iGEM 2019 — Team Ruperto_Carola

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# Team:Ruperto Carola/Notebook

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  Home](https://2019.igem.org/Team%3ARuperto_Carola)
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  Team

  [Team Members](https://2019.igem.org/Team%3ARuperto_Carola/Team)
  [Attributions](https://2019.igem.org/Team%3ARuperto_Carola/Attributions)
  [Sponsors](https://2019.igem.org/Team%3ARuperto_Carola/Sponsors)
* ![](https://static.igem.org/mediawiki/2019/9/9b/T--Ruperto_Carola--Project.png)
  Project

  [Description](https://2019.igem.org/Team%3ARuperto_Carola/Description)
  [Notebook](https://2019.igem.org/Team%3ARuperto_Carola/Notebook)
  [Contribution](https://2019.igem.org/Team%3ARuperto_Carola/Contribution)
  [Results](https://2019.igem.org/Team%3ARuperto_Carola/Results)
  [Demonstrate](https://2019.igem.org/Team%3ARuperto_Carola/Demonstrate)
  [Improve](https://2019.igem.org/Team%3ARuperto_Carola/Improve)
* ![](https://static.igem.org/mediawiki/2019/5/57/T--Ruperto_Carola--Parts.png)
  Parts

  [Parts Overview](https://2019.igem.org/Team%3ARuperto_Carola/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ARuperto_Carola/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ARuperto_Carola/Composite_Part)
* [![](https://static.igem.org/mediawiki/2019/2/25/T--Ruperto_Carola--Safety.svg)
  Safety](https://2019.igem.org/Team%3ARuperto_Carola/Safety)
* ![](https://static.igem.org/mediawiki/2019/1/11/T--Ruperto_Carola--Human.png)
  Human Practices

  [Overview](https://2019.igem.org/Team%3ARuperto_Carola/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/HP/integrated)
  [Education & Engagement](https://2019.igem.org/Team%3ARuperto_Carola/Public_Engagement)
  [Expert Interviews](https://2019.igem.org/Team%3ARuperto_Carola/HP/Expert_Interviews)
  [Collaborations](https://2019.igem.org/Team%3ARuperto_Carola/Collaborations)
* ![](https://static.igem.org/mediawiki/2019/7/73/T--Ruperto_Carola--Modelling.png)
  In Silico

  [Model](https://2019.igem.org/Team%3ARuperto_Carola/Model)
  [Software](https://2019.igem.org/Team%3ARuperto_Carola/Software)
* [![](https://static.igem.org/mediawiki/2019/0/0c/T--Ruperto_Carola--Form.png)
  JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Ruperto_Carola)

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* Team

  [Team Members](https://2019.igem.org/Team%3ARuperto_Carola/Team)
  [Attributions](https://2019.igem.org/Team%3ARuperto_Carola/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3ARuperto_Carola/Description)
  [Design](https://2019.igem.org/Team%3ARuperto_Carola/Design)
  [Experiments](https://2019.igem.org/Team%3ARuperto_Carola/Experiments)
  [Notebook](https://2019.igem.org/Team%3ARuperto_Carola/Notebook)
  [Contribution](https://2019.igem.org/Team%3ARuperto_Carola/Contribution)
  [Results](https://2019.igem.org/Team%3ARuperto_Carola/Results)
  [Demonstrate](https://2019.igem.org/Team%3ARuperto_Carola/Demonstrate)
  [Improve](https://2019.igem.org/Team%3ARuperto_Carola/Improve)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ARuperto_Carola/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ARuperto_Carola/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ARuperto_Carola/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ARuperto_Carola/Part_Collection)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/HP/integrated)
  [Education & Engagement](https://2019.igem.org/Team%3ARuperto_Carola/Public_Engagement)
  [Expert Interviews](https://2019.igem.org/Team%3ARuperto_Carola/HP/Expert_Interviews)
  [Collaborations](https://2019.igem.org/Team%3ARuperto_Carola/HP/Collaborations)
* In Silico

