The advent of the genomic era has made elucidating gene function at large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources towards this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5,099 ORFs cloned in a Gateway‚ĄĘ donor vector, representing 83% of the currently annotated coding sequences of C. albicans.
All ORFs marked as “cloned” by the ORFeome project can be obtained through the Centre International de Ressources Microbiennes (CIRM) .
Work flow of the C. albicans ORFeome project. Shown is the overall scheme for high-throughput recombinational cloning of the C. albicans ORFs using the Gateway system. Quality control is monitored and documented using PCR and DNA sequencing. The main steps are briefly summarized on the right.
Step 1: Pairs of primers specific for C. albicans predicted ORFs are designed. 5-prime primers include the ORF start codon while 3-prime primers do not include the stop codon. Primers are synthesized by groups of 96 with ORFs of increasing size, plate 1 including the smallest ORFs and plate 65 including the largest ORFs.

Step 2: The ORF are amplified from genomic DNA of C. albicans strain SC5314 using high fidelity polymerase. PCR products are gel verified for size and quantity and subsequently transferred into the pDONR207 donor vector using BP clonase.

Step 3: BP clonase reactions are transformed into E. coli and one transformant per ORF is stored as well as the remainder of the transformation mixture. This will allow the future identification of additional pDONR plasmids for each ORF corresponding to the second allele of the ORF.

Step 4: pDONR plasmids are individually prepared and subjected to Sanger sequencing from the 5-prime end of the ORF in order to ensure proper clone allocation in 96-well plates. Furthermore, the pDONR plasmids are subjected to Solexa sequencing and the resulting sequencing data are compared to ORF sequences obtained from CGD and SNP data generated by Solexa sequencing of C. albicans strain SC5314. These data are used to decide whether to proceed to Step 5 or reiterate the procedure, eg if mutations in the cloned ORF are not validated from Solexa sequencing data from C. albicans strain SC5314.

Step 5: ORFs in pDONR plasmids are individually transferred into a barcoded destination vector CIp10-TETp-GTW, using LR clonase, resulting in the CIp-Tet::orf19.xxxx plasmid.

Step 6: CIp-Tet::orf19.xxxx plasmids are individually prepared and gel verified following a EcoRV-digest.

Step 7: StuI or I-SceI-digested CIp-Tet::orf19.xxxx plasmids are transformed in a C. albicans strain constitutively expressing a C. albicans-adapted reverse Tet-dependent transactivator, allowing doxycycline-inducible expression of the ORF in the CIp-Tet::orf19.xxxx plasmid. Integration of the CIp-Tet::orf19.xxxx plasmid at the RPS1 locus is verified by colony PCR.

Acknowledgements:

The Candida albicans ORFeome collection is made possible through the collaboration between the d’Enfert lab (Fungal Biology and Pathogenicity Unit, Institut Pasteur, Paris, France) and the Munro lab (Aberdeen Fungal Group, University of Aberdeen, Scotland).