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.

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:

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.

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