **X3A** | **Ass2A** | **Ass2B** | **Ass2C** | **X3C** | **Status** |
| CD19 | For succellular localization assay of sgp130(D1-D6)-Cub | sgp130(D1-D6)-Cub-sfGFP (2) | empty | empty | empty | empty | Confirmed |
| CD20 | For succellular localization assay of sgp130(D1-D6)-Nub | empty | empty | empty | sgp130(D1-D6)-Nub-sfGFP (4) | empty | no |
| CD21 | For succellular localization assay of sgp130(D1-D3)-Cub | sgp130(D1-D3)-Cub-sfGFP (8) | empty | empty | empty | empty | Confirmed |
| CD22 | For succellular localization assay of sIL-6R-Nub | empty | empty | empty | sIL-6R-Nub-sfGFP (10) | empty | Confirmed |
| CD23 | For succellular localization assay of sIL-1R2-Cub | sIL-1R2-Cub-sfGFP (12) | empty | empty | empty | empty | Confirmed |
| CD24 | For succellular localization assay of sIL-1RAcP-Nub | empty | empty | empty | sIL-1RAcP-Nub-sfGFP (14) | empty | no |
| CD25 | For succellular localization assay of sIL-10R1-Cub | sIL-10R1-Cub-sfGFP (16) | empty | empty | empty | empty | Confirmed |
| CD26 | For succellular localization assay of sIL-10R2-Nub | empty | empty | empty | sIL-10R2-Nub-sfGFP (18) | empty | Confirmed |
| CD27 | For succellular localization assay of sgp130(D1-D6)-cTEV | sgp130(D1-D6)-cTEV-sfGFP (22) | empty | empty | empty | empty | Confirmed |
| CD28 | For succellular localization assay of sgp130(D1-D6)-nTEV | empty | empty | empty | sgp130(D1-D6)-nTEV-sfGFP (23) | empty | Confirmed |
| CD29 | For succellular localization assay of sgp130(D1-D3)-cTEV | sgp130(D1-D3)-cTEV-sfGFP (26) | empty | empty | empty | empty | no |
| CD30 | For succellular localizationassay of sIL-6R-nTEV | empty | empty | empty | sIL-6R-nTEV-sfGFP (27) | empty | Confirmed |
| CD31 | For succellular localization assay of sIL-1R2-cTEV | sIL-1R2-cTEV-sfGFP (30) | empty | empty | empty | empty | no |
| CD32 | For succellular localization assay of sIL-1RAcP-nTEV | empty | empty | empty | sIL-1RAcP-nTEV-sfGFP (31) | empty | Confirmed |
| CD33 | For succellular localization assay of sIL-10R1-cTEV | sIL-10R1-cTEV-sfGFP (34) | empty | empty | empty | empty | no |
| CD34 | For succellular localization assay of sIL-10R2-nTEV | empty | empty | empty | sIL-10R2-nTEV-sfGFP (35) | empty | Confirmed |
| CD35 | For succellular localization assay of sIL-6R | empty | empty | empty | sIL-6R-sfGFP (6) | empty | no |
| CD36 | For succellular localization assay of TMD-LexA-VP16 | empty | TMD-TF-sfGFP (46) | empty | empty | empty | no |

Characterizing modified Gαs

For our third design to work, we needed to modify the endogenous g-protein subunit α, GPA1, from *S. cerevisiae.* These are the strains we needed to test the functionality of the different modified GPA1 proteins we designed.

