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Toxicity of EG Graph for MG1655 and DH5a
We observed that EG is non-toxic to both DH5a and MG1655 cells, as evident from the growth of the two strains. Both strains were exposed to LB media containing varying amounts of EG, ranging from 0 mM to 150 mM.
E-15 EG3 in Ethylene Glycol Graph
Knowing that Strain E-15 EG3 utilizes ethylene glycol, we devised an experiment to test the optimal amount of ethylene glycol the strain would thrive in. From the graph, we deduced that 30 mM of ethylene glycol showed the highest growth rate, which also matched previous data provided in the referenced paper [1].
Arabinose Optimization for MG1655 and E-15 EG3
The point of this Tecan experiment was to see the optimal amount of arabinose in the different strains. In MG1655, it seems that all concentrations of arabinose give about the same maximum OD as the control with no arabinose. Strain E-15 EG3 shows a higher deviation in growth for 2 µM, relative to the 8 µM and 10 µM samples. However, all amounts of arabinose had a significant increase in OD due to the arabinose induction, compared to the no arabinose control. From our data, we see that the 2 µM, 8 µM, and 10 µM are the top three concentrations of arabinose in terms of the maximum OD. The K206000 data page shows that 10 µM has the maximum induction with arabinose, similar to our data observed from this experiment. These two sources demonstrated that 10 µM of arabinose had high induction, leading us to use that concentration in following experiments. The parts registry’s data page did not show optimal induction for 2 µM, but it had the highest maximum OD in our experiment, which is why we included it in our subsequent experiments too.
Directed Evolution of Strain E-15 EG3
For directed evolution, when we repassaged Strain E-15 EG3 in new media, we saw a general increase in growth rate over time. We ran two trials at the same time in two separate tubes, which we are going to call Tube 1 and Tube 2. For Tube 1, we saw a 74.8% increase in growth rate from the original strain and for Tube 2, we saw a 227.84 % increase, both over a time period of 285.95 hours.
We tested colonies #2, #23, and #26 from the repassaging experiments with multiple replicates to see if the increase in OD was by chance or a true increase in growth rate. From this graph, you can see that the groth rate of these evolved colonies was indeed higher than the original E-15 EG3.
After further testing the highlighted colonies (#2, #23, and #26) with more replicates, we concluded that repassaging the cells increased the growth rate of Strain E-15 EG3. This matches our previous assumption that directed evolution through repassaging can enhance growth rate.
Ethyl Methanesulfonate Results
The maximum OD was plotted for individual colonies of Strain E-15 EG3. The graph to the left shows no exposure to EMS, while the graph on the right shows exposure. We can see that EMS introduces unfavorable base changes, usually deleterious, thus decreasing the overall growth rate (shown in the graph to the right).However, there seems to be a few colonies, highlighted in red, that show a higher growth rate due to exposure to the treatment.
For MG1655, the no exposure graph still yields observable growth. Although we previously assumed that no wild type E. coli could utilize ethylene glycol, the data suggests that this phenotype may not be too difficult to achieve.
The colonies highlighted in red were subjected to more rigorous testing to confirm for enhanced ethylene glycol utilization.
From the graph, we cannot see any distinct differences between EMS treatment and no treatment. Rather, due to EMS, we can see that the untreated colonies showed a higher maximum OD, leading us to confirm that the EMS treatment most likely introduced more harm to the cells than improved it. Another approach would be to expose the cells to EMS for a shorter period of time, in hopes to search for a better ethylene glycol utilizing cell.
Construct Testing
The left bar in yellow represents the no plasmid control for the E-15 EG3, which we will use as a baseline for comparison with our constructs. The peach and purple bars are with just one enzyme – dehydrogenase. We wanted to see if one enzyme’s expression was sufficient to increase the utilization of ethylene glycol. Here, we see that it either hinders or marginally increases the growth. The next two bars, in dark purple and teal, represent the whole construct that we made. We expect to see an increase in ethylene glycol utilization with both constructs, and we see that this is true. Both of them have an increase over the E-15 EG3 no plasmid control. The J23101 (constitutive) variation of the construct had a 28% increase in ethylene glycol utilization, relative to the no plasmid control. From this data, we can say that we were able to increase the ethylene glycol degradation in the University of Barcelona’s E-15 EG3.
