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Directed Evolution

Evolution occurs naturally by selection pressure, but in an overall slow pace. To speed up the process for certain desired traits, it is possible to re-passage cells to grow on a certain type of media, or expose certain mutagens to increase the variation in the cells to select for surviving mutants. Our team has carried out both of these selection pressures in hopes to isolate an ethylene glycol utilizing bacterium.

History

Mutants of E. coli able to grow on propylene glycol were selected for ethylene glycol enzymatic breakdown after ethyl methane sulfonate (EMS) mutagenesis. The culture conditions strongly selected for ethylene glycol utilizing mutants, by having ethylene glycol as the sole carbon source in liquid culture. Through directed evolution, colonies were continuously streaked on ethylene glycol plates for three more generations, in order to isolate ethylene glycol degrading mutants. Through spectrophotometric assays, increased activities of glycolaldehyde reductase and glycolaldehyde dehydrogenase were observed in the ethylene glycol isolates.

Genome Sequencing

In progress ...

Tecan Experiments

References

1. Boronat, Albert, Caballero, Estrella, and Juan Aguilar. “Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli.” Journal of Bacteriology, Vol. 153 No. 1, pp. 134-139, January 1983.
2. Andrianantoandro, Ernesto, Subhayu Basu, David K. Karig, and Ron Weiss. "Synthetic biology: new engineering rules for an emerging discipline." Nature - Molecular Systems Biology. (2006): n. page. Web. 29 Aug. 2012. .

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 <h1>Tecan Experiments</h1>
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-The Tecan experiments with MG1655 and DH5α show us that the ethylene glycol does not hinder the growth and development of the strains, as long as it is mixed with LB media. The growth curves all had the same shape, independent of the amount of ethylene glycol in solution. We chose a broad, nearly exponential range of ethylene glycol concentrations to allow a broad range to test the toxicity. We attempted to find the lower limit of toxicity due to a saturation of ethylene glycol. However, we had not reached it. In our engineered strain, we will not expect to see a concentration of ethylene glycol above 150mM, so we can expect our strain to be able to live in an environment with a concentration as high as that.
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-After seeing that ethylene glycol does not pose a threat to MG1655 and DH5a, we subjected the Barcelona strain to the same broad range of ethylene glycol. We sought out to find the most efficient concentration of ethylene glycol for this strain, as a guideline for the efficient concentration of EG for our engineered strain. While analyzing the data, we realized that we have to define efficiency more clearly. Efficiency can mean faster growth on low amounts of ethylene glycol or it could mean a higher optical density after a certain amount of time, where it reaches the stationary phase. We saw that once the ethylene glycol concentration reaches a certain threshold (49.34 mM), the growth curves are all the same in terms of time when the stationary phase has been reached. We saw that some of the E. coli were efficient at low concentrations, making us focus on the fast growth efficiency at lower concentrations of EG because the LC-cutinase will not degrade quickly enough to produce 49.34 mM in a cell’s solution. Now, we have an ongoing experiment where we re-passage cells between ethylene glycol media at 30 mM. We discuss this in more detail in our directed evolution section.
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Team:UC Davis/Project/Directed Evolution

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