Figure 2

The T. cruzi inducible expression vectors pTcINDEX and pTcINDEX-C-myc. (A) Map of pTcINDEX. The grey box adjacent to the multiple cloning site (MCS) indicates the ribosomal protein P2β splice acceptor site [25]. The hatched box indicates the T. cruzi actin intergenic region. The black box (T) is the T7 transcriptional terminator. Black flags represent T7 promoters and the oval identifies the location of the tet operator. R-NTS/P is the ribosomal non-transcribed spacer and promoter region used to target the construct. Roman numerals I and II indicate the two halves of the targeting sequence which are cloned in the opposite order to their position in the genome (see Fig. 3A). The white flag indicates the location of the pol I transcription start site [24]. Following insertion of a gene of interest into the MCS, the construct can be linearised with Spe I (dotted line) to facilitate targeting to the rRNA non-transcribed spacer region. The vector is built on a pUC19 backbone (not illustrated for clarity) and confers ampicillin resistance on E. coli. The sequence of the MCS is shown above the map indicating useful restriction sites. Nae I and Nru I were incorporated as blunt end sites to facilitate cloning of genes which contain the other restriction sites. (B) Map of pTcINDEX-C-myc. The inducible cassette alone is shown for clarity. The rest of the vector is identical to pTcINDEX. Features are as shown in A, except that the BPP1-myc fusion gene has been inserted into the Bam HI/Nru I sites of pTcINDEX (Methods). The white box labelled "stuffer" indicates the dispensable BPP1 ORF [48]. This can be removed by digestion with one of the MCS enzymes and Eco RV and replaced with the gene of interest. The c-myc epitope tag is indicated by a green box. The Eco RV cleavage site and the translated sequence of the c-myc tag (underlined) are indicated to allow easy design of in-frame fusions with the epitope tag. Note the Nru I site is absent in this plasmid.