  [Model](https://2019.igem.org/Team%3ARuperto_Carola/Model)
  [Software](https://2019.igem.org/Team%3ARuperto_Carola/Software)
* [Safety](https://2019.igem.org/Team%3ARuperto_Carola/Safety)
* [JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Ruperto_Carola)

# Weekly reports


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# Team:Ruperto Carola/Experiments

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  Home](https://2019.igem.org/Team%3ARuperto_Carola)
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  Team

  [Team Members](https://2019.igem.org/Team%3ARuperto_Carola/Team)
  [Attributions](https://2019.igem.org/Team%3ARuperto_Carola/Attributions)
  [Sponsors](https://2019.igem.org/Team%3ARuperto_Carola/Sponsors)
* ![](https://static.igem.org/mediawiki/2019/9/9b/T--Ruperto_Carola--Project.png)
  Project

  [Description](https://2019.igem.org/Team%3ARuperto_Carola/Description)
  [Notebook](https://2019.igem.org/Team%3ARuperto_Carola/Notebook)
  [Contribution](https://2019.igem.org/Team%3ARuperto_Carola/Contribution)
  [Results](https://2019.igem.org/Team%3ARuperto_Carola/Results)
  [Demonstrate](https://2019.igem.org/Team%3ARuperto_Carola/Demonstrate)
  [Improve](https://2019.igem.org/Team%3ARuperto_Carola/Improve)
* ![](https://static.igem.org/mediawiki/2019/5/57/T--Ruperto_Carola--Parts.png)
  Parts

  [Parts Overview](https://2019.igem.org/Team%3ARuperto_Carola/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ARuperto_Carola/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ARuperto_Carola/Composite_Part)
* [![](https://static.igem.org/mediawiki/2019/2/25/T--Ruperto_Carola--Safety.svg)
  Safety](https://2019.igem.org/Team%3ARuperto_Carola/Safety)
* ![](https://static.igem.org/mediawiki/2019/1/11/T--Ruperto_Carola--Human.png)
  Human Practices

  [Overview](https://2019.igem.org/Team%3ARuperto_Carola/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/HP/integrated)
  [Education & Engagement](https://2019.igem.org/Team%3ARuperto_Carola/Public_Engagement)
  [Expert Interviews](https://2019.igem.org/Team%3ARuperto_Carola/HP/Expert_Interviews)
  [Collaborations](https://2019.igem.org/Team%3ARuperto_Carola/Collaborations)
* ![](https://static.igem.org/mediawiki/2019/7/73/T--Ruperto_Carola--Modelling.png)
  In Silico

  [Model](https://2019.igem.org/Team%3ARuperto_Carola/Model)
  [Software](https://2019.igem.org/Team%3ARuperto_Carola/Software)
* [![](https://static.igem.org/mediawiki/2019/0/0c/T--Ruperto_Carola--Form.png)
  JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Ruperto_Carola)

[![](https://static.igem.org/mediawiki/2019/8/8a/T--Ruperto_Carola--logo_navbar_test1.png)](https://2019.igem.org/Team%3ARuperto_Carola)

* Team

  [Team Members](https://2019.igem.org/Team%3ARuperto_Carola/Team)
  [Attributions](https://2019.igem.org/Team%3ARuperto_Carola/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3ARuperto_Carola/Description)
  [Design](https://2019.igem.org/Team%3ARuperto_Carola/Design)
  [Experiments](https://2019.igem.org/Team%3ARuperto_Carola/Experiments)
  [Notebook](https://2019.igem.org/Team%3ARuperto_Carola/Notebook)
  [Contribution](https://2019.igem.org/Team%3ARuperto_Carola/Contribution)
  [Results](https://2019.igem.org/Team%3ARuperto_Carola/Results)
  [Demonstrate](https://2019.igem.org/Team%3ARuperto_Carola/Demonstrate)
  [Improve](https://2019.igem.org/Team%3ARuperto_Carola/Improve)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ARuperto_Carola/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ARuperto_Carola/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ARuperto_Carola/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ARuperto_Carola/Part_Collection)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/HP/integrated)
  [Education & Engagement](https://2019.igem.org/Team%3ARuperto_Carola/Public_Engagement)
  [Expert Interviews](https://2019.igem.org/Team%3ARuperto_Carola/HP/Expert_Interviews)
  [Collaborations](https://2019.igem.org/Team%3ARuperto_Carola/HP/Collaborations)
* In Silico

