Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.
Food waste disposal and transportation of commodity chemicals to the point-of-need are substantial challenges in military environments. Here, we propose addressing these challenges via the design of a microbial consortium for the fermentation of food waste to hydrogen. First, we simulated the exchange metabolic fluxes of monocultures and pairwise co-cultures using genome-scale metabolic models on a food waste proxy. We identified that one of the top hydrogen producing co-cultures comprised Clostridium beijerinckii NCIMB 8052 and Yokenella regensburgei ATCC 43003. A consortium of these two strains produced a similar amount of hydrogen gas and increased butyrate compared to the C. beijerinckii monoculture, when grown on an artificial garbage slurry. Increased butyrate production in the consortium can be attributed to cross-feeding of lactate produced by Y. regensburgei. Moreover, exogenous lactate promotes the growth of C. beijerinckii with or without a limited amount of glucose. Increasing the scale of the consortium fermentation proved challenging, as two distinct attempts to scale-up the enhanced butyrate production resulted in different metabolic profiles than observed in smaller scale fermentations. Though the genome-scale metabolic model simulations provided a useful starting point for the design of microbial consortia to generate value-added products from waste materials, further model refinements based on experimental results are required for more robust predictions.
SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment.
The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory exercise uses size exclusion chromatography (SEC) and ion exchange (IEX) chromatography to separate a mixture of four different proteins. Students use an SEC chromatogram and corresponding SDS-PAGE gel to understand how protein conformations change under different conditions (i.e. native and nonnative). Students explore strategies to separate co-eluting proteins by IEX chromatography. Using either cation or anion exchange, one protein is bound to the column while the other is collected in the flow-through. In this exercise, undergraduate students gain hands-on experience with experimental design, buffer and sample preparation, and implementation of instrumentation that is commonly used by experienced researchers while learning and applying the fundamental concepts of protein structure, protein purification, and SDS-PAGE. V C 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):60-68, 2017.
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