pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

Abstract

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzi genes and is freely available for scientific community.

Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

“…Aiming to produce transfectant cell lines of T. cruzi epimastigotes expressing TcPATs plus a FLAG tag at the C terminus (FLAGC tagged TcPAT), the genes were amplified using specific primers ( Table I ) with recombination sites for the Gateway cloning platform (Thermo Fischer Scientific, Waltham, MA, USA) by using the entry plasmid vector pDONR 221 and the destination T. cruzi vector pTcGWFLAGC ( Batista et al 2010 , Kugeratski et al 2015 ). All genes were cloned, except TcPAT6 and TcPAT1 (already characterised).…”

Characterising ISWI chromatin remodeler in Trypanosoma cruzi

“…In addition to offering a platform to the determination of subcellular localisation, co-localisation assays and expression of fusion proteins in T. cruzi, this new upgrade, like previous versions, ensures a fast and efficient cloning system with the advantage of allowing easy construction of cassettes for gene knockout by homologous recombination. Besides, the old intergenic regions, present in pTcGW versions 1.0 and 1.1, (15,16) were replaced by three new ones aiming to increase the possibility of tagged proteins expression in all life stages of T. cruzi. Nevertheless, this was not addressed here.…”

“…Phyre2 (http:// www.sbg.bio.ic.ac.uk/phyre2/), a tool for remote homology detection and 3D structure prediction, was used to analyse the TcISWI protein sequence. pTcGW plasmid vectors version 2.0 for expression of tagged proteins in T. cruzi -An upgraded version of the pTcGW vectors (15,16) was used in this study. To construct these plasmids, the cassettes for expressing proteins tagged at N-terminal or C-terminal end with FLAG tag were synthesised (GenScript) and cloned into pBlue Script II KS+.…”

Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

“…Plasmids pTcGW-3xHA-N and pTcGW-3xHA-C encode three HA epitopes in tandem (3 × HA) for N- and C-terminal tagging, respectively, while plasmid pTcGFP-NH contains the EGFP gene for N-terminal tagging. These expression plasmids are modified versions of the pTcGW 1.1 series Gateway expression vectors constructed for constitutive expression and selection in T. cruzi 33 . T. cruzi Y strain parasites were transfected with 25 µg of pTcGW-Polθ-3xHA-N, pTcGW-Polθ-3xHA-C or pTcGW-Polθ-GFP-N and selected in LIT medium containing 500 µg ml-1 G418 (Sigma-Aldrich, St. Louis, MO) as previously described 34 .…”