  [Model](https://2019.igem.org/Team%3ARuperto_Carola/Model)
  [Software](https://2019.igem.org/Team%3ARuperto_Carola/Software)
* [Safety](https://2019.igem.org/Team%3ARuperto_Carola/Safety)
* [JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Ruperto_Carola)

# Protocols

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

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Ruperto Carola/Results

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* [![](https://static.igem.org/mediawiki/2019/e/e7/T--Ruperto_Carola--Home.png)
  Home](https://2019.igem.org/Team%3ARuperto_Carola)
* ![](https://static.igem.org/mediawiki/2019/a/a6/T--Ruperto_Carola--Team.png)
  Team

  [Team Members](https://2019.igem.org/Team%3ARuperto_Carola/Team)
  [Attributions](https://2019.igem.org/Team%3ARuperto_Carola/Attributions)
  [Sponsors](https://2019.igem.org/Team%3ARuperto_Carola/Sponsors)
* ![](https://static.igem.org/mediawiki/2019/9/9b/T--Ruperto_Carola--Project.png)
  Project

  [Description](https://2019.igem.org/Team%3ARuperto_Carola/Description)
  [Notebook](https://2019.igem.org/Team%3ARuperto_Carola/Notebook)
  [Contribution](https://2019.igem.org/Team%3ARuperto_Carola/Contribution)
  [Results](https://2019.igem.org/Team%3ARuperto_Carola/Results)
  [Demonstrate](https://2019.igem.org/Team%3ARuperto_Carola/Demonstrate)
  [Improve](https://2019.igem.org/Team%3ARuperto_Carola/Improve)
* ![](https://static.igem.org/mediawiki/2019/5/57/T--Ruperto_Carola--Parts.png)
  Parts

  [Parts Overview](https://2019.igem.org/Team%3ARuperto_Carola/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ARuperto_Carola/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ARuperto_Carola/Composite_Part)
* [![](https://static.igem.org/mediawiki/2019/2/25/T--Ruperto_Carola--Safety.svg)
  Safety](https://2019.igem.org/Team%3ARuperto_Carola/Safety)
* ![](https://static.igem.org/mediawiki/2019/1/11/T--Ruperto_Carola--Human.png)
  Human Practices

  [Overview](https://2019.igem.org/Team%3ARuperto_Carola/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/HP/integrated)
  [Education & Engagement](https://2019.igem.org/Team%3ARuperto_Carola/Public_Engagement)
  [Expert Interviews](https://2019.igem.org/Team%3ARuperto_Carola/HP/Expert_Interviews)
  [Collaborations](https://2019.igem.org/Team%3ARuperto_Carola/Collaborations)
* ![](https://static.igem.org/mediawiki/2019/7/73/T--Ruperto_Carola--Modelling.png)
  In Silico

  [Model](https://2019.igem.org/Team%3ARuperto_Carola/Model)
  [Software](https://2019.igem.org/Team%3ARuperto_Carola/Software)
* [![](https://static.igem.org/mediawiki/2019/0/0c/T--Ruperto_Carola--Form.png)
  JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Ruperto_Carola)

[![](https://static.igem.org/mediawiki/2019/8/8a/T--Ruperto_Carola--logo_navbar_test1.png)](https://2019.igem.org/Team%3ARuperto_Carola)

