A new and improved host-independent plasmid system for RK2-based conjugal transfer
Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4...
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Published in | PloS one Vol. 9; no. 3; p. e90372 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Public Library of Science
03.03.2014
Public Library of Science (PLoS) |
Subjects | |
Online Access | Get full text |
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Summary: | Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT) are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Conceived and designed the experiments: TAS RL KFD SV. Performed the experiments: TAS RL KFD ML. Analyzed the data: TAS KFD SV. Contributed reagents/materials/analysis tools: TAS RL KFD ML. Wrote the paper: TAS RL SV. Competing Interests: The authors have declared that no competing interests exist. |
ISSN: | 1932-6203 1932-6203 |
DOI: | 10.1371/journal.pone.0090372 |