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| **Assembly nr.** | **Description** | **X3A** | **Ass2A** | **Ass2B** | **Ass2C** | **X3C** | **Status** |
| CD12.01 | A2A biosensor with GPA1-mutation 1 | A2A (R199A) | GPA1-mutation 1 | LexA-Ste12 | empty | LexAop-nanoluc | no |
| CD12.02 | A2A biosensor with GPA1-mutation 2 | A2A (R199A) | GPA1-mutation 2 | LexA-Ste12 | empty | LexAop-nanoluc | no |
| CD12.03 | A2A biosensor with GPA1-mutation 3 | A2A (R199A) | GPA1-mutation 3 | LexA-Ste12 | empty | LexAop-nanoluc | no |
| CD12.04 | A2A biosensor with GPA1-mutation 4 | A2A (R199A) | GPA1-mutation 4 | LexA-Ste12 | empty | LexAop-nanoluc | no |
| CD12.05 | A2A biosensor with GPA1-mutation 5 | A2A (R199A) | GPA1-mutation 5 | LexA-Ste12 | empty | LexAop-nanoluc | no |
| CD13 | A2A biosensor | A2A (R199A) | GPA1 | LexA-Ste12 | empty | LexAop-nanoluc | no |
| CD14.01 | A2A biosensor with GPA1-mutation 1 and inducible TEV protease | A2A (R199A) | GPA1-mutation1 | LexA-Ste12 | scGal1-TEV | LexAop-nanoluc | Confirmed |
| CD14.02 | A2A biosensor with GPA1-mutation 2 and inducible TEV protease | A2A (R199A) | GPA1-mutation2 | LexA-Ste12 | scGal1-TEV | LexAop-nanoluc | Confirmed |
| CD14.03 | A2A biosensor with GPA1-mutation 3 and inducible TEV protease | A2A (R199A) | GPA1-mutation3 | LexA-Ste12 | scGal1-TEV | LexAop-nanoluc | Confirmed |
| CD14.04 | A2A biosensor with GPA1-mutation 4 and inducible TEV protease | A2A (R199A) | GPA1-mutation4 | LexA-Ste12 | scGal1-TEV | LexAop-nanoluc | no |
| CD14.05 | A2A biosensor with GPA1-mutation 5 and inducible TEV protease | A2A (R199A) | GPA1-mutation5 | LexA-Ste12 | scGal1-TEV | LexAop-nanoluc | no |
| CD15 | A2A biosensor and inducible TEV protease | A2A (R199A) | GPA1 | LexA-Ste12 | scGal1-TEV | LexAop-nanoluc | Confirmed |

Strains for other experiments

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| **Assembly nr.** | **Description** | **X3A** | **Ass2A** | **Ass2B** | **Ass2C** | **X3C** | **Status** |
| CD10 | TMD-LexA-VP16 cleaved by inducible TEV | empty | TMD-LexA-VP16 (44) | empty | scGal1-TEV | LexAop-nanoluc | Confirmed |
| CD37 | Leucinezippers (K3 and E3) with partially optimised cTEV as in Bba\_K1159102 | (36.1) | TMD-LexA-VP16 (44) | empty | (37.1) | LexAop-nanoluc | no |
| CD38 | Leucinezippers (K3 and E3) with most optiimsed cTEV | (38.1) | TMD-LexA-VP16 (44) | empty | (37.1) | LexAop-nanoluc | no |
| CD39 | Leucinezippers (K3 and E3) with Wt cTEV as in Bba\_K1965020 | (39.1) | TMD-LexA-VP16 (44) | empty | (37.1) | LexAop-nanoluc | no |
| CD40 | Leucinezippers (ZR and ZE) with partially optimised cTEV as in Bba\_K1159102 | (36.2) | TMD-LexA-VP16 (44) | empty | (37.2) | LexAop-nanoluc | no |
| CD41 | Leucinezippers (ZR and ZE) with optimised cTEV | (38.2) | TMD-LexA-VP16 (44) | empty | (37.2) | LexAop-nanoluc | no |
| CD42 | Leucinezippers (ZR and ZE) with Wt cTEV as in Bba\_K1965020 | (39.2) | TMD-LexA-VP16 (44) | empty | (37.2) | LexAop-nanoluc | no |

Other strains:

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| **Assembly nr.** | **Description** | **X3A** | **Ass2A** | **Ass2B** | **Ass2C** | **X3C** | **Status** |
| CD11 | control for reporter leakage | empty | empty | empty | empty | LexAop-nanoluc | no |

Table 2: Yeast strains

![](https://static.igem.org/mediawiki/2020/4/4c/T--UCopenhagen--fold_tables.png)

## Minimal biosensor design

All our biosensor designs are based on the association of extracellular domains of receptor proteins in the presence of interleukins at the plasma membrane of *S. cerevisiae.*

In the minimal design, the extracellular Interleukin-receptors are fused with either a C- or N-terminal part of a split-ubiquitin protein. Upon receptor association in the presence of IL-molecules, the two halves reconstitute resulting in full-length ubiquitin that can be recognized by a deubiquitinase leading to cleavage of the protein and release of a LexA-VP16 synthetic transcription factor. This would further result in the expression of the luciferase reporter gene (nanoluc).