* Team

  [Team Members](https://2019.igem.org/Team%3ARuperto_Carola/Team)
  [Attributions](https://2019.igem.org/Team%3ARuperto_Carola/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3ARuperto_Carola/Description)
  [Design](https://2019.igem.org/Team%3ARuperto_Carola/Design)
  [Experiments](https://2019.igem.org/Team%3ARuperto_Carola/Experiments)
  [Notebook](https://2019.igem.org/Team%3ARuperto_Carola/Notebook)
  [Contribution](https://2019.igem.org/Team%3ARuperto_Carola/Contribution)
  [Results](https://2019.igem.org/Team%3ARuperto_Carola/Results)
  [Demonstrate](https://2019.igem.org/Team%3ARuperto_Carola/Demonstrate)
  [Improve](https://2019.igem.org/Team%3ARuperto_Carola/Improve)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ARuperto_Carola/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ARuperto_Carola/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ARuperto_Carola/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ARuperto_Carola/Part_Collection)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3ARuperto_Carola/HP/integrated)
  [Education & Engagement](https://2019.igem.org/Team%3ARuperto_Carola/Public_Engagement)
  [Expert Interviews](https://2019.igem.org/Team%3ARuperto_Carola/HP/Expert_Interviews)
  [Collaborations](https://2019.igem.org/Team%3ARuperto_Carola/HP/Collaborations)
* In Silico

  [Model](https://2019.igem.org/Team%3ARuperto_Carola/Model)
  [Software](https://2019.igem.org/Team%3ARuperto_Carola/Software)
* [Safety](https://2019.igem.org/Team%3ARuperto_Carola/Safety)
* [JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Ruperto_Carola)

# Results

Here we present to you the results of our project. Have fun reading :)

Darwinian evolution is a machine for innovation, incredibly powerful in generating enormous landscapes of possible lifeforms but a bit disadvantageous if you want to employ it not at the universe timescale, but rather at a timescale of an iGEM project. As you may have noticed, evolution takes time. Like A LOT.
We, therefore, have joined the global effort of numerous other synthetic biologists to make evolution malleable to human manipulation within the lab setting and its efficiency available for obtaining desired outcomes and products.
We (and an army of much more talented and rich synthetic biologists – self-proclaimed and real) are wasting a lot of our personal life-time to change that and thus make lab life easier for future generations. Especially now, with climate change arising, and people cutting funding for research in renewable energies and other useful stuff that could help us, we are kinda running low on time to adapt to the rapidly and unpredictably changing world. Thus, we really need to develop tools for controllable and efficient directed evolution.
Having this great goal in our minds, we, the iGEM team Ruperto-Carola decided to make our first little steps towards a better life and start small by evolving a very old friend of us humans, especially of the world-famous German breweries and also many Nobel laureates in physiology and medicine (2001, 2006, 2009, 2013, and 2016) – the Brewer’s Yeast (also known as S. cerevisiae).

Thereby we first started looking for a promising structure to evolve and quickly landed at the ste2 receptor (you can find nearly the entire story – with only a ton painful facts left out – at the project description). Disclaimer: it started with pork.

But we are at the results section here – ‘pork-free zone’, so let's talk yeast.

To start evolving our receptor we decided to search for alternatives to generation of gene blocks, which besides high cost can also lead to some experimental challenges like high amounts of clonings and transformations that need to establish a functional mutant library as well as difficulties with parallel selection (generation of such a library could take several items to complete). To tackle the last issue we decided to focus on in vivo mutagenesis. One of the relatively new and already well-described methods is the so-called OthoRep. This method developed by the Liu lab relies on an engineered error-prone DNA-polymerase that specifically replicates an orthogonal cytoplasmic high-copy plasmid (P1) completely sparing the yeast chromosomes and hence adding no survival disadvantage. The P1 plasmid is a stabilized cytoplasmic linear plasmid, into which genes can be introduced to be subjected to the elevated mutation rate and therefore accelerated evolution. Ravikumar2018. The Cheng Liu lab at the University of California in Irvine kindly provided us with a yeast strain F102, that contained the whole OrthoRep system.

