Gene replacement in mycobacteria by using incompatible plasmids
A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2003 |
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| Erschienen: |
2003 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:69 |
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| Enthalten in: |
Applied and environmental microbiology - 69(2003), 1 vom: 01. Jan., Seite 517-23 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Pashley, Carey A [VerfasserIn] |
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| Themen: |
Bacterial Proteins |
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| Anmerkungen: |
Date Completed 21.03.2003 Date Revised 26.05.2021 published: Print Citation Status MEDLINE |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM122966392 |
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| 100 | 1 | |a Pashley, Carey A |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Gene replacement in mycobacteria by using incompatible plasmids |
| 264 | 1 | |c 2003 | |
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| 337 | |a ohne Hilfsmittel zu benutzen |b n |2 rdamedia | ||
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| 500 | |a Date Completed 21.03.2003 | ||
| 500 | |a Date Revised 26.05.2021 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis | ||
| 650 | 4 | |a Evaluation Study | |
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a Bacterial Proteins |2 NLM | |
| 700 | 1 | |a Parish, Tanya |e verfasserin |4 aut | |
| 700 | 1 | |a McAdam, Ruth A |e verfasserin |4 aut | |
| 700 | 1 | |a Duncan, Ken |e verfasserin |4 aut | |
| 700 | 1 | |a Stoker, Neil G |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Applied and environmental microbiology |d 1976 |g 69(2003), 1 vom: 01. Jan., Seite 517-23 |w (DE-627)NLM000057142 |x 1098-5336 |7 nnns |
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