We are sequencing the K-12 Strain E-15 EG3 genome 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 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 the prep of the sequencing library, so it is still a work in progress as of now.  

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 K-12 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 K-12 Strain E-15 EG3 growth.