edits

Author: CamDavidsonPilon

_posts/2020-09-18-building-a-bioreactor-introduction.md

@@ -24,7 +24,7 @@ While there a many uses of a bioreactor, our goal is going to be a technique cal
 
 Today, lab directed evolution is a tool used in modern microbiology. Importantly, because of the extremely short generation time of microbes, evolution occurs on the order of days to weeks (vs centuries for plants and animals). The engineers of lab directed evolution have a specific goal in mind, expressed as the phenotype of the organism, and they reverse engineer what the environment should look like to reach that goal. For example, if the desired phenotype is heat tolerance, the engineers will slowly expose the microbes to higher and higher temperatures: the microbes that happen to be more heat tolerant will survive more often and continue to reproduce. However, if the engineers are not careful, they will inadvertently select for other traits too. Consider that the microbes are always competing for nutrients as well, and very quickly the initial nutrients will be consumed and the microbes will enter their stationary phase. So in the above scheme, if they only raised the temperature slowly over time, they would select for both heat tolerance *and* ability to survive well in the stationary phase. This may not be what is wanted. Instead, the culture is kept at a constant *cell  density.* This is typically a density where there is an abundance of nutrients for each microbe, so no selection occurs in this dimension (that's a lie: there is always a selection for increasing growth rate). To accomplish a constant cell density, periodically a small amount of liquid is removed and replaced by new media. Tuned correctly, this keeps the volume constant and the culture at a constant density. 
 
-An important point is that lab directed evolution is not *rational* design - the engineers don't care about the mechanisms of heat tolerance (yet), but they are allowing random gene-flipping plus selection to find a solution. Rational design would be something like finding a heat-tolerant phenotype in another organism, translating that into DNA, and inserting the DNA into the microbe. Sometimes, the rational design path is too complicated or difficult, and lab directed evolution feels like cheating. Directed evolution does the exploration (sheer population size) and exploitation (selective pressure) for us! On the other hand, often the rational design is sometimes easier: evolving a brand new enzyme has about 0 probability of occurring in a person's lifetime, but adding it to a microbe can be trivial. So both lab directed evolution and rational design are tools, and not replacements of each other.  
+An important point is that lab directed evolution is not *rational* design - the engineers don't care about the mechanisms of heat tolerance (yet), but they are allowing random gene-flipping plus selection to find a solution. Rational design would be something like finding a heat-tolerant phenotype in another organism, translating that into DNA, and inserting the DNA into the microbe. Sometimes, the rational design path is too complicated or difficult, and lab directed evolution feels like cheating. Directed evolution does the exploration (sheer population size) and exploitation (selective pressure) for us! On the other hand, often the rational design is sometimes easier: evolving a brand new enzyme has about 0 probability of occurring in a person's lifetime, but adding the enzyme's DNA to a microbe can be trivial. So both lab directed evolution and rational design are tools, and not replacements of each other.  
 
 Finally, my interest in lab directed evolution is that it is just so much in the spirit of "Controlled Mold" - literally controlling the phenotype of the mold! In the list below are some examples, including with molds, of more lab directed evolution applications:
 
@@ -34,15 +34,15 @@ I've been collecting the really cool uses of lab directed evolution, and use the
 
 1. Evolving a traditional brewer's yeast to thrive in new brewing environments. New environments for brewer's yeast could be higher/lower temperature, higher (alcohol, IBU, caffeine), concentration, lower pH, salt %.
 2. Yeast can only ferment a short list of carbohydrates. By slowly depleting yeast's traditional carbon sources, it forces the yeast to adapt to new carbon sources, like lactose. See [Attfield, 2006]
-3. Similarly, filamentous fungi can be evolved to consume new carbon sources, like raffinose, a sugar which is a cause of digestive discomfort after eating soybean *tempeh*.
+3. Similarly, filamentous fungi can be evolved to consume new carbon sources, like raffinose, a sugar which is a cause of digestive discomfort after eating soybean tempeh.
 4. Lactobacillus, used in sour beer production, can be evolved to be more alcohol, pH, or IBU tolerant.
 5. Some algae are facultative heterotrophs. Directed evolution can be used to evolve a stronger and faster heterotrophic metabolism.
 6. Triton Algae Innovations has used directed evolution to evolve heme in algae. They accelerated the process by flashing the microbes with UV light which caused a higher mutation rate.
 7. Algae can be evolved to produce more carotenoids by changing the light conditions, see [Fu, 2013]
 8. Improving yeast culture density, as demonstrated in [Wong, 2018]
 9. Improving growth rates after rational design. When modifying the genes of a microorganism though modern genetic engineering, the growth rate is typically lowered due to new proteins or metabolites being constructed. By subjecting the organism to an environment with abundant nutrients, over time, the population will evolve to increase its growth rate.
 10. Improving metabolite production after rational design. After adding the genes of carotenoid production to yeast but wanting a higher yield, [Reyes, 2013] exploited the antioxidant of carotenoids. They exposed the yeast to high levels of hydrogen peroxide. The yeast evolved to counteract the hydrogen peroxide by producing more carotenoids.
-11. The original inventors of the morbidostat [Toprak, 2013] were interested in antibiotic resistance in bacteria. They subjected E. coli to a slowly increasing level of antibiotics, and after two weeks, the bacteria had grown resistance to the highest antibiotic concentration in their experiment design.
+11. The original inventors of the morbidostat [Toprak, 2013] were interested in antibiotic resistance in bacteria. They subjected *E. coli* to a slowly increasing level of antibiotics, and after two weeks, the bacteria had grown resistance to the highest antibiotic concentration in their experiment design.
 12. In [Ekkers, 2020], the authors hint at evolving an *anticipatory* response. Amazing!
 
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