![](https://static.igem.org/mediawiki/2020/1/10/T--UCopenhagen--Ubiquitin2.png)

**Figure 8: Minimal biosensor design;** Split ubiquitin complementation leading to release of C-ub bound LexA-VP16 to initiate gene expression.

#### Induction assays

In order to test whether our first design of the biosensor works, induction assays with dilution series of the corresponding interleukins were carried out.

Biosensors for IL-10 and IL-6, corresponding to assembly 1 and 2 in table 2, were tested. The two strains were grown to an OD600=0,5 and incubated at 30°C for a set of different incubation times with different concentrations of the respective biomarker. Next, luciferase assays were performed with the strains (figures 9 and 10).

![](https://static.igem.org/mediawiki/parts/6/63/T--UCopenhagen--results-IL6assay.jpg)

**Figure 9: Luciferase assay of the minimal interleukin-6 biosensor.** The amount of luminescence observed after incubation with a dilution series of interleukin-6 for 1, 3, 14 and 22 hours.

![](https://static.igem.org/mediawiki/parts/0/07/T--UCopenhagen--results-IL10assay.jpg)

**Figure 10: Luciferase assay of the minimal interleukin-10 biosensor.** The amount of luminescence observed after incubation with a dilution series of interleukin-10 for 1, 3, 14, 22 and 44 hours.

No correlation between interleukin concentration and luminescence was observed for either strain at any incubation time. Thus, the biosensors did not function at the given interleukin concentrations in the dilution series, under the conditions of the experiment.

Interestingly, we noticed that the level of luminescence obtained for the IL-6 biosensor is 3-10 times higher than for the IL-10 biosensor. This suggests that the IL-6 biosensor might be constitutively signalling. One possible explanation is that there was affinity between the two receptor proteins even when the interleukin was not present. This would further infer that the two receptor proteins were localized to the same subcellular compartments. Alternatively, the receptor protein with the transcription factor had been partially degraded which would allow the transcription factor to activate expression of the reporter.

For both biosensors, the amount of luminescence varied over time with the highest intensity recorded after 14 hours of incubation and lower intensity at shorter and longer incubation times. This might reflect that the cells were in different growth phases at the different times of the luciferase assays.

#### Subcellular localization of receptors

In order for our biosensors to work, the receptor proteins need to be localized to the plasma membrane of *S. cerevisiae*. To investigate whether the subcellular localization of the proteins could be the explanation for the dysfunction of our biosensors, confocal fluorescence microscopy was carried out.

Yeast strains that constitutively express super-folding green fluorescent protein (sfGFP) linked to the C-terminal of the receptor proteins were constructed (see table with strains for localization assays) to track the localization of the proteins. After confirming the strains for chromosomal integration of the receptor-GFP fusion constructs, the strains were grown in liquid media and observed with confocal fluorescence microscopy.

##### IL-6 receptor proteins

 Flourescence

 Brightfield

sIL-6R-Nub-sfGFP

![](https://static.igem.org/mediawiki/parts/7/73/T--UCopenhagen--results-loca22_1flour.jpg)

sgp130(D1-D3)-Cub

![](https://static.igem.org/mediawiki/parts/a/a9/T--UCopenhagen--results-loca21fluor.jpg)

**Figure 11a: Subcellular localization of sgp130(D1-D3)-Cub** Confocal laser scanning microscopy suggests possible localization of sgp130(D1-D3)-Cub in the endoplasmic reticulum (a), with some protein also localizing to the plasma membrane (b). Inclusion bodies/vacuoles (c) are visible on the brightfield image.

**Figure 11b: Subcellular localization of sIL-6R-Nub**  The majority of sIL-6R-Nub-sfGFP localizes to inclusion bodies (a) near the plasma membrane of *Saccharomyces cerevisiae* cells. Little to none of the protein can be found localized to other subcellular compartments, with only faint localization uniformly in the cytosol (c) and plasma membrane (b).

