Modification of Salmonella phage φSan23 using BRED
Foodborne illnesses caused by Salmonella species are common and represent a significant public health challenge. Identifying the sources of contamination by these microorganisms is crucial for controlling and preventing infections. While various tests have been proposed for this purpose, many requir...
- Autores:
-
Fajardo Poveda, Johan David
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2025
- Institución:
- Universidad de los Andes
- Repositorio:
- Séneca: repositorio Uniandes
- Idioma:
- eng
- OAI Identifier:
- oai:repositorio.uniandes.edu.co:1992/75938
- Acceso en línea:
- https://hdl.handle.net/1992/75938
- Palabra clave:
- Salmonella
Biosensors
Bacteriophage
φSan23
Genetics
BRED
Microbiología
- Rights
- openAccess
- License
- Attribution 4.0 International
| id |
UNIANDES2_6fdd132b2534776c1939378299edf2a0 |
|---|---|
| oai_identifier_str |
oai:repositorio.uniandes.edu.co:1992/75938 |
| network_acronym_str |
UNIANDES2 |
| network_name_str |
Séneca: repositorio Uniandes |
| repository_id_str |
|
| dc.title.eng.fl_str_mv |
Modification of Salmonella phage φSan23 using BRED |
| title |
Modification of Salmonella phage φSan23 using BRED |
| spellingShingle |
Modification of Salmonella phage φSan23 using BRED Salmonella Biosensors Bacteriophage φSan23 Genetics BRED Microbiología |
| title_short |
Modification of Salmonella phage φSan23 using BRED |
| title_full |
Modification of Salmonella phage φSan23 using BRED |
| title_fullStr |
Modification of Salmonella phage φSan23 using BRED |
| title_full_unstemmed |
Modification of Salmonella phage φSan23 using BRED |
| title_sort |
Modification of Salmonella phage φSan23 using BRED |
| dc.creator.fl_str_mv |
Fajardo Poveda, Johan David |
| dc.contributor.advisor.none.fl_str_mv |
Vives Flórez, Martha Josefina |
| dc.contributor.author.none.fl_str_mv |
Fajardo Poveda, Johan David |
| dc.contributor.researchgroup.none.fl_str_mv |
Facultad de Ciencias::Microbiologia Ambiental y Bioprospeccion |
| dc.subject.keyword.eng.fl_str_mv |
Salmonella Biosensors Bacteriophage φSan23 Genetics BRED |
| topic |
Salmonella Biosensors Bacteriophage φSan23 Genetics BRED Microbiología |
| dc.subject.themes.spa.fl_str_mv |
Microbiología |
| description |
Foodborne illnesses caused by Salmonella species are common and represent a significant public health challenge. Identifying the sources of contamination by these microorganisms is crucial for controlling and preventing infections. While various tests have been proposed for this purpose, many require complex and time-consuming procedures. Although in-situ tests exist, they often lack the capacity for quantitative detection. Phage-based biosensors have been proposed as a promising alternative for identifying pathogens causing these foodborne diseases. However, they face technical limitations, such as the difficulty in properly attaching bacteriophages to the biosensor. Genetically modified bacteriophages have been suggested as a potential solution to overcome these limitations. In this work, we explore the genetic modification of the bacteriophage φSan23 using the BRED technique. To achieve this, we aimed to identify structural proteins of the bacteriophage head and fuse them with a His-tag and a marker protein, such as GFP. Additionally, we sought to produce this GFP marker protein inside the bacterial host. We were able to annotate and model the structural and non-structural proteins of the bacteriophage and found an efficient method for electroporating exogenous DNA into Salmonella. However, the modification of the bacteriophage φSan23 using the BRED technique was not successful. Additional approaches are needed to achieve this goal in future studies. |
| publishDate |
2025 |
| dc.date.accessioned.none.fl_str_mv |
2025-01-31T19:56:26Z |
| dc.date.available.none.fl_str_mv |
2025-01-31T19:56:26Z |
| dc.date.issued.none.fl_str_mv |
2025-01-29 |
| dc.type.none.fl_str_mv |
