![]() Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from more » commercial cocktails or from extracts of novel microorganisms. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. Lastly, we anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. Here, we demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). Integration of these steps minimizes the loss of reagents and products more » compared to that with conventional methods, which require multiple pipetting steps. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow. The system will be expanded in the future to include colony picking and/or actual sequencing. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The entire system will be integrated with one central controller that will direct each machine and the robot. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. A technician handled all movement of the 96-well sample plates between machines. Following purification and thermal cycling, an automated sequencing machine was more » used for the sequencing. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. ![]() This facility is novel because its development is based on existing standard biology laboratory equipment thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research.
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