Generating genomic platforms to study Candida albicans pathogenesis

Mélanie Legrand; Sophie Bachellier-Bassi; Keunsook K. Lee; Yogesh Chaudhari; Hélène Tournu; Laurence Arbogast; Hélène Boyer; Murielle Chauvel; Vitor Cabral; Corinne Maufrais; Audrey Nesseir; Irena Maslanka; Emmanuelle Permal; Tristan Rossignol; Louise A. Walker; Ute Zeidler; Sadri Znaidi; Floris Schoeters; Charlotte Majgier; Renaud A. Julien; Laurence Ma; Magali Tichit; Christiane Bouchier; Patrick Van Dijck; Carol A. Munro; Christophe d’Enfert

Epitope tags. We then inserted in frame tags either upstream of attR1 or downstream of attR2 to allow N-terminal or C-terminal protein tagging, respectively. For the cloning of N-terminal tags, we used pUC-attR1-CmR, containing a PciI-BglII fragment from CIp-GTW cloned into the PciI and BamHI sites of pUC18. CIp-GTW was obtained after deleting P TET from CIp-P TET -GTW with Acc65I and HpaI. For cloning of the 3xHA coding sequence, two oligonucleotides (oligo1_PciHABsrG and oligo2_PciHABsrG) containing three HA epitopes were hybridized, gel purified, and cloned into pUC-attR1-CmR cut with PciI and BsrGI. The resulting plasmid was then used as a PCR template with primers GTW02 and GTW03. The resulting PCR product was cut with BspEI and HpaI, and cloned into the same sites of pCA-Dest1100 to yield pCA-Dest1110. For cloning of the GFP tag, the GFP gene was amplified from pFA-GFP-URA3 (35) with primers GTW04 and GTW05. The PCR product was cut with PciI and EcoRV and ligated into the same sites of pUC-attR1-CmR. The resulting plasmid was then used as a PCR template with primers GTW07 and GTW03. The resulting PCR product was cut with ScaI and BspEI and ligated into pCA-Dest1100 cut with HpaI and BspEI, yielding pCA-Dest1120. For cloning of the TAP-tag, the TAP-tag coding region was PCR amplified from pFA-TAP-URA3 (11) with primers GTW13 and GTW14. The resulting PCR product was then cut with PciI and BsrGI, and ligated into the same sites of pUC-attR1-CmR. The resulting plasmid was then digested with EcoRV and BspEI and the smallest restriction fragment was ligated into pCA-Dest1100 cut with HpaI and BspEI to yield pCA-Dest1130. For the cloning of C-terminal tags, we generated pUC-attR2 by inserting a SalI-HindIII fragment from pCA-Dest1100 into the same sites of pUC18. For cloning of the 3xHA epitope, pCaMPY-3xHA (36) was used as a PCR template with primers GTW20 and GTW21, and the resulting PCR product cut with BsrGI and NsiI was ligated in the same sites of pUC-attR2. The resulting plasmid was cut with SalI and NsiI and the fragment cloned in the same sites of pCA-Dest1100, yielding pCA-Dest1101. For cloning the GFP tag, the GFP gene was amplified from pFA-GFP-URA3 (35) with primers GTW11 and GTW12. The resulting PCR product was cut with NsiI and EcoRV and ligated into the same sites of pUC-attR2. The resulting plasmid was then cut with SalI and NsiI and the GFP-bearing fragment ligated into the same sites of pCA-Dest1100, yielding pCA-Dest1102. For cloning of the TAP-tag, the TAP-tag coding region was amplified from CIp10-P PCK1 -GTW-TAPtag (11) with primers GTW15 and GTW16. The PCR product was cut with BsrGI and NsiI and cloned in the same sites of pUC-attR2. The resulting plasmid was digested with SalI and NsiI, and the TAP-containing fragment ligated in the same sites of pCA-Dest1100, yielding pCA-Dest1103.