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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, we repassaged Strain E-15 EG3 25 times in media that contained only ethylene glycol as the sole carbon source. We repassaged two trials, shown below as Tube 1 and Tube 2. Over the course of the experiment, we observed the growth rates to increase by 74.8% and 227.84% for each respective replicate.
After observing this increase in growth rate in the repassaged Strain E-15 EG3, we wondered if there were individual clones within each population that could potentially be "super" ethylene glycol utilizers. To test this, we took the final repassaging set and plated it on an ethylene glycol media plate. We then took the fastest growing colonies and plucked them to run in a plate reader. Of these colonies we see a diverse range of maximum OD600s, shown below. The ones where we observed the highest growth are highlighted in red.
We took the fastest growing colonies (highlighted in red) and did a more rigorous growth assay on them. After testing the highlighted colonies (#2, #23, and #26) with more replicates, we see a higher growth rate and maximum growth yield (up until stationary phase) as compared to the original Strain E-15 EG3 cells. We can conclude that repassaging the cells increased the growth rate and at least initially, the maximum growth yield 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 (below), the no exposure graph (0 min EMS exposure, left panel) 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.
To more accurately check the growth profile of the EMS mutants, we took the colonies highlighted in red and subjected them to a more rigorous test to confirm for enhanced ethylene glycol utilization.
After regrowing each "high achieving" mutant with multiple duplicates from the EMS graphs above, we cannot see any distinct differences between EMS treatment and no treatment. Rather, due to EMS, we can see that most 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.
Library Construction of Strain E-15 EG3
We decided to sequence Strain E-15 EG3 to see once and for all what the actual mutations/changes were in this strain that allowed it to utilize ethylene glycol at an efficient rate. To do this we created an transposase mediated Illumina library as described in our protocols section. Below we display our gel from our size selection step, and the Agilent Bioanalyzer trace during the quality checking steps of our library.
After construction and quality checking, we were sad to discover that our library was not at a high enough concentration to run on the Illumina MiSeq. Our total yield represented about 200ng, while the sequencing core required at least 1µg. We plan to repeat this process to try to achieve a higher yield so that we can correctly sequence this strain.
