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-# Background
+# Background
+
+
+## Phage-Assisted continuous evolution
+Life on earth was shaped by evolution by which the different species adapted to their environment over the course of countless generations and millions of years. In the lab it is possible to speed up this process and guide it towards a user-defined goal, which is called directed evolution. Here, the goal is usually to steer proteins or nucleic acids towards adapting new functions or improve their main features like the catalysis of a certain reaction. Directed evolution of biomolecules generally involves three main steps. First, we take the molecule of interest and introduce mutations—small changes that have the potential to improve, or impair, its function. From this pool of mutants, we then need to identify those that perform better, either by screening—measuring how well they carry out their function, and picking those that perform best—or by selection, where only the mutants that perform their function well enough are allowed to pass. Finally, once we've identified promising variants, we repeat the cycle again and again. If this sounds like a long and tedious process, that's because it is. Each of these steps can take several days, requiring the constant attention of scientists. 
+
+To make directed evolution more efficient without compromising any steps, researchers have turned to a natural system that thrives on mutation, selection, and iteration—viruses. More specifically, the spotlight is on viruses that infect bacteria, known as bacteriophages. Like viruses that infect humans and animals, bacteriophages invade their host cells, hijack the bacterial machinery, and produce new viral particles to continue the infection cycle. During replication, some phages naturally acquire mutations because the bacterial replication process is prone to errors. For example, the bacteriophage M13, which infects E. coli (a common model bacterium), generates a mutation in about 1 out of every 200 phages due to these replication errors (10.1534/genetics.109.106005).
+Now, imagine if we could link the replication of a phage to the activity of our biomolecule of interest. We could then leverage the phage's natural traits to drive the continuous evolution of the biomolecule. Phages carrying better-performing variants would replicate faster, infect more bacteria, and increase the presence of those improved variants. Meanwhile, the replication errors would ensure that new variants constantly enter the evolutionary race.
+
+Fortunately, scientists figured out how to do this back in 2011 creating the Phage-Assisted Continuous Evolution (PACE) approach and sparing us the need to reinvent the wheel [^pace_paper]. They used the fact that M13 phage needs a specific protein, pIII, to infect bacteria. Phages without pIII replicate poorly, while those that produce more pIII generate more infectious particles. This created an opportunity: by coupling the activity of our biomolecule to pIII production, phages carrying more active biomolecule variants would produce more pIII and, as a result, replicate more efficiently.
+
+The key to this system is linking the activity of the biomolecule to the expression of a gene coding for pIII. This challenge, central to synthetic biology, has been successfully addressed for a range of biomolecular functions, including DNA, RNA, and protein binding, as well as enzyme activity (10.1093/nar/27.4.919, 10.1073/pnas.262420099). To achieve this, we use an accessory plasmid (AP), which carries a regulatory circuit that ties the biomolecule’s activity to the expression of pIII. This plasmid is placed in the host bacteria, allowing for controlled expression of pIII based on the biomolecule's performance. At the same time, we introduce the gene encoding the biomolecule of interest into the genome of the phage, which we call a selection phage (SP). To ensure that only phages with active biomolecules can propagate, we remove the native gene for pIII from the phage genome. This means the phage can only replicate if the biomolecule inside the phage activates the regulatory circuit in the host cell, leading to pIII production. This clever setup ensures that only phages carrying functional or improved biomolecule variants can replicate and propagate, effectively driving the selection process.
+
+To make this a self-sustaining evolution system, a few more adjustments are needed. We provide the phages with a steady supply of fresh bacteria in a "lagoon" - a chamber where bacteria and phages grow. This ensures that newly generated phages always have hosts to infect. Simultaneously, we flush out old bacteria and phages, ensuring that only those phages that replicate quickly enough can survive. In this setup, better-performing variants will thrive and outcompete others, driving evolution forward. To speed up this process, a special mutation plasmid (MP) is introduced into the bacteria. This plasmid increases the already high mutation rate of the phages, significantly accelerating the pace of evolution and allowing researchers to quickly refine biomolecules. Over time, phages with beneficial mutations accumulate and become dominant in the population. These mutations can then be identified by sequencing the phages. Finally, one often uses orthogonal methods to confirm the activity of the evolved biomolecule.
+
+Sometimes, the activity of the biomolecule we're evolving is too weak, resulting in slow phage propagation that makes it difficult to run PACE effectively. In such cases, we use a modified approach called Phage-Assisted Non-Continuous Evolution (PANCE). Unlike PACE, where phages and bacteria are continuously refreshed in a bioreactor (lagoon), PANCE works in a batch culture. In this method, phages are introduced into a batch of bacteria and allowed to propagate over a set period. Once the cycle is complete, a sample of the culture is taken and used to inoculate a new batch of bacteria, repeating the process. This non-continuous setup eliminates the risk of phages being removed from the culture faster than they can propagate, enabling evolution even when the activity of the biomolecule is low. PANCE is often used as a preliminary step before PACE, allowing us to first evolve the biomolecule to a higher activity level, which is then sufficient for running continuous evolution in the PACE system.
+
+## References
+[^pace_paper]:Esvelt KM, Carlson JC, Liu DR. A system for the continuous directed evolution of biomolecules. Nature. 2011;472: 499–503. doi:10.1038/nature09929
+