Team:Ruperto Carola/Results
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.
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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Cell-Cell Communication
Orthogonal receptor-ligand pairs may be utilized for directed Co-Evolution. Different cells exchange information whether a ligand was bound triggering a response in the ligand producing cell. Hence, they communicate with each other. The idea is to not stop there but cultivate the general mechanism to create a network of single cells being able to interact with one another, receiving and transmitting information. We want to develop several receptor-ligand pairs or similar tools to create pathways that can be equipped, or so to say programmed, into the system.
By coupling peptide binding to the production of another distinct ligand the signal can be transmitted to another cell, which in turn also induces the expression of this additional ligand allowing for signal amplification by lateral cell-cell communication. Accordingly, the signal is amplified across the network. At the same time, sensitivity and selectivity may be increased by a cell receiving several inputs at once. The binding of multiple different peptides o their distinct receptors can also be coupled to a specific signal that indicates high specificity. A single cell receiving this combination of ligands results in the output signal, which in turn, may be amplified. While the detection of ASF related epitopes represent the input of data, the system is to be programmed to give a detectable signal as an output. This is one of many possibilities for how to program the network.
But once a signal is transmitted, the process is finished, and valuable information may be lost. Therefore, it is preferable to have the cell undergo some kind of more permanent change after receiving information in order to retain it. The system is to become something more sophisticated. Similar to abrain it should be able to memorize what kind of information it received and adapt future behavior accordingly. Speaking of cellular memory normally refers to epigenetic changes passed on to the next generation. Even though we want to stay in the same generation it may be possible to utilize epigenetic principles: A gene product can regulate its own expression by binding to a regulatory element of the operon thereby creating a positive feedback loop. If the protein is, in turn, faster expressed than degraded a steady-state is acquired.
On the other hand, there is something called structural inheritance which refers to the heritage of spatial cell structures from previous generations. In our case, we want to alter structures connected to the receiving organ, the membrane. This module would also allow us to investigate the idea of a body memory being separated from the brain but instead existing in changes inside the cells of the human body themselves- A topic hotly debated in Psychology.