Trabajo de grado - Pregrado |
| dc.type.driver.none.fl_str_mv |
info:eu-repo/semantics/bachelorThesis |
| dc.type.version.none.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| dc.type.coar.none.fl_str_mv |
http://purl.org/coar/resource_type/c_7a1f |
| dc.type.content.none.fl_str_mv |
Text |
| dc.type.redcol.none.fl_str_mv |
http://purl.org/redcol/resource_type/TP |
| format |
http://purl.org/coar/resource_type/c_7a1f |
| status_str |
acceptedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://hdl.handle.net/1992/75938 |
| dc.identifier.instname.none.fl_str_mv |
instname:Universidad de los Andes |
| dc.identifier.reponame.none.fl_str_mv |
reponame:Repositorio Institucional Séneca |
| dc.identifier.repourl.none.fl_str_mv |
repourl:https://repositorio.uniandes.edu.co/ |
| url |
https://hdl.handle.net/1992/75938 |
| identifier_str_mv |
instname:Universidad de los Andes reponame:Repositorio Institucional Séneca repourl:https://repositorio.uniandes.edu.co/ |
| dc.language.iso.none.fl_str_mv |
eng |
| language |
eng |
| dc.relation.references.none.fl_str_mv |
Al-Hindi, R. R. et al. (2022). Bacteriophage-based biosensors: A platform for detection of foodborne bacterial pathogens from food and environment. Biosensors, 12(10), 905. Andes, U. (2015). Composition comprising bacteriophage for reducing, eliminating and/or preventing Salmonella enteritidis, Salmonella typhimurium and Salmonella paratyphi b. Colombia. Bernardinelli, G., & Högberg, B. (2017). Entirely enzymatic nanofabrication of DNA-protein conjugates. Nucleic Acids Research, 45(18), e160. https://doi.org/10.1093/nar/gkx707 Bio-Rad. (n.d.-a). Micropulser electroporator: Instruction manual and applications guide [Catalog #1652100]. https://www.bio-rad.com/sites/default/files/2022-01/10000148532.pdf Bio-Rad. (n.d.-b). Pglo plasmid map resources [Accessed: 2025-01-25]. https://www.bio-rad.com/es-co/applications-technologies/pglo-plasmid-map-resources?ID=NISQOC15 Chinchilla Sarmiento, S. (2023). Aproximaciones al desarrollo de un bacteriófago recombinante reportero de Salmonella enteritidis y Salmonella typhimurium, mediante la refactorización in-vitro del genoma de φSan23. Comeau, A. M., & Krisch, H. M. (2008). The capsid of the T4 phage superfamily: The evolution, diversity, and structure of some of the most prevalent proteins in the biosphere. Molecular Biology and Evolution, 25(7), 1321–1332. Costa, A. R., Azeredo, J., & Pires, D. P. (2023). Synthetic biology to engineer bacteriophage genomes. In Bacteriophage therapy: From lab to clinical practice (pp. 261–277). Springer US. Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences, 97(12), 6640–6645. Dong, X. et al. (2022). Genetic manipulation of the human gut bacterium Eggerthella lenta reveals a widespread family of transcriptional regulators. Nature Communications, 13(1), 7624. https://doi.org/10.1038/s41467-022-33576-3 Fang, Z., Feng, T., Zhou, H., & Chen, M. (2022). DeepVP: Identification and classification of phage virion proteins using deep learning. Gigascience, 11, giac076. Farooq, U., Yang, Q., Ullah, M. W., & Wang, S. (2019). Principle and development of phage-based biosensors. Biosens. Environ. Monit, 1, 1–18. Flamholz, Z. N., Li, C., & Kelly, L. (2024). Improving viral annotation with artificial intelligence. mBio, 15(10), e03206–23. Jensen, S. I. et al. (2015). Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress. Scientific Reports, 5, 17874. https://doi.org/10.1038/srep17874 Jiménez Sánchez, A. (2015). Caracterización y evaluación de la eficiencia in vitro de bacteriófagos nativos contra Salmonella, causante de salmonelosis en Colombia. Universidad de los Andes. Khambhati, K. et al. (2023). Phage engineering and phage-assisted CRISPR-Cas delivery to combat multidrug-resistant pathogens. Bioengineering Translational Medicine, 8(2), e10381. Kim, S., Kim, M., & Ryu, S. (2014). Development of an engineered bioluminescent reporter phage for the sensitive detection