Confocal laser scanning microscopy suggests that both sgp130-Cub and IL-6R-Nub exhibit low degrees of localization to the plasma membrane, yet none of them localize there perfectly. Both localize in greater abundance in the endoplasmic reticulum. IL-6R-Nub seemed to have a higher preference for localization in inclusion bodies or cytosol. This apparent overlap in localization of sgp13-Cub and IL-6R-Nub could confirm the hypothesis inspired by results from luciferase assays, i.e., that both proteins are localized in the same compartment, which would be a necessary prerequisite for them to bind even in absence of IL-6.

However, findings from the confocal fluorescence microscopy cannot lead to rejection of the other hypothesis – that the system is always signalling due to the partial degradation of one of the proteins, thus, allowing the transcription factor to reach the nucleus at all times. Results from the confocal fluorescence microscopy indicate that the problems with the dysfunctional biosensor might be caused by unsatisfactory localization to the plasma membrane.

##### IL-10 receptor proteins

 Flourescence

 Brightfield

sIL-10R1-Cub-sfGFP

![](https://static.igem.org/mediawiki/parts/c/c6/T--UCopenhagen--results-loca25fluor.jpg)

sIL-10R2-Nub-sfGFP

![](https://static.igem.org/mediawiki/parts/c/c0/T--UCopenhagen--results-loca26fluor.jpg)

**Figure 12a: subcellular localization of sIL-10R1-Cub.** sIL-10R1-Cub-sfGFP is shown to primarily localize to the plasma membrane (a) of *S. cerevisiae* cells and faintly to moderately in the endoplasmic reticulum (b). Small inclusion bodies (c) can be observed near the plasma membrane.

**Figure 12b subcellular localization of sIL-10R2-Nub-sfGFP.** sIL-10R2-Nub-sfGFP shows localization within the entirety (a) of the *S. cerevisiae* cell and the plasma membrane (b) without distinction of which subcellular compartments that the protein resides in.

According to confocal microscopy, sIL-10R1-Cub seemed to localize in the endoplasmic reticulum, plasma membrane and inclusion bodies. However, localization of sIL-10R2-Nub in the cell was uniform, or with a preference for the cytosolic and nuclear compartments. According to the luciferase assay, the biosensor was not functioning. Localization of sIL-10R2-Nub is a highly likely reason for dysfunction, as both sIL-10R1-Cub and sIL-10R2-Nub must be present at the plasma membrane in order to associate in the presence of IL-10 and trigger intracellular signalling.

As a conclusion, confocal fluorescence microscopy, together with luciferase assays, show that improving protein localization of the IL-10 receptor could be a plausible way to improve the IL-10 biosensor. However, causes of spontaneous signalling of the IL-6 biosensor need to be elucidated to guide further modifications of the biosensor.

## Intermediate biosensor design

In this design, further amplification of the biosensor signal is achieved by adding an enzymatic step to the signaling pathway. Whereas in the minimal design, one pair of activated receptors can only release one transcription factor, in this design one activated receptor pair can lead to the release of multiple membrane bound transcription factors. Here, the extracellular IL-receptors were fused with the Wsc1 transmembrane domain and either the C- or N-terminal part of a split-TEV protease. Upon receptor association in the presence of IL molecules, the two halves reconstitute to a functioning TEV protease. This protease recognizes the membrane bound transcription factor LexA-VP16 and releases it from the membrane.

![](https://static.igem.org/mediawiki/2020/0/04/T--UCopenhagen--TMDesign.png)

**Figure 13: Intermediate biosensor design.** Split-TEV protease complementation leading to cleavage of a TEV-recognition sequence near LexA-VP16, thereby, releasing LexA-VP16 to initiate gene expression.

#### Localization of the receptors

In order for the receptor proteins of the intermediate and the advanced design to work, they needed to be localized to the plasma membrane of *S. cerevisiae.* Subcellular localization seemed to be part of the cause that the biosensors of the minimal design did not work. Therefore, localization assays of the receptor proteins for the intermediate and advanced design were carried out to gauge the chance of success for these designs.

