Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP
- Autores
- Silva, Joas L. da; Piuri, Mariana; Broussard, Gregory; Marinelli, Laura J.; Bastos, Gisele M.; Hirata, Rosario D. C.; Hatfull, Graham F.; Hirata, Mario H.
- Año de publicación
- 2013
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc2155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.
Fil: Silva, Joas L. da. Universidade de Sao Paulo; Brasil
Fil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Fil: Broussard, Gregory. University Of Pittsburgh; Estados Unidos
Fil: Marinelli, Laura J.. University Of Pittsburgh; Estados Unidos
Fil: Bastos, Gisele M.. Universidade de Sao Paulo; Brasil
Fil: Hirata, Rosario D. C.. Universidade de Sao Paulo; Brasil
Fil: Hatfull, Graham F.. University Of Pittsburgh; Estados Unidos
Fil: Hirata, Mario H.. Universidade de Sao Paulo; Brasil - Materia
-
Mycobacterium
Bacteriophage
Recombineering
Green Fluorescent Protein - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/21239
Ver los metadatos del registro completo
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Application of BRED technology to construct recombinant D29 reporter phage expressing EGFPSilva, Joas L. daPiuri, MarianaBroussard, GregoryMarinelli, Laura J.Bastos, Gisele M.Hirata, Rosario D. C.Hatfull, Graham F.Hirata, Mario H.MycobacteriumBacteriophageRecombineeringGreen Fluorescent Proteinhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc2155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.Fil: Silva, Joas L. da. Universidade de Sao Paulo; BrasilFil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Broussard, Gregory. University Of Pittsburgh; Estados UnidosFil: Marinelli, Laura J.. University Of Pittsburgh; Estados UnidosFil: Bastos, Gisele M.. Universidade de Sao Paulo; BrasilFil: Hirata, Rosario D. C.. Universidade de Sao Paulo; BrasilFil: Hatfull, Graham F.. University Of Pittsburgh; Estados UnidosFil: Hirata, Mario H.. Universidade de Sao Paulo; BrasilWiley2013-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/21239Silva, Joas L. da; Piuri, Mariana; Broussard, Gregory; Marinelli, Laura J.; Bastos, Gisele M.; et al.; Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP; Wiley; FEMS Microbiology Letters; 344; 2; 6-2013; 166-1720378-1097CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1111/1574-6968.12171info:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1111/1574-6968.12171/fullinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:31:37Zoai:ri.conicet.gov.ar:11336/21239instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:31:37.688CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP |
title |
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP |
spellingShingle |
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP Silva, Joas L. da Mycobacterium Bacteriophage Recombineering Green Fluorescent Protein |
title_short |
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP |
title_full |
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP |
title_fullStr |
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP |
title_full_unstemmed |
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP |
title_sort |
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP |
dc.creator.none.fl_str_mv |
Silva, Joas L. da Piuri, Mariana Broussard, Gregory Marinelli, Laura J. Bastos, Gisele M. Hirata, Rosario D. C. Hatfull, Graham F. Hirata, Mario H. |
author |
Silva, Joas L. da |
author_facet |
Silva, Joas L. da Piuri, Mariana Broussard, Gregory Marinelli, Laura J. Bastos, Gisele M. Hirata, Rosario D. C. Hatfull, Graham F. Hirata, Mario H. |
author_role |
author |
author2 |
Piuri, Mariana Broussard, Gregory Marinelli, Laura J. Bastos, Gisele M. Hirata, Rosario D. C. Hatfull, Graham F. Hirata, Mario H. |
author2_role |
author author author author author author author |
dc.subject.none.fl_str_mv |
Mycobacterium Bacteriophage Recombineering Green Fluorescent Protein |
topic |
Mycobacterium Bacteriophage Recombineering Green Fluorescent Protein |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc2155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages. Fil: Silva, Joas L. da. Universidade de Sao Paulo; Brasil Fil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Broussard, Gregory. University Of Pittsburgh; Estados Unidos Fil: Marinelli, Laura J.. University Of Pittsburgh; Estados Unidos Fil: Bastos, Gisele M.. Universidade de Sao Paulo; Brasil Fil: Hirata, Rosario D. C.. Universidade de Sao Paulo; Brasil Fil: Hatfull, Graham F.. University Of Pittsburgh; Estados Unidos Fil: Hirata, Mario H.. Universidade de Sao Paulo; Brasil |
description |
Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc2155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013-06 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://purl.org/coar/resource_type/c_6501 info:ar-repo/semantics/articulo |
format |
article |
status_str |
publishedVersion |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/11336/21239 Silva, Joas L. da; Piuri, Mariana; Broussard, Gregory; Marinelli, Laura J.; Bastos, Gisele M.; et al.; Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP; Wiley; FEMS Microbiology Letters; 344; 2; 6-2013; 166-172 0378-1097 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/21239 |
identifier_str_mv |
Silva, Joas L. da; Piuri, Mariana; Broussard, Gregory; Marinelli, Laura J.; Bastos, Gisele M.; et al.; Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP; Wiley; FEMS Microbiology Letters; 344; 2; 6-2013; 166-172 0378-1097 CONICET Digital CONICET |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/doi/10.1111/1574-6968.12171 info:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1111/1574-6968.12171/full |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Wiley |
publisher.none.fl_str_mv |
Wiley |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
reponame_str |
CONICET Digital (CONICET) |
collection |
CONICET Digital (CONICET) |
instname_str |
Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.name.fl_str_mv |
CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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13.070432 |