CAN Teams: Univ. of Illinois, Univ. of Montana, USC, Univ. of Wisconsin, JPL, Univ. of Colorado, U.C.-Riverside, NASA Goddard
The GeoBioCell is a microfluidics based synthetic ecology, which allows the experimenter delicate control over nutrient flow and gradients, while at the same time spatially partitioning microbes into demes where they can mix and interact.
The goal of these carefully controlled experiments is to quantify how environmental interactions can drive genome instability and evolution in populations, on laboratory time scales.
Newly designed and fabricated microfluidic devices with porous media (called GeoBioCells) are being used to determine the effects of chemical stress fields on enhancement rates of microbial adaptation and evolution. The GeoBioCells were fabricated using standard photolithography methods. They each consist of a hexagonal well array with interconnecting channels, bound on either side by channels that deliver nutrients and stressor chemicals. The boundary channels are separated from the well array by either a nanoporous barrier wall etched in silicon, or a polymerized hydrogel wall emplaced in a PDMS microfluidic well array. These walls allow passage of chemicals, but prevent bacteria from leaving the interior well array and entering the boundary channels. Transport in the well array is diffusion controlled, so chemicals introduced in the boundary channels diffuse into the well array and establish a concentration gradient.
Two strategically chosen bacteria were chosen for evaluation, Escherichia coli and Shewanella oneidensis. The stressor antibiotic ciprofloxacin (Cipro) was chosen. Along with nutrients, it is being delivered to one boundary channel at different concentrations between 4X and 100X the minimum inhibitory concentration. The growth of E. coli and S. oneidensis are being evaluated in separate experiments along the concentration gradients of Cipro and nutrients as a function of time. Microbes that appear to adapt and grow at higher Cipro concentrations over time are being evaluated for genomic changes. Specifically, specific DNA segments where single nucleotide polymorphism (SNPs) mutations might occur (e.g., gyrA, mexF) are being sequenced.
Preliminary observations suggest that the number of E. coli and S. oneidensis cells increase over time in regions with lower Cipro concentration. In regions with higher antibiotic concentrations, cell numbers initially decrease and then fluctuate. The overall distribution of biomass in the porous media shows good correlation with the linear gradient of stress and nutrients at steady state. The area with the most abundant bacteria is the most optimal environment in the system and might indicate microchemical conditions that enhance survival and evolution. We are now evaluating effects of other environmental stresses, including pH, oxygen availability, and other antibiotics on microorganisms adaptation.
We initiated a collaborative research project with Dr. Elbert Branscomb (Illinois NAI), and Dr. Laurie Barge and Dr. Mike Russell at the NASA Jet Propulsion Laboratory in applying the GeoBioCell to test key predictions of Russell’s alkaline hydrothermal vent model. These predictions have to do with the ability of minerals in the precipitates formed by the collision between vent fluids and the Hadean ocean, and providing a membranous interface between them, to covert the thermodynamic disequilibria that are geophyscially established across those membranes into the critical chemical ‘internal’ disequilibria required to initiate life’s emergence – acting that is as nitrate and carbon dioxide reductases, and as pyrophosphate synthetases.