Strains constitutively expressing superfolder green fluorescent protein linked to the C-terminal of the receptor proteins were created (table 2), grown and observed with confocal laser scanning microscopy.

##### Receptor proteins

 Flourescence

 Brightfield

sIL-6R-nTEV-sfGFP

![](https://static.igem.org/mediawiki/parts/0/05/T--UCopenhagen--results-loca30fluor.jpg)

sIL-1RAcP-nTEV-sfGFP

![](https://static.igem.org/mediawiki/parts/a/a8/T--UCopenhagen--results-loca32fluor.jpg)

sIL-10R2-nTEV-sfGFP

![](https://static.igem.org/mediawiki/parts/e/eb/T--UCopenhagen--results-loca34fluor.jpg)

**Figure 14a subcellular localization of sIL-6R-nTEV.** sIL-6R-nTEV-sfGFP can be observed to likely localize primarily to the plasma membrane (a), both in small and medium-sized inclusion bodies (b). Some protein localizes in the endoplasmic reticulum (c).

**Figure 14b subcellular localization of sIL-1RAcP-nTEV** sIL-1RAcP-nTEV-sfGFP seemingly localizes uniformly throughout the cytosol (a) of the cell in what looks like inclusion bodies, seemingly without localizing, or localizing very little, to the plasma membrane (b).

**Figure 14c subcellular localization of sIL-10R2-nTEV** sIL-10R2-nTEV-sfGFP localizes primarily to inclusion bodies (a) near the plasma membrane of the *S. cerevisiae cells.* This is particularly evident in the top right and bottom cells. However, much of the protein is also localized within the cytosol (b). While unclear, it may seem that some of the protein localizes within the endoplasmic reticulum (c).

sIL-6R-nTEV localized preferentially to the plasma membrane, unlike sIL-1RAcP-nTEV which had a preference for the cytosolic and nuclear compartments. sIL-10R2-nTEV localized to the membrane in the form of inclusion bodies, but also localized within the cytosol.

sIL-6R-nTEV seems to localize as desired and could be possibly used as part of a IL-6 biosensor. In contrast, improvement in the localization of sIL-10R2-nTEV may be needed before it can perform its role in a IL-10 biosensor. Likewise, localization of sIL-1RAcP-nTEV needs to be significantly improved before it can function in a biosensor for IL-1.

#### Cleavage of membrane bound transcription factor

The results from the subcellular localization assays and the induction assays of the minimal design indicated that the receptor proteins of the intermediate and advanced design would have a poor chance of success without further optimization of subcellular localization and the design of the receptor proteins in general. Due to limited time, we chose to focus on characterizing the accessory proteins of the intermediate and advanced design, i.e. the membrane bound transcription factor in the intermediate design and the modified GPA1 proteins in the advanced design.

In the intermediate biosensor design, a split TEV protease releases transcription factors from the membrane upon receptor association and TEV reconstitution. For this, we constructed a transmembrane protein - an accessory protein with a transcription factor linked to a transmembrane domain through a flexible linker that had a cut site for the TEV protease. This experiment was designed in order to verify that a TEV protease could induce signalling by cleaving the flexible linker and releasing the transcription factor. For the experiment, we integrated the constitutively expressed accessory protein, an inducible TEV protease and a nanoLuc luciferase under the control of a synthetic LexAop promoter into *S. cerevisiae* as described above (see assembly 10 in table 2). The strain was grown to an OD600 of 5.1 and incubated at 30°C for 17 hours in either glucose or galactose + raffinose media to repress or activate the transcription of the TEV protease, respectively. Then, a luciferase assay was carried out with the culture.

![](https://static.igem.org/mediawiki/parts/e/ed/T--UCopenhagen--results-TMD-TFassay.jpg)

**Figure 15: Luminescence assay based on cleavage of membrane bound transcription factor.** Cells were expressed without (glucose media) and with (galactose+raffinose media) induction of the TEV protease. A 1300-fold increase in luminescence was seen upon induction of the TEV protease. Data shown is the average of three biological replicates.

