Cargando…

Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome

BACKGROUND: A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi...

Descripción completa

Detalles Bibliográficos
Autores principales: Cox, Mandy M, Layton, Sherryll L, Jiang, Tieshan, Cole, Kim, Hargis, Billy M, Berghman, Luc R, Bottje, Walter G, Kwon, Young Min
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2096622/
https://www.ncbi.nlm.nih.gov/pubmed/17875218
http://dx.doi.org/10.1186/1472-6750-7-59
_version_ 1782138244474863616
author Cox, Mandy M
Layton, Sherryll L
Jiang, Tieshan
Cole, Kim
Hargis, Billy M
Berghman, Luc R
Bottje, Walter G
Kwon, Young Min
author_facet Cox, Mandy M
Layton, Sherryll L
Jiang, Tieshan
Cole, Kim
Hargis, Billy M
Berghman, Luc R
Bottje, Walter G
Kwon, Young Min
author_sort Cox, Mandy M
collection PubMed
description BACKGROUND: A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome. RESULTS: The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of S. enteritidis. CONCLUSION: We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.
format Text
id pubmed-2096622
institution National Center for Biotechnology Information
language English
publishDate 2007
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-20966222007-11-28 Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome Cox, Mandy M Layton, Sherryll L Jiang, Tieshan Cole, Kim Hargis, Billy M Berghman, Luc R Bottje, Walter G Kwon, Young Min BMC Biotechnol Methodology Article BACKGROUND: A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome. RESULTS: The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of S. enteritidis. CONCLUSION: We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed. BioMed Central 2007-09-17 /pmc/articles/PMC2096622/ /pubmed/17875218 http://dx.doi.org/10.1186/1472-6750-7-59 Text en Copyright © 2007 Cox et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Article
Cox, Mandy M
Layton, Sherryll L
Jiang, Tieshan
Cole, Kim
Hargis, Billy M
Berghman, Luc R
Bottje, Walter G
Kwon, Young Min
Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome
title Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome
title_full Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome
title_fullStr Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome
title_full_unstemmed Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome
title_short Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome
title_sort scarless and site-directed mutagenesis in salmonella enteritidis chromosome
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2096622/
https://www.ncbi.nlm.nih.gov/pubmed/17875218
http://dx.doi.org/10.1186/1472-6750-7-59
work_keys_str_mv AT coxmandym scarlessandsitedirectedmutagenesisinsalmonellaenteritidischromosome
AT laytonsherrylll scarlessandsitedirectedmutagenesisinsalmonellaenteritidischromosome
AT jiangtieshan scarlessandsitedirectedmutagenesisinsalmonellaenteritidischromosome
AT colekim scarlessandsitedirectedmutagenesisinsalmonellaenteritidischromosome
AT hargisbillym scarlessandsitedirectedmutagenesisinsalmonellaenteritidischromosome
AT berghmanlucr scarlessandsitedirectedmutagenesisinsalmonellaenteritidischromosome
AT bottjewalterg scarlessandsitedirectedmutagenesisinsalmonellaenteritidischromosome
AT kwonyoungmin scarlessandsitedirectedmutagenesisinsalmonellaenteritidischromosome