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.

Identification of TOEFAZ1‐interacting proteins reveals key regulators of Trypanosoma brucei cytokinesis

“…BioID relies on the proximity of interacting proteins to the BirA* fusion, so assuring that the fusion protein is correctly localized is essential (Kim et al ., ). We titrated the expression level of the Ty1‐BirA*‐TOEFAZ1 fusion compared to a cell line where both endogenous TOEFAZ1 alleles had been tagged with 3×‐Ty1 (McAllaster et al ., ). We found that expression of the BirA*‐TOEFAZ1 fusion at 40 ng ml −1 of doxycycline was very similar to the double‐tagged endogenous levels of protein (Supporting Information Fig.…”

“…We included proteins that had been previously identified as TOEFAZ1 interactors but whose localizations were not reported (Zhou et al, 2016b). Each gene selected for analysis was initially N-terminally tagged at its endogenous locus with three copies of the Ty1 epitope using a Gibson Assembly TM strategy (McAllaster et al, 2016). Clonal cell lines for each protein were tested for correct insertion of the endogenous tag using PCR on isolated genomic DNA with primers specific for each locus and anti-Ty1 western blotting to test the size of the tagged protein.…”

“…We depleted KPP1 using a tetracycline-inducible long hairpin RNAi (lhRNAi) in cells where one KPP1 allele was tagged with Ty1 so we could follow depletion of the protein by western blotting (McAllaster et al, 2016). Cells were treated with doxycycline to induce lhRNAi expression or a vehicle control, and cell growth was monitored over the course of 8 days (Supporting Information Fig.…”

“…All DNA constructs were assembled using PCR amplification of inserts followed by either restriction-ligation or Gibson Assembly as previously described (McAllaster et al, 2016). Constructs were transfected into cells using an electroporator (GenePulser xCell, Bio-Rad, Hercules, CA).…”

“…Target sequences for RNAi were obtained using the RNAit software (trypanofan.bioc.cam.ac.uk/software/RNAit.html) and assembled into a modified pLEW100 vector as previously described (McAllaster et al, 2016;Redmond et al, 2003). 400-600 bp of the coding region were targeted for each of the proteins selected for RNAi: bp 1311-1772 for KLIF, bp 97-543 for PAVE1 and bp 994-1576 for KPP1.…”