of viable Salmonella typhimurium. Analytical Chemistry, 86(12), 5858–5864. Klucar, L., Stano, M., & Hajduk, M. (2010). Phisite: Database of gene regulation in bacteriophages. Nucleic Acids Research, 38(suppl1), D366–D370. Lamas, A. et al. (2018). A comprehensive review of non-enterica subspecies of Salmonella enterica. Microbiological Research, 206, 60–73. McNair, K. et al. (2019). PHANOTATE: A novel approach to gene identification in phage genomes. Bioinformatics, 35(22), 4537–4542. Paczesny, J., Richter, L., & Hołyst, R. (2020). Recent progress in the detection of bacteria using bacteriophages: A review. Viruses, 12(8), 845. Paddison, P. et al. (1998). The roles of the bacteriophage T4 r genes in lysis inhibition and fine-structure genetics: A new perspective. Genetics, 148(4), 1539–1550. Pires, D. P. et al. (2016). Genetically engineered phages: A review of advances over the last decade. Microbiology and Molecular Biology Reviews, 80(3), 523–543. |
| dc.rights.en.fl_str_mv |
Attribution 4.0 International |
| dc.rights.uri.none.fl_str_mv |
http://creativecommons.org/licenses/by/4.0/ |
| dc.rights.accessrights.none.fl_str_mv |
info:eu-repo/semantics/openAccess |
| dc.rights.coar.none.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
| rights_invalid_str_mv |
Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ http://purl.org/coar/access_right/c_abf2 |
| eu_rights_str_mv |
openAccess |
| dc.format.extent.none.fl_str_mv |
30 páginas |
| dc.format.mimetype.none.fl_str_mv |
application/pdf |
| dc.publisher.none.fl_str_mv |
Universidad de los Andes |
| dc.publisher.program.none.fl_str_mv |
Microbiología |
| dc.publisher.faculty.none.fl_str_mv |
Facultad de Ciencias |
| dc.publisher.department.none.fl_str_mv |
Departamento de Ciencias Biológicas |
| publisher.none.fl_str_mv |
Universidad de los Andes |
| institution |
Universidad de los Andes |
| bitstream.url.fl_str_mv |
https://repositorio.uniandes.edu.co/bitstreams/1d20fe8b-501d-466f-879f-9a7e678a5633/download https://repositorio.uniandes.edu.co/bitstreams/c4dfdc8e-6349-43f5-89bf-026f7352a06f/download https://repositorio.uniandes.edu.co/bitstreams/0e50cb4e-3649-4cb9-8ba3-f67196f571aa/download https://repositorio.uniandes.edu.co/bitstreams/60cc877a-5947-4566-a3c1-ee7957b400e3/download https://repositorio.uniandes.edu.co/bitstreams/42bfad1c-4b39-42fd-ae88-41906e6b2545/download https://repositorio.uniandes.edu.co/bitstreams/43083852-eac9-41d0-ac9d-475e0b62ce11/download https://repositorio.uniandes.edu.co/bitstreams/4f8a020a-8351-4b4b-b4d9-f0b065371740/download https://repositorio.uniandes.edu.co/bitstreams/a8ac01ee-b91c-4831-94d3-b27cb2fc8e7a/download |
| bitstream.checksum.fl_str_mv |
cd6d97eaf3952f394e9dc2e26468695a 8c8c1e5eff79ac8238f84b06ac345398 0175ea4a2d4caec4bbcc37e300941108 ae9e573a68e7f92501b6913cc846c39f 4e440b51763f920a75ef6288ec4d22a2 bd82ae98f1ff209af2a12d2a86a69efa 6e8c0818851cbf3b6899048f563bcd75 f598b5e1ffa1269617f1d9a3236a4f59 |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 MD5 MD5 MD5 MD5 |
| repository.name.fl_str_mv |
Repositorio institucional Séneca |
| repository.mail.fl_str_mv |
adminrepositorio@uniandes.edu.co |
| _version_ |
1831927759277916160 |
| spelling |
Vives Flórez, Martha Josefinavirtual::23022-1Fajardo Poveda, Johan DavidFacultad de Ciencias::Microbiologia Ambiental y Bioprospeccion2025-01-31T19:56:26Z2025-01-31T19:56:26Z2025-01-29https://hdl.handle.net/1992/75938instname:Universidad de los Andesreponame:Repositorio Institucional Sénecarepourl:https://repositorio.uniandes.edu.co/Foodborne illnesses caused by Salmonella species are common and represent a significant public health challenge. Identifying the sources of contamination by these microorganisms is crucial for controlling and preventing infections. While various tests have been proposed for this purpose, many require complex and time-consuming procedures. Although in-situ tests exist, they often lack the capacity for quantitative detection. Phage-based