Molecular-based methods for the mark-recapture of microorganisms
Abstract
Microorganisms are the most abundant and diverse forms of life on Earth. While some are the causative agents of human and agricultural diseases, many microorganisms are beneficial and ensure the stability and function of engineered, environmental, and host-associated ecosystems. Over the past decade, bioinformatic tools have advanced to the point where it is now possible to describe the genetic diversity of microorganisms in a range of habitats. Yet, it is still difficult to track the fate of a lineage, which limits our ability to understand fundamental aspects of microbial biology that are of societal importance. For example, there are very few tools that can be used to estimate the dispersal of cells, the metabolic cost of antibiotic resistance, or the response of microbes to climate change, especially when focal organisms are embedded in diverse communities containing thousands of species. In this proposal, we describe a molecular-based mark-recapture approach that will overcome some of these challenges. Our pipeline involves the insertion of unique barcodes into conserved and non-essential regions of bacterial genomes. These labeled strains will then be used in simple competition trials in test tubes as well as multi-species experiments in complex habitats like soil and the gut. In this DURIP proposal, we demonstrate proof-of-concept with preliminary data and outline a request for equipment that will refine our method by increasing efficiency and quantitative sensitivity. The necessary equipment includes a clean bench, fluorometer, and a liquid handling robot that will be used for the engineering and maintenance of barcoded bacterial strains. We are also requesting funds to purchase a digital droplet PCR machine, which will allow us to determine absolute counts of barcodes with a high degree of accuracy and precision. DURIP support will enhance the quality of other DoD-funded projects. Specifically, the mark-recapture pipeline will allow us to 1) measure the fitness cost associated with virus resistance, 2) track probiotic bacteria in 3D-printed gut bioreactors, and 3) quantify the persistence of spore-forming bacteria in warming permafrost samples. DURIP funds will also enhance research-related education by supporting early career scientists, including a DoD SMART fellow. Development of the mark-recapture technology is directly relevant to the DoD mission as it can be readily adopted for the purpose of biosecurity and biodefense, including improved containment and tracking of pathogenic microorganisms.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Feb 09, 2023
- Source ID
- W911NF2310054
Entities
People
- Jay T. Lennon
Organizations
- Army Contracting Command
- Indiana University
- United States Army