Grant: $132,375 - National Institutes of Health - Aug. 13, 2009
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Award Description: Our Specific Aims are to assess the roles that effective population size (Ne) and nutrient availability play in IS element expansion in intracellular bacteria. We are testing these hypotheses with a laboratory evolution experiment. We have 12 Escherichia coli populations that have been evolving in batch culture for ?3100 generations (575 days to date) under four experimental treatments: (1) low Ne/rich medium, (2) high Ne/rich medium, (3) low Ne/minimal medium, and (4) high Ne/minimal medium. Therefore, we are evolving three replicate ?intracellular? bacterial populations (treatment #1), three ?free-living? populations (treatment #4), and six ecologically intermediate populations (treatments #2 and #3). We expect that IS elements will be significantly more prevalent in the small populations than the large populations (because natural selection will be less effective at purging high IS load genotypes from the small populations, due to enhanced genetic drift), and in the rich medium populations than the minimal medium populations (because some biosynthetic genes will be superfluous in rich medium, and thus selectively neutral territory for IS insertion). IS elements have been active in the evolving populations during the first 3000 generations, although neither population size nor diet consistently correlate to IS load differences among populations. However, the genomes of intracellular bacteria often shrink following host restriction. Since we are evolving ?intracellular? bacteria in the lab, they too may be experiencing genome shrinkage. If so, then our absolute IS load estimates under represent the actual contribution of ISs in these populations. Therefore, the ARRA Administrative Supplement will allow us to size the genomes of isolates from each evolving population. This information, combined with the IS load estimates, will be used to calculate the proportion of each genome that is comprised of IS elements, thus providing a richer understanding of the evolutionary dynamics of IS elements in the small populations and nutrient rich environments of intracellular symbionts and pathogens.
Project Description: We have ordered and received the pulsed-field gel electrophoresis apparatus (Bio-Rad CHEF-DR II, purchased with funds from Fordham University), which will be used to estimate genome sizes in the evolving populations. We are currently getting the apparatus set up, ordering the necessary expendables (e.g., restriction enzymes, DNA ladders, agarose), and finalizing the protocol details.
Jobs Summary: Job title: Laboratory technician (full-time). Job description: Manage the on-going long-term E. coli evolution experiment. This includes overseeing: (1) the day-to-day duties of the experiment (e.g., the daily bacterial transfers, aliquoting media), (2) the three undergraduate students who participate in various aspects of the experiment, and (3) the collection of data from the evolving populations (i.e., quantifying the IS element copy numbers, sizing the genomes from bacterial isolates, and assessing the prevalence of intragenomic recombination). (Total jobs reported: 0)
Project Status: Not Started
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