Since we are a slightly paranoid team, we began our labwork by further characterizing the strain we received for growth in the relevant conditions to our planned experiments. Therefore we first studied the growth of our newly obtained yeast strains in different media - the rich YPD, the full synthetic (similar to YPD in a sense like protein shakes resemble actual food - they might contain everything you need to survive, but after a month of such a diet you might end up somewhat unhappy). The original F102 strain harbors 3 auxotrophies: histidine, leucine, and uracil. To start preparations for our actual experiments with mutation characterization we used the linearized FDP plasmid containing the ura3 and leu2 genes as an integration cassette into the P1 plasmid. This would enable us to select for strains with successful integration via synthetic dropout media lacking uracil and leucine.

The following graph presents the growth experiments for the original strain and the FDP containing strain in different media:

![](https://static.igem.org/mediawiki/2019/7/73/T--Ruperto_Carola--img-Ex4.png)

The graphs clearly demonstrate, that our yeasts are similar to sloths when it comes to productivity: they grow visibly slower if they have to produce nucleotides or amino-acids themselves. Funfact: for some reasons the yeasts did not appreciate full synthetic supplement medium. The reason is either the age of the medium (several generations of iGEMers have probably seen it standing there in the darkest corner of the lab) or their personal religious beliefs. Nevertheless, this is also an issue often mentioned in literature.

To further characterize our OrthoRep strain we’ve subjected it to another challenge – growth in media lacking only leucine and media missing both - leucine and uracil. The following result (see figure 2) finally proves that yeasts are unmotivated beasts – even the deletion of one nucleotide from the medium significantly reduces its growth rate.

![](https://static.igem.org/mediawiki/2019/d/db/T--Ruperto_Carola--img-Characterization.png)

Now with the complete knowledge of what our yeasts need to happily grow and reproduce we decided to take it from them and see how they deal with it. Some people may call it playing God, we call it directed evolution. To verify the capability of OrthoRep to mutagenize and diversify target genes and to determine the mutation rate by the orthogonal polymerase we decided to go a familiar route and first made our yeast mutagenize Ura3 under selective pressure. For that we used 5-FOA, a chemical substance which gives you a lot of genotoxic stress if you are a yeast and just happen to use your intact Ura3 gene. The mechanism here is very simple – in the normal form the 5-FOA is not harming anyone but being activated (or metabolized to 5-fluorouacil) by Ura3, it starts killing your replication system, that is normally really useful if you want to continue living and proliferating. Here, the colonies were kept in the delta Leu medium for minimizing the selective pressure on ura3, thus forcing them to mutate the gene instead of losing the plasmid. To test the limits of our yeasts and kindly encourage them to evolve loss of function of their ura3, we treated them with increasing concentrations of 5-FOA – in human terms, everything from a light squeeze to the pressure needed to turn coal into diamonds.

And become diamonds they did (as you can see from the following graphs).

![](https://static.igem.org/mediawiki/2019/b/b7/T--Ruperto_Carola--img-c_foa_1.png)

![Second slide](https://static.igem.org/mediawiki/2019/b/b5/T--Ruperto_Carola--img-05_foa_1.png)

![Third slide](https://static.igem.org/mediawiki/2019/8/8a/T--Ruperto_Carola--img-06_foa_1.png)

![Fourth slide](https://static.igem.org/mediawiki/2019/a/a7/T--Ruperto_Carola--img-07_foa_1.png)

![Fifth slide](https://static.igem.org/mediawiki/2019/2/27/T--Ruperto_Carola--img-08_foa_1.png)

![Sixth slide](https://static.igem.org/mediawiki/2019/5/57/T--Ruperto_Carola--img-09_foa_1.png)

