Integrated Instrumentation to Advance Army Center for Synthetic Biology Workflows
Abstract
Publicly Releasable Project Summary/Abstract Filamentous fungi make up one of the largest and most diverse contributors of biomass on earth. They are common in soil and are critical for the delivery of nutrients to crops and are the major biological contributor to the corrosion of textiles, infrastructure (cement, wood, etc), and metals (by degrading rust-protective coatings). They are also harnessed in biotechnology, including biomanufacturing (pharmaceuticals, fuels, chemicals, food), materials (artificial leather, engineered living materials), and bioremediation (plastics, toxins). Despite their potential, they represent one of the most underutilized resources in biology. Modern techniques to study and engineer microorganisms for biotechnological purposes have been well established for bacteria and yeast but not for filamentous fungi. Most biotechnological efforts in filamentous fungi have been limited to discovering new natural products in a handful of species for pharmaceutical purposes. While new fungi are being discovered that have potential across many applications, the challenge lies in that only a few model species are tractable and most filamentous fungi are difficult to engineer, not least due to the lack of genetic engineering tools and measured genetic parts. Typically, the characterization of genetic parts is done using fluorescent reporters and flow cytometry. Cytometry is the method of choice over microcopy because 100,000s of cells can be evaluated in a single experiment, thus providing critical cell-to-cell variation. However, standard flow cytometers are not suitable to analyze fungal samples because the filamentous mycelia can clog the nozzle of standard flow cytometers and are sensitive to the sheer stresses of the flow. Fungi are also multicellular structures, which cannot be analyzed through cytometry. Sometimes, this problem is addressed by treating fungal multicellular structures to break them up, but this loses important spatial and functional information, does not work for all species, makes the cell sick, and still results in clumps and experimental variability. Different regions of mycelial structures can also exhibit significant differences in gene expression. The only flow cytometer capable of quantifying fluorescence in various morphologies of an organism in high-throughput manner is the COPAS Vision, a large-particle flow cytometry manufactured by Union Biometrica. The fungal mycelia are encapsulated in microgels prior to the flow allowing entire intact mycelia to be visualized and used for sorting. The COPAS Vision can image and sort particles of up to 1500 ?m, and bright field and fluorescence images can be captured for each event. COPAS is the only equipment that can enable the characterization of genetic parts and circuits in filamentous fungi, making the first step to render filamentous fungi tractable for various synthetic biology applications. This equipment has not yet been applied to synthetic biology research, but we have performed proof-of-principle experiments with the company with species of relevance. This piece of equipment will be housed at MIT and will be used as part of the Army Center for Synthetic Biology (PreMADE/CHARMME), of which 11 universities (MIT, Stanford, Northwestern, UT-Austin, U Southern Mississippi, Kansas State, Johns Hopkins, UIUC, Columbia, Caltech, U Colorado) with broad regional representation participate along with DoD national labs. This Center provides a structure by which protocols and training will be made available to DoD researchers. Further, the protocols, strains, and genetic engineering tools that result from this work will be broadly disseminated.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Aug 02, 2023
- Source ID
- W911NF2310331
Entities
People
- Christopher Voigt
Organizations
- Army Contracting Command
- Massachusetts Institute of Technology
- United States Army