## Advanced biosensor design

In the advanced design, amplification of the biosensor signal is achieved by hijacking the yeast pheromone pathway through a modified Gα subunit. The IL-receptors were fused with halves of a split-TEV protease that can cleave a modified GPA1 subunit. Upon association of the receptors, the two halves will reconstitute and cleave Gα, releasing the beta-gamma subunit that transduces the signal.

![](https://static.igem.org/mediawiki/2020/8/8b/T--UCopenhagen--Gpa1.png)

**Figure 16: Advanced biosensor design.** Split-TEV protease complementation leading to cleavage of a TEV protease cut site in GPA1, thereby, initiating gene expression through the yeast pheromone pathway.

#### Testing functionality of modified GPA1

For the third design to work, the modified GPA1 must be cleaved by the TEV protease, dissociate from the Gβγ complex and induce signalling through the pheromone pathway. This experiment was designed to test whether expression of a TEV protease, induced by galactose media, could activate signalling through the pheromone pathway. The experiment was further designed to investigate whether the native functions of GPA1 were conserved in the modified GPA1 proteins either with or without induction of a TEV protease. For more information about the different versions of GPA1, visit the  [modeling](https://2020.igem.org/Team%3AUCopenhagen/Model)and  [engineering success](https://2020.igem.org/Team%3AUCopenhagen/Engineering)pages.

The different GPA1 mutants were tested in the context of an adenosine biosensor employing the human adenosine GPCR receptor A2A(R199A) (unpublished) in *S. cerevisiae*, previously developed in the Kampranis lab. Compared to the original biosensor, the strains in this experiment had an inducible TEV protease and the wt GPA1 was exchanged with the modified GPA1 proteins (see table 2, strains 14.1-14.3 and 15). The strains were grown to an OD600 of 0.4 and incubated in either glucose or galactose + raffinose media at 30°C for 17 hours. Next, the amount of signalling was measured with a luciferase assay.

![](https://static.igem.org/mediawiki/parts/e/ed/T--UCopenhagen--results-modifiedGPA1assays.jpg)

**Figure 17: Characterization of modified GPA1 proteins in adenosine biosensor.** The biosensors were grown in either glucose media or galactose + raffinose media which repressed or activated expression of a TEV protease. The biosensors were further grown with a dilution series of adenosine.

For all versions of GPA1, and at all concentrations of adenosine, the amount of luminescence was higher when cells were incubated in galactose + raffinose media compared to glucose media. This was also the case for the biosensor without any TEV cleavage site introduced to the GPA1 sequence. This infers that the rise in luminescence when the media was changed might be caused by other mechanisms than the cleavage of GPA1. Thus, the experiments neither confirmed nor rejected that the TEV protease was able to activate signalling through the cleavage of the GPA1 mutants.

Biosensors with version one and three of the GPA1 subunit showed very high amounts of luminescence compared to the biosensor with wt GPA1 even in glucose media. This might suggest that these versions of GPA1 are always causing signalling through the pheromone pathway. Mutation 2 showed very low amounts of luminescence compared to the biosensor with wt GPA1 even in galactose media. This might suggest that the Gα strongly inhibits all signalling, that the pheromone pathway has been shut down, or that the yeast has mutated so that the reporter expression is uncoupled from the pheromone pathway.

For the biosensor with wt GPA1, a correlation between adenosine concentration and luminescence was observed at a level that reproduced earlier results from the Kampranis lab when glucose was used as a carbon source. A similar tendency was not observed for any of the mutant GPA1 strains neither with glucose nor galactose + raffinose media. This infers that all the mutations cause a loss-of-function in the sense that the GPA1 is not able to transduce the signal from the A2A GPCR.

The comparative experiment of effects of glucose media and galactose + raffinose media on mutant GPA1 strains and the wt GPA1 strain yielded mixed results. It was not possible to confirm nor reject whether TEV protease can trigger signalling via cleavage of mutant GPA1. However, the experiment did show that mutant GPA1 strains lost the ability to transduce signal from the A2A GPCR, while it was maintained in the wildtype GPA1.

