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Chapter 34 : Cool Tools 5: The ORFeome Project

Authors: Mé Legrand1, Carol Munro2, Christophe d’Enfert3
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Affiliations: 1: Unité Biologie et Pathogénicité Fongiques, Département Génomes et Génétique, Institut Pasteur, and USC2019, INRA, F-75015 Paris, France; 2: Aberdeen Fungal Group, Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, United Kingdom; 3: Unité Biologie et Pathogénicité Fongiques, Département Génomes et Génétique, Institut Pasteur, and USC2019, INRA, F-75015 Paris, France
Content Type: Monograph
Publication Year: 2012

Category: Fungi and Fungal Pathogenesis; Clinical Microbiology

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Abstract:

This chapter presents the strategy developed to generate a ORFeome collection in a versatile Gateway vector, which allows the transfer of the cloned genes into a variety of Gateway-compatible expression vectors. The development of collections of over-expression strains is facilitated by the pre-establishment of a complete set of cloned open reading frames (ORFs), or ORFeome. Importantly, ORFeomes represent useful resources for the implementation of other approaches used to elucidate gene function apart from gene deletion or overexpression. Individual characterization of mutants on the genome-wide scale can be slow and laborious. In this respect, signature-tagged mutagenesis (STM) provides an attractive alternative. In STM, each mutant is tagged with a different DNA sequence, allowing all tags to be amplified from the DNA of mixed populations of mutants in a single PCR. In the ORFeome, the start codon of the 6,205 ORFs has been included, whereas the stop codon has been excluded to allow insertion of C-terminal tags. The authors have favored C-terminal tagging since tags at the amino termini may possibly interfere with targeting of proteins to the secretory pathway. A panel of libraries of strains expressing tagged proteins should facilitate the systematic execution of high-throughput biochemical and microscopic assays of the proteome. The construction of the ORFeome could pave the way to the generation of the promoterome (library of gene promoters) in order to better characterize expression profiles.

Citation: Legrand M, Munro C, d’Enfert C. 2012. Cool Tools 5: The ORFeome Project, p 505-510. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch34
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Cool Tools 5: The Candida albicans ORFeome Project
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Citation: Legrand M, Munro C, d’Enfert C. 2012. Cool Tools 5: The ORFeome Project, p 505-510. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch34
/content/10.1128/9781555817176.ch34.ch34fig01
FIGURE 1
Workflow of the ORFeome project. Shown is the overall scheme for high-throughput recombinational cloning of the 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 in the order they are carried out. doi:10.1128/9781555817176.ch34.f1
10.1128/9781555817176/f0506-01_thmb.gif
10.1128/9781555817176/f0506-01.gif
Image of FIGURE 1
FIGURE 1

Workflow of the ORFeome project. Shown is the overall scheme for high-throughput recombinational cloning of the 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 in the order they are carried out. doi:10.1128/9781555817176.ch34.f1

Citation: Legrand M, Munro C, d’Enfert C. 2012. Cool Tools 5: The ORFeome Project, p 505-510. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch34
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/content/10.1128/9781555817176.ch34.ch34fig02
FIGURE 2
Plasmids used in this work. (A) Schematic map of the CIp10-TETp-GTW destination vector used for overexpression of ORFs. ORFs cloned in a Gateway donor vector can be transferred through Gateway-mediated recombination at and of CIp10-TETp-GTW overexpression vectors. The gene is used for selection of transformants. Derivatives of these overexpression vectors can be targeted to the locus when linearized with StuI or I-SceI. Expression of the cloned ORFs is achieved when cells are grown in the presence of doxycycline, which binds to the tetracycline-dependent transactivator rtTA, allowing transcriptional activation at the Tetp promoter. Each expression plasmid is tagged with a unique 12-bp barcode, enabling growth phenotypes of individual strains to be analyzed in parallel. (B) Structure of the tetracycline-dependent transactivator cassette contained in plasmid pNIMX. Unique restriction sites to excise the entire cassette are indicated. , basal promoter of ; , nourseothricin resistance gene; -adapted reverse tetracycline-dependent transactivator gene. doi:10.1128/9781555817176.ch34.f2
10.1128/9781555817176/f0508-01_thmb.gif
10.1128/9781555817176/f0508-01.gif
Image of FIGURE 2
FIGURE 2

Plasmids used in this work. (A) Schematic map of the CIp10-TETp-GTW destination vector used for overexpression of ORFs. ORFs cloned in a Gateway donor vector can be transferred through Gateway-mediated recombination at and of CIp10-TETp-GTW overexpression vectors. The gene is used for selection of transformants. Derivatives of these overexpression vectors can be targeted to the locus when linearized with StuI or I-SceI. Expression of the cloned ORFs is achieved when cells are grown in the presence of doxycycline, which binds to the tetracycline-dependent transactivator rtTA, allowing transcriptional activation at the Tetp promoter. Each expression plasmid is tagged with a unique 12-bp barcode, enabling growth phenotypes of individual strains to be analyzed in parallel. (B) Structure of the tetracycline-dependent transactivator cassette contained in plasmid pNIMX. Unique restriction sites to excise the entire cassette are indicated. , basal promoter of ; , nourseothricin resistance gene; -adapted reverse tetracycline-dependent transactivator gene. doi:10.1128/9781555817176.ch34.f2

Citation: Legrand M, Munro C, d’Enfert C. 2012. Cool Tools 5: The ORFeome Project, p 505-510. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch34
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