| Abstrakt: |
A Hudson Control Group, Inc. ProLink Express™ robotic workcell to conduct plasmid-based functional proteomics is being developed for optimization of protein open reading frames (ORF). The initial phase of this project is to design and assemble a Xantus liquid handler from Sias, Inc. modified by Hudson so that a workcell track component can be placed within the Xantus® gripper tool work area. The liquid handler is designed to produce plasmids using the Qiagen Turbo® plasmid preparation kit. This design allows processing of up to four 96-well plates in one run. The procedure eliminates disposable tips and provides an advanced wash system to prevent cross contamination. To evaluate liquid handler operation, a mutagenized cellulase F ORF plasmid library was prepared from wild-type cellulase F (Chen, H.; Li, X.-L.; Blum, D. L.; Ximenes, E. A.; Ljungdahl, L. G. CelF of Orpinomyces PC-2 has an intron and encodes a cellulase (CelF) containing a carbohydrate-binding module. Applied Biochemistry and Biotechnology 2003, 105–108, 775–785; Li, X.-L.; Chen, H.; Ljungdahl, L. G. Two cellulases, CelA and CelC, from the polycentric anaerobic fungus Orpinomyces strain PC-2 contain N-terminal docking domains for a cellulase–hemicellulase complex. Applied and Environmental Microbiology 1997, 63(12), 4721–4728) using a novel Invitrogen Gateway® cloning strategy. For the automated reproducibility run, the average yield of plasmid was 5.35μg per well from 1.347mL of starting culture. Four plates were processed automatically on the liquid handler in 374min compared to at least 441min for the same plate operations performed manually. The quality and quantity of plasmids prepared on the liquid handler made the implementation of the following workcell protocols possible: DNA sequencing, in vitro transcription/translation, and transformation of bacterial and yeast strains for protein expression. [Copyright &y& Elsevier] |
