A high-throughput cloning system for reverse genetics in Trypanosoma cruzi
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2010 |
| Outros Autores: | , , , , , , , , |
| Tipo de documento: | Artigo |
| Idioma: | por |
| Título da fonte: | Repositório Institucional da FIOCRUZ (ARCA) |
| Texto Completo: | https://www.arca.fiocruz.br/handle/icict/28670 |
Resumo: | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. |
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Batista, MichelMarchini, Fabrício KleryntonCeledon, Paola Alejandra FioraniFragoso, Stenio PerdigãoProbst, Christian MacagnanPreti, HenriqueOzaki, Luiz ShozoBuck, Gregory A.Goldenberg, SamuelKrieger, Marco Aurélio2018-09-12T13:02:58Z2018-09-12T13:02:58Z2010BATISTA, Michel et al. A high-throughput cloning system for reverse genetics in Trypanosoma cruzi. BMC Microbiology, v. 10, n. 259, p. 1-12, 2010.1471-2180https://www.arca.fiocruz.br/handle/icict/2867010.1186/1471-2180-10-2591471-2180porSpringer NatureTrypanosoma cruziCloningGenetic VectorsPlasmidsReverse GeneticsClonación MolecularVectores GenéticosPlásmidosGenética InversaClonagem MolecularVetores GenéticosPlasmídeosGenética ReversaA high-throughput cloning system for reverse genetics in Trypanosoma cruziinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Center for the Study of Biological Complexity. Virginia Commonwealth University. Richmond, Virginia, USA.Center for the Study of Biological Complexity. Virginia Commonwealth University. Richmond, Virginia, USA.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a highthroughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches. How Resulted, we constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed. This way, we constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-83084https://www.arca.fiocruz.br/bitstream/icict/28670/1/license.txt783568c2893d2e25a99990b126be1772MD51ORIGINALBMCMicrobiol147110-259.pdfBMCMicrobiol147110-259.pdfapplication/pdf2335112https://www.arca.fiocruz.br/bitstream/icict/28670/2/BMCMicrobiol147110-259.pdf84dc094ee7f8a4887c0aac47a8295b88MD52TEXTBMCMicrobiol147110-259.pdf.txtBMCMicrobiol147110-259.pdf.txtExtracted texttext/plain54648https://www.arca.fiocruz.br/bitstream/icict/28670/3/BMCMicrobiol147110-259.pdf.txt0e408fa7088a40e632742accb599b1c7MD53icict/286702019-11-23 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| dc.title.pt_BR.fl_str_mv |
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi |
| title |
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi |
| spellingShingle |
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi Batista, Michel Trypanosoma cruzi Cloning Genetic Vectors Plasmids Reverse Genetics Clonación Molecular Vectores Genéticos Plásmidos Genética Inversa Clonagem Molecular Vetores Genéticos Plasmídeos Genética Reversa |
| title_short |
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi |
| title_full |
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi |
| title_fullStr |
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi |
| title_full_unstemmed |
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi |
| title_sort |
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi |
| author |
Batista, Michel |
| author_facet |
Batista, Michel Marchini, Fabrício Klerynton Celedon, Paola Alejandra Fiorani Fragoso, Stenio Perdigão Probst, Christian Macagnan Preti, Henrique Ozaki, Luiz Shozo Buck, Gregory A. Goldenberg, Samuel Krieger, Marco Aurélio |
| author_role |
author |
| author2 |
Marchini, Fabrício Klerynton Celedon, Paola Alejandra Fiorani Fragoso, Stenio Perdigão Probst, Christian Macagnan Preti, Henrique Ozaki, Luiz Shozo Buck, Gregory A. Goldenberg, Samuel Krieger, Marco Aurélio |
| author2_role |
author author author author author author author author author |
| dc.contributor.author.fl_str_mv |
Batista, Michel Marchini, Fabrício Klerynton Celedon, Paola Alejandra Fiorani Fragoso, Stenio Perdigão Probst, Christian Macagnan Preti, Henrique Ozaki, Luiz Shozo Buck, Gregory A. Goldenberg, Samuel Krieger, Marco Aurélio |
| dc.subject.other.pt_BR.fl_str_mv |
Trypanosoma cruzi |
| topic |
Trypanosoma cruzi Cloning Genetic Vectors Plasmids Reverse Genetics Clonación Molecular Vectores Genéticos Plásmidos Genética Inversa Clonagem Molecular Vetores Genéticos Plasmídeos Genética Reversa |
| dc.subject.en.pt_BR.fl_str_mv |
Cloning Genetic Vectors Plasmids Reverse Genetics |
| dc.subject.es.pt_BR.fl_str_mv |
Clonación Molecular Vectores Genéticos Plásmidos Genética Inversa |
| dc.subject.decs.pt_BR.fl_str_mv |
Clonagem Molecular Vetores Genéticos Plasmídeos Genética Reversa |
| description |
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. |
| publishDate |
2010 |
| dc.date.issued.fl_str_mv |
2010 |
| dc.date.accessioned.fl_str_mv |
2018-09-12T13:02:58Z |
| dc.date.available.fl_str_mv |
2018-09-12T13:02:58Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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publishedVersion |
| dc.identifier.citation.fl_str_mv |
BATISTA, Michel et al. A high-throughput cloning system for reverse genetics in Trypanosoma cruzi. BMC Microbiology, v. 10, n. 259, p. 1-12, 2010. |
| dc.identifier.uri.fl_str_mv |
https://www.arca.fiocruz.br/handle/icict/28670 |
| dc.identifier.issn.pt_BR.fl_str_mv |
1471-2180 |
| dc.identifier.doi.none.fl_str_mv |
10.1186/1471-2180-10-259 |
| dc.identifier.eissn.none.fl_str_mv |
1471-2180 |
| identifier_str_mv |
BATISTA, Michel et al. A high-throughput cloning system for reverse genetics in Trypanosoma cruzi. BMC Microbiology, v. 10, n. 259, p. 1-12, 2010. 1471-2180 10.1186/1471-2180-10-259 |
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Springer Nature |
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Springer Nature |
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