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Once we discovered the potential toxicity of our project, we searched high and low to find ways to eliminate ethylene glycol (EG) as much as possible. In the scientific literature, we found that no wild-type E. coli can utilize EG (1). However, we did find an E. coli mutant, called Strain E-15 EG3, from the University of Barcelona, that is able to grow on ethylene glycol alone (1). While the University of Barcelona's paper on Strain E-15 EG3 was published nearly 30 years ago, we contacted the authors and asked for the strain to perform our own testing. The researchers were able to find the strain in one of their freezers and ship it to our lab. We have dubbed the rediscovery of the strain "Freezer Archaeology".
By performing various experiments on MG1655 including directed evolution, which selects the cells with the most fitness in a population, and ethyl methylsulfonate (EMS), which introduces random mutations to a population, the scientists learned that the main contributors in the degradation were propanediol oxidoreductase and glycolaldehyde dehydrogenase. These two enzymes are expressed at low levels in MG1655 but not at all in DH5α. We worked to overexpress these enzymes in MG1655 through directed strain engineering, and also clone the enzymes from MG1655 for more control, by performing our own directed evolution, EMS, rational engineering, and site-directed mutagenesis experiments.