biosensors have been proposed as a promising alternative for identifying pathogens causing these foodborne diseases. However, they face technical limitations, such as the difficulty in properly attaching bacteriophages to the biosensor. Genetically modified bacteriophages have been suggested as a potential solution to overcome these limitations. In this work, we explore the genetic modification of the bacteriophage φSan23 using the BRED technique. To achieve this, we aimed to identify structural proteins of the bacteriophage head and fuse them with a His-tag and a marker protein, such as GFP. Additionally, we sought to produce this GFP marker protein inside the bacterial host. We were able to annotate and model the structural and non-structural proteins of the bacteriophage and found an efficient method for electroporating exogenous DNA into Salmonella. However, the modification of the bacteriophage φSan23 using the BRED technique was not successful. Additional approaches are needed to achieve this goal in future studies.PregradoBacteriofagos30 páginasapplication/pdfengUniversidad de los AndesMicrobiologíaFacultad de CienciasDepartamento de Ciencias BiológicasAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Modification of Salmonella phage φSan23 using BREDTrabajo de grado - Pregradoinfo:eu-repo/semantics/bachelorThesisinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_7a1fTexthttp://purl.org/redcol/resource_type/TPSalmonellaBiosensorsBacteriophageφSan23GeneticsBREDMicrobiologíaAl-Hindi, R. R. et al. (2022). Bacteriophage-based biosensors: A platform for detection of foodborne bacterial pathogens from food and environment. Biosensors, 12(10), 905.Andes, U. (2015). Composition comprising bacteriophage for reducing, eliminating and/or preventing Salmonella enteritidis, Salmonella typhimurium and Salmonella paratyphi b. Colombia.Bernardinelli, G., & Högberg, B. (2017). Entirely enzymatic nanofabrication of DNA-protein conjugates. Nucleic Acids Research, 45(18), e160. https://doi.org/10.1093/nar/gkx707Bio-Rad. (n.d.-a). Micropulser electroporator: Instruction manual and applications guide [Catalog #1652100]. https://www.bio-rad.com/sites/default/files/2022-01/10000148532.pdfBio-Rad. (n.d.-b). Pglo plasmid map resources [Accessed: 2025-01-25]. https://www.bio-rad.com/es-co/applications-technologies/pglo-plasmid-map-resources?ID=NISQOC15Chinchilla Sarmiento, S. (2023). Aproximaciones al desarrollo de un bacteriófago recombinante reportero de Salmonella enteritidis y Salmonella typhimurium, mediante la refactorización in-vitro del genoma de φSan23.Comeau, A. M., & Krisch, H. M. (2008). The capsid of the T4 phage superfamily: The evolution, diversity, and structure of some of the most prevalent proteins in the biosphere. Molecular Biology and Evolution, 25(7), 1321–1332.Costa, A. R., Azeredo, J., & Pires, D. P. (2023). Synthetic biology to engineer bacteriophage genomes. In Bacteriophage therapy: From lab to clinical practice (pp. 261–277). Springer US.Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences, 97(12), 6640–6645.Dong, X. et al. (2022). Genetic manipulation of the human gut bacterium Eggerthella lenta reveals a widespread family of transcriptional regulators. Nature Communications, 13(1), 7624. https://doi.org/10.1038/s41467-022-33576-3Fang, Z., Feng, T., Zhou, H., & Chen, M. (2022). DeepVP: Identification and classification of phage virion proteins using deep learning. Gigascience, 11, giac076.Farooq, U., Yang, Q., Ullah, M. W., & Wang, S. (2019). Principle and development of phage-based biosensors. Biosens. Environ. Monit, 1, 1–18.Flamholz, Z. N., Li, C., & Kelly, L. (2024). Improving viral annotation with artificial intelligence. mBio, 15(10), e03206–23.Jensen, S. I. et al. (2015). Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress. Scientific Reports, 5, 17874. https://doi.org/10.1038/srep17874Jiménez Sánchez, A. (2015). Caracterización y evaluación de la eficiencia in vitro de bacteriófagos nativos contra Salmonella, causante de salmonelosis en Colombia. Universidad de los Andes.Khambhati, K. et al. (2023). Phage engineering and phage-assisted CRISPR-Cas delivery to combat multidrug-resistant pathogens. Bioengineering Translational Medicine, 8(2), e10381.Kim, S., Kim, M., & Ryu, S. (2014). Development of an engineered bioluminescent reporter phage for the sensitive detection of viable Salmonella typhimurium. Analytical Chemistry, 86(12), 5858–5864.Klucar, L., Stano, M., & Hajduk, M. (2010). Phisite: Database of gene regulation in bacteriophages. Nucleic Acids Research, 38(suppl1), D366–D370.Lamas, A. et al. (2018). A comprehensive review of non-enterica subspecies of Salmonella enterica. Microbiological Research, 206, 60–73.McNair, K. et al. (2019). PHANOTATE: A novel approach to gene identification in phage genomes. Bioinformatics, 35(22), 4537–4542.Paczesny, J., Richter, L., & Hołyst, R. (2020). Recent progress in the detection of bacteria using bacteriophages: A review. Viruses, 12(8), 845.Paddison, P. et al. (1998). The roles of the bacteriophage T4 r genes in lysis inhibition and fine-structure genetics: A new perspective. Genetics, 148(4), 1539–1550.Pires, D. P. et al. (2016). Genetically engineered phages: A review of advances over the last decade. Microbiology and Molecular Biology Reviews, 80(3), 523–543.202111390Publicationhttps://scholar.google.es/citations?user=FmskIBcAAAAJvirtual::23022-10000-0001-7795-1494virtual::23022-1https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000190195virtual::23022-1324db0c6-0b71-49b7-846b-c997885dbe42virtual::23022-1324db0c6-0b71-49b7-846b-c997885dbe42virtual::23022-1ORIGINAL202111390_ForAutEntTesis_TraGraSisBib_202420.pdf202111390_ForAutEntTesis_TraGraSisBib_202420.pdfHIDEapplication/pdf216145https://repositorio.uniandes.edu.co/bitstreams/1d20fe8b-501d-466f-879f-9a7e678a5633/downloadcd6d97eaf3952f394e9dc2e26468695aMD51Modification of Salmonella phage San23 using BRED.pdfModification of Salmonella phage San23 using BRED.pdfapplication/pdf4170112https://repositorio.uniandes.edu.co/bitstreams/c4dfdc8e-6349-43f5-89bf-026f7352a06f/download8c8c1e5eff79ac8238f84b06ac345398MD52CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8908https://repositorio.uniandes.edu.co/bitstreams/0e50cb4e-3649-4cb9-8ba3-f67196f571aa/download0175ea4a2d4caec4bbcc37e300941108MD53LICENSElicense.txtlicense.txttext/plain; charset=utf-82535https://repositorio.uniandes.edu.co/bitstreams/60cc877a-5947-4566-a3c1-ee7957b400e3/downloadae9e573a68e7f92501b6913cc846c39fMD54TEXT202111390_ForAutEntTesis_TraGraSisBib_202420.pdf.txt202111390_ForAutEntTesis_TraGraSisBib_202420.pdf.txtExtracted texttext/plain1429https://repositorio.uniandes.edu.co/bitstreams/42bfad1c-4b39-42fd-ae88-41906e6b2545/download4e440b51763f920a75ef6288ec4d22a2MD55Modification of Salmonella phage San23 using BRED.pdf.txtModification of Salmonella phage San23 using BRED.pdf.txtExtracted texttext/plain34736https://repositorio.uniandes.edu.co/bitstreams/43083852-eac9-41d0-ac9d-475e0b62ce11/downloadbd82ae98f1ff209af2a12d2a86a69efaMD57THUMBNAIL202111390_ForAutEntTesis_TraGraSisBib_202420.pdf.jpg202111390_ForAutEntTesis_TraGraSisBib_202420.pdf.jpgGenerated Thumbnailimage/jpeg10603https://repositorio.uniandes.edu.co/bitstreams/4f8a020a-8351-4b4b-b4d9-f0b065371740/download6e8c0818851cbf3b6899048f563bcd75MD56Modification of Salmonella phage San23 using BRED.pdf.jpgModification of Salmonella phage San23 using BRED.pdf.jpgGenerated Thumbnailimage/jpeg8139https://repositorio.uniandes.edu.co/bitstreams/a8ac01ee-b91c-4831-94d3-b27cb2fc8e7a/downloadf598b5e1ffa1269617f1d9a3236a4f59MD581992/75938oai:repositorio.uniandes.edu.co:1992/759382025-03-05 09:39:13.728http://creativecommons.org/licenses/by/4.0/Attribution 4.0 Internationalopen.accesshttps://repositorio.uniandes.edu.coRepositorio institucional Sénecaadminrepositorio@uniandes.edu.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 |