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
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/21239
Acceder
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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling 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 Técnicas2025-10-22T12:04:02Zoai: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:34982025-10-22 12:04:03.172CONICET 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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score 12.982451

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