title: A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly.
abstract: Trypanosoma brucei is the causative agent of human African trypanosomiasis and nagana in cattle. Recent advances in high throughput phenotypic and interaction screens have identified a wealth of novel candidate proteins for diverse functions such as drug resistance, life cycle progression, and cytoskeletal biogenesis. Characterization of these proteins will allow a more mechanistic understanding of the biology of this important pathogen and could identify novel drug targets. However, methods for rapidly validating and prioritizing these potential targets are still being developed. While gene tagging via homologous recombination and RNA interference are available in T. brucei, a general strategy for creating the most effective constructs for these approaches is lacking. Here, we adapt Gibson assembly, a one-step isothermal process that rapidly assembles multiple DNA segments in a single reaction, to create endogenous tagging, overexpression, and long hairpin RNAi constructs that are compatible with well-established T. brucei vectors. The generality of the Gibson approach has several advantages over current methodologies and substantially increases the speed and ease with which these constructs can be assembled.
authors: Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, 02912, United States.
authors: Michael R
authors: MR
authors: McAllaster
authors: Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, 02912, United States.
authors: Amy N
authors: AN
authors: Sinclair-Davis
authors: Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, 02912, United States.
authors: Nicholas A
authors: NA
authors: Hilton
authors: Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, 02912, United States. Electronic address: Christopher_degraffenried@Brown.edu.
authors: Christopher L
authors: CL
authors: de Graffenried
mesh_terms: D020121:5' Untranslated Regions; D003001:Cloning, Molecular; D018503:Genome, Protozoan; D023281:Genomics / Q000379:methods; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D014346:Trypanosoma brucei brucei / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions
keywords: Gibson assembly; RNAi; T. brucei