Factors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains

ABSTRACT An important risk factor for acquiring Clostridioides difficile infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Sev...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Disha Bhattacharjee, Joseph A. Sorg
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Clostridioides difficile electroporation genetics transformation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/2ae69dfd530e46639539d8ea0a0fe661
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:2ae69dfd530e46639539d8ea0a0fe661
record_format	dspace
spelling	oai:doaj.org-article:2ae69dfd530e46639539d8ea0a0fe6612021-11-15T15:29:15ZFactors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains10.1128/mSphere.00941-192379-5042https://doaj.org/article/2ae69dfd530e46639539d8ea0a0fe6612020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00941-19https://doaj.org/toc/2379-5042ABSTRACT An important risk factor for acquiring Clostridioides difficile infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Several genetic systems are available to study C. difficile in the laboratory environment, and all rely on stably replicating or segregationally unstable plasmids. Currently, the transfer of plasmids into C. difficile can only be performed by conjugation using Escherichia coli or Bacillus subtilis as conjugal donors. Here we report a method to introduce plasmid DNA into C. difficile using electroporation and test factors that might contribute to higher transformation efficiencies: osmolyte used to stabilize weakened cells, DNA concentration, and recovery time postelectroporation. Depending on the C. difficile strain and plasmid used, this transformation protocol achieves between 20 and 200 colonies per microgram of DNA and is mostly influenced by the recovery time postelectroporation. Based on our findings, we recommend that each strain be tested for the optimum recovery time in each lab. IMPORTANCE Understanding the underlying biology of pathogens is essential to develop novel treatment options. To drive this understanding, genetic tools are essential. In recent years, the genetic toolbox available to Clostridioides difficile researchers has expanded significantly but still requires the conjugal transfer of DNA from a donor strain into C. difficile. Here we describe an electroporation-based transformation protocol that was effective at introducing existing genetic tools into different C. difficile strains.Disha BhattacharjeeJoseph A. SorgAmerican Society for MicrobiologyarticleClostridioides difficileelectroporationgeneticstransformationMicrobiologyQR1-502ENmSphere, Vol 5, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Clostridioides difficile electroporation genetics transformation Microbiology QR1-502
spellingShingle	Clostridioides difficile electroporation genetics transformation Microbiology QR1-502 Disha Bhattacharjee Joseph A. Sorg Factors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains
description	ABSTRACT An important risk factor for acquiring Clostridioides difficile infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Several genetic systems are available to study C. difficile in the laboratory environment, and all rely on stably replicating or segregationally unstable plasmids. Currently, the transfer of plasmids into C. difficile can only be performed by conjugation using Escherichia coli or Bacillus subtilis as conjugal donors. Here we report a method to introduce plasmid DNA into C. difficile using electroporation and test factors that might contribute to higher transformation efficiencies: osmolyte used to stabilize weakened cells, DNA concentration, and recovery time postelectroporation. Depending on the C. difficile strain and plasmid used, this transformation protocol achieves between 20 and 200 colonies per microgram of DNA and is mostly influenced by the recovery time postelectroporation. Based on our findings, we recommend that each strain be tested for the optimum recovery time in each lab. IMPORTANCE Understanding the underlying biology of pathogens is essential to develop novel treatment options. To drive this understanding, genetic tools are essential. In recent years, the genetic toolbox available to Clostridioides difficile researchers has expanded significantly but still requires the conjugal transfer of DNA from a donor strain into C. difficile. Here we describe an electroporation-based transformation protocol that was effective at introducing existing genetic tools into different C. difficile strains.
format	article
author	Disha Bhattacharjee Joseph A. Sorg
author_facet	Disha Bhattacharjee Joseph A. Sorg
author_sort	Disha Bhattacharjee
title	Factors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains
title_short	Factors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains
title_full	Factors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains
title_fullStr	Factors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains
title_full_unstemmed	Factors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains
title_sort	factors and conditions that impact electroporation of <named-content content-type="genus-species">clostridioides difficile</named-content> strains
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/2ae69dfd530e46639539d8ea0a0fe661
work_keys_str_mv	AT dishabhattacharjee factorsandconditionsthatimpactelectroporationofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentstrains AT josephasorg factorsandconditionsthatimpactelectroporationofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentstrains
_version_	1718427874432647168

Factors and Conditions That Impact Electroporation of <named-content content-type="genus-species">Clostridioides difficile</named-content> Strains

Ejemplares similares