Development of an integrative vector for the expression of antisense RNA in Clostridium difficile

Adam P. Roberts; Claire Hennequin; Michael Elmore; Anne Collignon; Tuomo Karjalainen; Nigel Minton; Peter Mullany

doi:10.1016/S0167-7012(03)00200-8

Development of an integrative vector for the expression of antisense RNA in Clostridium difficile

Adam P. Roberts^*, Claire Hennequin, Michael Elmore, Anne Collignon, Tuomo Karjalainen, Nigel Minton, Peter Mullany

^*Corresponding author for this work

Research & Technical Staff

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Abstract

A method was developed to use the conjugative transposon Tn916 as a vector for introducing recombinant DNA into Clostridium difficile. This was used to introduce antisense RNA for the adhesin encoding gene cwp66 into C. difficile 79-685. RT-PCR demonstrated that cwp66 specific antisense RNA was produced. However, there was no statistically significant difference in the protein expression or in the adherence of recombinant C. difficile strains. This may be due to the amount of transcripts of the wild-type (sense) cwp66 outnumbering the antisense transcripts or secondary structures present within the cwp66 mRNA. Unlike in other strains of C. difficile, where Tn916 inserts into the genome at highly preferred sites, in C. difficile 79-685, it integrates into multiple sites opening up the possibility of using Tn916 as a mutagen in this strain.

Original language	English
Pages (from-to)	617-624
Number of pages	8
Journal	Journal of Microbiological Methods
Volume	55
Issue number	3
DOIs	https://doi.org/10.1016/S0167-7012(03)00200-8
Publication status	Published - Dec 2003

Bibliographical note

Funding Information:
CH was supported partly by a Mari Curie training fellowship. We gratefully acknowledge the BBSRC (grant number 346/E13746) for funding this work.

Keywords

Antisense RNA
Clostridium difficile
Conjugative vector
Tn916

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10.1016/S0167-7012(03)00200-8

Cite this

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title = "Development of an integrative vector for the expression of antisense RNA in Clostridium difficile",

abstract = "A method was developed to use the conjugative transposon Tn916 as a vector for introducing recombinant DNA into Clostridium difficile. This was used to introduce antisense RNA for the adhesin encoding gene cwp66 into C. difficile 79-685. RT-PCR demonstrated that cwp66 specific antisense RNA was produced. However, there was no statistically significant difference in the protein expression or in the adherence of recombinant C. difficile strains. This may be due to the amount of transcripts of the wild-type (sense) cwp66 outnumbering the antisense transcripts or secondary structures present within the cwp66 mRNA. Unlike in other strains of C. difficile, where Tn916 inserts into the genome at highly preferred sites, in C. difficile 79-685, it integrates into multiple sites opening up the possibility of using Tn916 as a mutagen in this strain.",

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author = "Roberts, {Adam P.} and Claire Hennequin and Michael Elmore and Anne Collignon and Tuomo Karjalainen and Nigel Minton and Peter Mullany",

note = "Funding Information: CH was supported partly by a Mari Curie training fellowship. We gratefully acknowledge the BBSRC (grant number 346/E13746) for funding this work.",

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AU - Roberts, Adam P.

AU - Hennequin, Claire

AU - Elmore, Michael

AU - Collignon, Anne

AU - Karjalainen, Tuomo

AU - Minton, Nigel

AU - Mullany, Peter

N1 - Funding Information: CH was supported partly by a Mari Curie training fellowship. We gratefully acknowledge the BBSRC (grant number 346/E13746) for funding this work.

PY - 2003/12

Y1 - 2003/12

N2 - A method was developed to use the conjugative transposon Tn916 as a vector for introducing recombinant DNA into Clostridium difficile. This was used to introduce antisense RNA for the adhesin encoding gene cwp66 into C. difficile 79-685. RT-PCR demonstrated that cwp66 specific antisense RNA was produced. However, there was no statistically significant difference in the protein expression or in the adherence of recombinant C. difficile strains. This may be due to the amount of transcripts of the wild-type (sense) cwp66 outnumbering the antisense transcripts or secondary structures present within the cwp66 mRNA. Unlike in other strains of C. difficile, where Tn916 inserts into the genome at highly preferred sites, in C. difficile 79-685, it integrates into multiple sites opening up the possibility of using Tn916 as a mutagen in this strain.

AB - A method was developed to use the conjugative transposon Tn916 as a vector for introducing recombinant DNA into Clostridium difficile. This was used to introduce antisense RNA for the adhesin encoding gene cwp66 into C. difficile 79-685. RT-PCR demonstrated that cwp66 specific antisense RNA was produced. However, there was no statistically significant difference in the protein expression or in the adherence of recombinant C. difficile strains. This may be due to the amount of transcripts of the wild-type (sense) cwp66 outnumbering the antisense transcripts or secondary structures present within the cwp66 mRNA. Unlike in other strains of C. difficile, where Tn916 inserts into the genome at highly preferred sites, in C. difficile 79-685, it integrates into multiple sites opening up the possibility of using Tn916 as a mutagen in this strain.

KW - Antisense RNA

KW - Clostridium difficile

KW - Conjugative vector

KW - Tn916

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ER -

Development of an integrative vector for the expression of antisense RNA in Clostridium difficile

Abstract

Bibliographical note

Keywords

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