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Team:UC Davis - 2012.igem.org

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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 methanesulfonate (EMS) mutagenesis by the University of Barcelona [1]. 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

We are sequencing the genome of Strain E-15 EG3 so that we can see what changes are present between the MG1655 and this strain. Aligning the sequences will show single nucleotide polymorphisms (SNPs), as well as any deletions or insertions. From this, we will be able to apply the knowledge to a hybrid approach with the rational engineering. However, we have been unsuccessful in preparing the sequencing library, so it is still a work in progress as of now.

Tecan Experiments

We plated the EMS experiments (which we completed at Arcadia Biosciences) on 30 mM ethylene glycol agar plates. The first 55 colonies that appeared were selected, added to a replicate plate, and liquid cultured in 30 mM ethylene glycol media until they reached an OD of 0.5. Then, they were placed into Tecan wells to be tested for the fastest growth. We also included a set of Strain E-15 EG3 standards in each plate, allowing a comparison across plates for relative growth rates. We expect that the EMS caused some variation in each of the populations. However, they are probably minute variations, and they are likely to be similar in efficiency.

We also plated the liquid culture directed evolution that we had re-passaged 25 times prior. With the colonies from the plates, we will load a Tecan plate similar to the EMS testing and procedure, allowing us to compare between the EMS results and the normal Strain E-15 EG3 growth.

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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