High-Throughput Sample Preparation Group
Significant technical and scientific challenges need to be overcome to develop a viable process that can utilize the potential of lignocellulosic biomass for production of liquid transportation fuel. Achieving these goals require the successful development and integration of numerous state of the art technologies. Our group develops and automates these processes to advance research in this area. We have implemented a variety of new technological capabilities, including high-throughput approaches for surveying enzyme diversity, production and functional analysis of enzyme systems, screening of thousands of natural and modified enzyme variants, and assessment of genetically selected or engineered microorganisms for integrated bioprocessing. Development of these integrated solutions/technologies in throughput form has led to higher efficiency in research related to plant cell wall structure and biosynthesis, biomass breakdown, and fermentation of monomers.
Bioreactors running in batch, chemostats, turbidostats, sequential processing, anaerobic, aerobic, microaerophilic, and extremophile culturing are being used for the diverse JBEI projects. Bench scale bioreactors are being used for controlled conditions for fluxomics, metabolomics, and fuels synthesis research and to produce large quantities of proteins when needed. OmniLog arrays are used for high-throughput phenotypic characterization with monitoring and dynamic controls allowing optimal biomass and fuel production with tuned media and conditions.
Many thousands of clones are required for JBEI's research needs. Gene synthesis is being used in concert with the development of efficient vectors and gene delivery methods for gene expression and characterization in prokaryotic and eukaryotic systems. Commercially available liquid handling robots were adapted to automate this process, which are capable of producing thousands of gene constructs per month.
No single surrogate host system can effectively express all proteins in active forms. Targets that are not tractable in established recombinant hosts are expressed in cell-free systems. Preparative reactions are carried out by In vitro transcription/translation (IVT) and wheat germ expression (WGEX). The IVT system is used for biochemical characterization and populating JBEI's microsystem-based protein characterization platform. Using affinity tags (via genetic tags), we purify hundreds of proteins per week. If a protein is not purified to homogeneity by affinity chromatography, gel filtration and ion exchange chromatography is used to follow-up and polishing. Electronic data capturing and applying quality control procedures, we have amassed a comprehensive database of preparative biochemical data, which is used for analysis of experimental results, troubleshooting, and protocol optimization.
Screening methods performed in research laboratories today involve low-throughput, tedious processes and require large amounts (several hundred micrograms) of enzymes or cells per experiment. We developed a high-throughput pipeline for rapidly screening thousands of natural and genetically modified enzymes at the microgram scale. This pipeline was constructed using commercially available components and is adaptable to be used across all research areas in JBEI. Exhaustive screening of naturally occurring and genetically-engineered enzymes, substrates, and products is being done by the Deconstruction Division; and, Feedstocks Division will assay thousands of carbohydrate active enzymes against hundreds of potential substrates to correlate and annotate activities. The Fuels Synthesis Division is evaluating enzymes that increase the efficiency of biofuels production.
