A Model For Selecting An Environmentally Responsive Trait: Evaluating Micro-scale Fitness Through UV-C Resistance and Exposure in Escherichia coli.

Abstract:

Experimental evolution of microorganisms in controlled microenvironments serves as a powerful tool for understanding the relationship between micro-scale microbial interactions as well as local-to global-scale environmental factors. In response to iterative and targeted environmental pressures, mutagenesis drives the emergence of novel phenotypes. Current methods to induce expression of these phenotypes require repetitive and time intensive procedures and do not allow for the continuous monitoring of conditions such as optical density, pH and temperature.

To address this shortcoming, an Automated Dynamic Directed Evolution Chamber is being developed. It will initially produce Escherichia coli cells with an elevated UV-C resistance phenotype that will ultimately be adapted for different organisms as well as studying environmental effects.

A useful phenotype and environmental factor for examining this relationship is UV-C resistance and exposure. In order to build a baseline for the device’s operational parameters, a UV-C assay was performed on six E. coli replicates with three exposure fluxes across seven iterations. The fluxes included a 0 second exposure (control), 6 seconds at 3.3 J/m2/s and 40 seconds at 0.5 J/m2/s. After each iteration the cells were regrown and tested for UV-C resistance. We sought to quantify the increase and variability of UV-C resistance among different fluxes, and observe changes in each replicate at each iteration in terms of variance.

Under different fluxes, we observed that the 0s control showed no significant increase in resistance, while the 6s/40s fluxes showed increased resistance as the number of iterations increased. A one-million fold increase in survivability was observed after seven iterations. Through statistical analysis using Spearman’s rank correlation, the 40s exposure showed signs of more consistently increased resistance, but seven iterations was insufficient to demonstrate statistical significance; to test this further, our experiments will include more iterations. Furthermore, we plan to sequence all the replicants. As adaptation dynamics under intense UV exposure leads to high rate of change, it would be useful to observe differences in tolerance-related and non-tolerance-related genes between the original and UV resistant strains.