title: Design and construction of a first-generation high-throughput integrated robotic molecular biology platform for bioenergy applications.
abstract: The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly clone and express heterologous gene open reading frames in bacteria and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving microbial strains for biofuel production. The process involves the production of full-length complementary DNA libraries as a source of plasmid-based clones to express the desired proteins in active form for determination of their functions. Proteins that were identified by high-throughput screening as having desired characteristics are overexpressed in microbes to enable them to perform functions that will allow more cost-effective and sustainable production of biofuels. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. This review describes the design and implementation of an automated integrated programmable robotic workcell capable of producing complementary DNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. These operations will allow tailoring microbial strains to use renewable feedstocks for production of biofuels, bioderived chemicals, fertilizers, and other coproducts for profitable and sustainable biorefineries.
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Renewable Product Technology Research Unit, Peoria, IL 61604, USA. Stephen.Hughes@ars.usda.gov
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hughes
authors.forename: Tauseef R
authors.initials: TR
authors.lastname: Butt
authors.forename: Scott
authors.initials: S
authors.lastname: Bartolett
authors.forename: Steven B
authors.initials: SB
authors.lastname: Riedmuller
authors.forename: Philip
authors.initials: P
authors.lastname: Farrelly
mesh_terms: D057005:Bioengineering / Q000295:instrumentation; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods; D008967:Molecular Biology / Q000295:instrumentation* / Q000379:methods; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D012371:Robotics / Q000295:instrumentation*; D058615:Synthetic Biology / Q000295:instrumentation* / Q000379:methods