## Conclusion/discussion

To sum up our achievements in the laboratory, we managed to:

* ✧ Test the functionality of our minimal design biosensor
* ✧ Investigate the subcellular localization of 7 of our receptor proteins
* ✧ Verify the functionality of the accessory protein, the membrane bound synthetic transcription factor, in the intermediate design.
* ✧ Test various functions of the modified GPA1 proteins for the advanced design, eg. the ability to signal after being cleaved by a TEV protease and the ability to transduce the signal from a GPCR.
* ✧ Clone 54 constructs
* ✧ Create 21 new yeast strains

Our induction assays showed that the IL-6 and IL-10 biosensors of our minimal design were not able to sense different concentrations of the respective biomarkers. For the interleukin-6 biosensor, the system seemed to be constantly signalling. This might indicate that the dysfunctionality could, to some extent, be explained by an affinity between the receptor proteins in the absence of IL-6. To understand why our biosensors were dysfunctional, the subcellular localization of our modified receptors and accessory proteins were investigated using confocal fluorescence microscopy. In order for our receptor proteins to work, they had to be localized at the plasma membrane of S. cerevisiae. While none of the modified proteins solely localized to the plasma membrane, they were all expressed to some extent, some more than others. For the minimal design, sgp130(D1-D3)-Cub-sfGFP only localized faintly to the plasma membrane, while the soluble sIL-6R-Nub-sfGFP almost solely was found in inclusion bodies. This might explain the dysfunctionality of the minimal IL-6 biosensor.

sIL-10R1-Cub-sfGFP localized primarily to the plasma membrane and endoplasmic reticulum but sIL-10R2-Nub-sfGFP localized unspecifically to the entire cell, mainly in the cytosolic compartment. This might, therefore, also explain the dysfunctionality of the minimal IL-10 biosensor.

The subcellular localization assays of sIL-6R-nTEV-sfGFP, sIL-1RAcP-nTEV-sfGFP, and sIL-10R2-nTEV-sfGFP all showed cytosolic localization and the presence of inclusion bodies, with only sIL-6R-nTEV-sfGFP localizing primarily to the plasma membrane in cells lacking inclusion bodies. However, this was seen for cells containing vacuoles, which may not be representative for the entire population of cells.

For the intermediate design, it was evident that the transcription factor in the form of LexA-VP16 containing a transmembrane domain and a TEV-recognition site in a flexible linker, was able to be cleaved by a TEV protease. Upon induction of TEV protease expression, and carrying out a luciferase assay, luminescence was increased over 1000-fold. This indicated that the protein functioned as intended. In hindsight, the experiment could have been designed to include a control strain without the inducible TEV protease as well as a strain without the membrane bound transcription factor. A western blot of a tagged version of the protein after incubation in either media could further verify that the protein is cleaved by the TEV protease. The next step in characterizing the membrane bound transcription factor would be to investigate whether it can be cleaved by a membrane bound split TEV protease.

For the advanced design, the functionality of the modified GPA1 proteins was compared to the functionality of the wt GPA1 in an adenosine biosensor. To test how cleavage of the modified GPA1 proteins affected signalling through the pheromone pathway, expression of a TEV protease was controlled by changing media from glucose to galactose. The signalling increased in all the modified GPA1 proteins as expected. Surprisingly, the biosensor with wt GPA1 also showed increased signalling upon change of the media. It is, therefore, inconclusive whether the GPA1 proteins functioned as intended. To shed further light on the functionality of the GPA1 proteins, the experiment would have to be designed differently. This could include making a control strain without the TEV protease or utilizing another method for inducing expression of the TEV protease where the carbon source is kept constant. One possible explanation for the elevated level of luminescence with galactose media is that the pPGK1 promoter, which regulates the expression of the GPA1 protein, might be less active when cells are grown in galactose media. Since GPA1 is inhibiting signalling through the pheromone pathway when bound to the Ste4-Ste18(Gβγ)-complex, less GPA1 present would lead to more signalling through the pathway.

Finally, the ability of the modified GPA1 proteins to transduce a signal from a GPCR was investigated. While the results showed that the adenosine biosensor with wt GPA1 responded to the adenosine concentrations as expected, this response was not observed for any of the adenosine biosensors with modified GPA1 proteins. Thus, the modifications introduced a loss-of-function in respect to signal transduction in GPCR-based biosensors. The investigations as to whether the modifications introduced a gain-of-function in regards to signalling through cleavage by the TEV protease were inconclusive.