![Seventh slide](https://static.igem.org/mediawiki/2019/6/64/T--Ruperto_Carola--img-1_foa_1.png)

[Previous](#carouselOne)
[Next](#carouselOne)

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![Control Assay 2](https://static.igem.org/mediawiki/2019/9/90/T--Ruperto_Carola--img-c_foa_2.png)

![Second slide](https://static.igem.org/mediawiki/2019/2/29/T--Ruperto_Carola--img-0.5_foa_2.png)

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[Previous](#carouselTwo)
[Next](#carouselTwo)

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![](https://static.igem.org/mediawiki/2019/f/fd/T--Ruperto_Carola--img-08_foa_2.png)

![](https://static.igem.org/mediawiki/2019/e/ee/T--Ruperto_Carola--img-09_foa_2.png)

![](https://static.igem.org/mediawiki/2019/a/a0/T--Ruperto_Carola--img-1_foa_2.png)

As one can see, from the presented results the complete inactivation of Ura3 even without selective pressure took multiple weeks to obtain. The colonies 1-3, 4-6 and 7-8 each lie 2 weeks apart from one another. So when the 1-6 at the end of the weeks 6-8 of growth began to adapt the latter still died by contact with low amounts of 5-FOA. But once achieved, the mutations remained relatively stable.

As one can see, from the presented results the complete inactivation of Ura3 even without selective pressure took multiple weeks to obtain. The colonies 1-3, 4-6 and 7-8 each lie 2 weeks apart from one another. So when the 1-6 at the end of the weeks 6-8 of growth began to adapt the latter still died by contact with low amounts of 5-FOA. But once achieved, the mutations remained relatively stable.

So far so good - even after some time we got adaptation. Nevertheless, you probably have noticed a few additional interesting moments in the graphs: first – not all the colonies adapted to the changing conditions, second – the colonies needed a relatively long time for the initial adaptation, third – the lower concentrations appear to be harder to adapt to. If you see the aforementioned points– congratulations! You are not the only one wondering about these results. We welcome you at the world of strange yeasts. You, just as we did a few weeks ago discovered that not all yeasts are fantastic. Thankfully, we will spare you of the weeks of crying in frustration trying to optimize the assay and direct you directly to the people who will gently guide you through this weird stuff - the mathematicians.

Disclaimer: the following calculations might contain some weird integrals and normalized spherical yeasts in vacuum. For the sake of theirs and your sanity all the calculations are done in the units of c=\bar{h}=\gamma=1.
Now, OrthoRep gives our yeast awesome mutagenesis superpowers. Sadly, it also gives us terrible mathematician-brain-melting episodes. To stop our brains from melting, we tried to grab a model from the literature. Here, we quickly found that there were no ready made models fitting our system. Instead, we had to rewrite our models in the language of evolutionary game theory – the theory of pitting lots of hapless individuals against each other in a cruel survival game (c.f. The Hunger Games {jup, we promised no more crossovers, but really this reference just pleads to be here!}).

Coming back to the main mathematical problem of this story – OrthoRep is a high copy plasmid and needs to be treated inside an evolving system, which is non trivial - for non-mathematicians: now we know why nearly nobody looked at this kind of system before. The closest analogy would be mixed-strategy evolutionary games - every cell keeps a probability distribution of strategies (genotypes), given by its set of plasmids. So we treat it as one while formulating our yeast population dynamics. The individual yeasts in our population play games against each other, which determine their fitness and thus, their propensity to mutate. The goal of the game in our situation is simple – can you survive under 5-FOA? Yes? Then you win, reproduce, mutate. No? Then you die, end of story. More complex games arise when there is significant interaction between individuals in the population, say if individual yeast were to communicate via quorum sensing. In our case, yeast are blissfully ignorant of each other, while feeling the pressure put on them by our 5-FOA. This makes things much easier for us and allows us to set up a model which we can actually – huge surprise for such a model – solve exactly. Fitting this model nicely explained the growth of our yeast without the selective pressure as well as the general adaptation to 5-FOA. However, it did not tackle the slow initial adaptation and the observed differences between the effects of the highest and the lowest concentration of 5-FOA.

