Articles by Jian Gao in JoVE
Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions Jian Gao1,2, Joelle Sasse1,2, Kyle M. Lewald1,2, Kateryna Zhalnina1,2, Lloyd T. Cornmesser1,2, Todd A. Duncombe3, Yasuo Yoshikuni2, John P. Vogel2, Mary K. Firestone4, Trent R. Northen1,2 1Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 2Joint Genome Institute, Department of Energy, 3Joint BioEnergy Institute, 4Department of Environmental Science Policy and Management, University of California This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.
Other articles by Jian Gao on PubMed
Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS Frontiers in Bioengineering and Biotechnology. | Pubmed ID: 26528471 Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design.
Application of Black Silicon for Nanostructure-Initiator Mass Spectrometry Analytical Chemistry. | Pubmed ID: 26741735 Nanostructure-initiator mass spectrometry (NIMS) is a matrix-free desorption/ionization technique with high sensitivity for small molecules. Surface preparation has relied on hydrofluoric acid (HF) electrochemical etching which is undesirable given the significant safety controls required in this specialized process. In this study, we examine a conventional and widely used process for producing black silicon based on sulfur hexafluoride/oxygen (SF6/O2) inductively coupled plasma (ICP) etching at cryogenic temperatures and we find it to be suitable for NIMS. A systematic study varying parameters in the plasma etching process was performed to understand the relationship of black silicon morphology and its sensitivity as a NIMS substrate. The results suggest that a combination of higher silicon temperature and oxygen flow rate gives rise to the formation of black silicon with fine pillar structures, whose aspect ratio are ∼ 8.7 and depth are
On-chip Integration of Droplet Microfluidics and Nanostructure-initiator Mass Spectrometry for Enzyme Screening Lab on a Chip. | Pubmed ID: 27957569 Biological assays often require expensive reagents and tedious manipulations. These shortcomings can be overcome using digitally operated microfluidic devices that require reduced sample volumes to automate assays. One particular challenge is integrating bioassays with mass spectrometry based analysis. Towards this goal we have developed μNIMS, a highly sensitive and high throughput technique that integrates droplet microfluidics with nanostructure-initiator mass spectrometry (NIMS). Enzyme reactions are carried out in droplets that can be arrayed on discrete NIMS elements at defined time intervals for subsequent mass spectrometry analysis, enabling time resolved enzyme activity assay. We apply the μNIMS platform for kinetic characterization of a glycoside hydrolase enzyme (CelE-CMB3A), a chimeric enzyme capable of deconstructing plant hemicellulose into monosaccharides for subsequent conversion to biofuel. This study reveals NIMS nanostructures can be fabricated into arrays for microfluidic droplet deposition, NIMS is compatible with droplet and digital microfluidics, and can be used on-chip to assay glycoside hydrolase enzyme in vitro.