Now, we have forgotten one important detail: 5-FOA wreaks havoc on our DNA replication. More 5-FOA means less plasmid. Less plasmid means less mutagenesis, means less adaptation, even more so, because our plasmid resides in the cytoplasm. Less nucleotides to work with then leaves us stranded. Possible effects contributing to this result might be first the inhibition of nucleotide synthesis by 5-FOA, which "conserves mutations", as well as the large threshold due to multiple plasmids being present in the cell at the same time. The more complete explanation with the corresponding calculations can be obtained from our model.

As our constant copy-number model cannot cope with inhibition of nucleotide synthesis - constant copy-number, duh! - we needed our model to adjust. Therefore, we moved our model from constant to arbitrary copy-numbers - yes, you’ve read that right, all the way up to infinity - and introduced a probability of plasmid replication dependent on the genotype. Of course, going up to infinity would not only melt our math-guy’s brain, but also his computer, so they had to “truncate their state-space”, whatever that means. The final model can indeed deal with inhibition of replication by 5-FOA as well as arbitrary other perturbations to DNA synthesis.

Thus using the obtained knowledge together with the calculations you can use such ura3 5-FOA titration assays to determine the mutation rate of the in vivo system in any lab and thus account for changes and optimize the assays.

Now having this issue sorted out one has enough information to start evolving the receptor towards detecting the target peptide. To provide the community with a suitable tool for that we complemented our theoretical and practical assessments of the OrthoRep system with an ste2 cloned into the FDP integration cassette, so that further generations of iGEMmers might work on closing the gap between the small and large peptide detection tools.

And become diamonds they did (as you can see from the following graphs).

### Cell-Cell Communication

Various types of cells can exchange information by producing ligands dependent on different surrounding stimuli. Hence, they communicate with each other. The idea is to cultivate the general mechanism to create a network of single cells being able to receive and transmit information with one another.
We utilized a cleavable, synthetic receptor tail, coupling peptide binding on the yeast cell to enable protein release from the membrane.
Receptor binding leads to a conformational change exposing the C-terminal tail for arrestin adapters and subsequently for a TEV Protease coupled to arrestin.
![](https://static.igem.org/mediawiki/2019/7/74/T--Ruperto_Carola--cccc.svg)

GPCRs can show different affinities for arrestin resulting from possible variations of earlier phosphorylation. Previous works showed, that it is possible to increase affinity for β arrestin by coupling the C-terminal AVPR2 GPCR tail (v2-tail) to the receptor of interest.
The part BBa\_K3173007 contains a NLS behind a protease cleavage site and the v2-tail. It was successfully assembled by extension PCR from oligos <60 bp.
Theoretically one can exchange any module, e.g. leaving out the NLS or specifying another protease cleavage site.
One can couple a variety of GPCR´s upstream BBa\_K3173007 and therefore different POI´s downstream are consequently being released by receptor binding.
We used it to build BBa\_K3173010 and BBa\_K3173014 coupling to couple Ste2 to GFP (BBa\_K3173010) and the controllable Transcription factor camTA (BBa\_K3173014).
The downstream protein is released by protease cleavage at the TCS. The protease itself is recruited to the membrane by coupling it to arrestin.
Because the yeast cells don’t express β arrestin it can be used as an orthogonal system. At the same time yeast do express α arrestins (Ldb19, Rod1, Rog3) binding to Ste2 in a similar manner.
We used BBa\_K3173011 to genomically tag the arrestins.
Because the protease binding is specific for receptor binding it can be used to target not a single but multiple synthetic BBa\_K3173014 like constructs. Only the receptor itself has to be exchanged to teach the yeast cell to understand a new word. To teach it speaking another word one can exchange the Protein downstream of BBa\_K3173007.