Recombinational cloning of large DNA fragments of bacterial chromosome by plasmid rescue
OBJECTIVE: To develop a new method to cline large DNA fragment by combining whole genome sequence information and the principle of plasmid rescue.
METHODS: First, we amplified a fragment downstream the region to be cloned and cloned the fragment into a suicide plasmid to construct targeting plasmid, which was then targeted into chromosome by homologous recombination. Then, genomic DNA was isolated, digested with appropriate enzyme, re-ligated and transformed into host bacteria to rescue the plasmid and the large DNA fragment. The rescued DNA fragment was sub-cloned into new plasmids for special purposes.
RESULTS: Using this method, we successfully cloned the 11kb virB operon of Brucella and constructed complementary plasmid. The transcription of the disrupted virB operon was restored with the complementary plasmid, validating the feasibility of the strategy.
CONCLUSION: This recombinational cloning strategy provides us a new method to modify large DNA fragment of bacteria.
| Medienart: |
Artikel |
|---|
| Erscheinungsjahr: |
2008 |
|---|---|
| Erschienen: |
2008 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:48 |
|---|---|
| Enthalten in: |
Wei sheng wu xue bao = Acta microbiologica Sinica - 48(2008), 4 vom: 16. Apr., Seite 532-8 |
| Sprache: |
Chinesisch |
|---|
| Beteiligte Personen: |
Wang, Yufei [VerfasserIn] |
|---|
| Themen: |
DNA, Bacterial |
|---|
| Anmerkungen: |
Date Completed 15.05.2009 Date Revised 09.04.2012 published: Print Citation Status MEDLINE |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM180556347 |
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| 041 | |a chi | ||
| 100 | 1 | |a Wang, Yufei |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Recombinational cloning of large DNA fragments of bacterial chromosome by plasmid rescue |
| 264 | 1 | |c 2008 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ohne Hilfsmittel zu benutzen |b n |2 rdamedia | ||
| 338 | |a Band |b nc |2 rdacarrier | ||
| 500 | |a Date Completed 15.05.2009 | ||
| 500 | |a Date Revised 09.04.2012 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a OBJECTIVE: To develop a new method to cline large DNA fragment by combining whole genome sequence information and the principle of plasmid rescue | ||
| 520 | |a METHODS: First, we amplified a fragment downstream the region to be cloned and cloned the fragment into a suicide plasmid to construct targeting plasmid, which was then targeted into chromosome by homologous recombination. Then, genomic DNA was isolated, digested with appropriate enzyme, re-ligated and transformed into host bacteria to rescue the plasmid and the large DNA fragment. The rescued DNA fragment was sub-cloned into new plasmids for special purposes | ||
| 520 | |a RESULTS: Using this method, we successfully cloned the 11kb virB operon of Brucella and constructed complementary plasmid. The transcription of the disrupted virB operon was restored with the complementary plasmid, validating the feasibility of the strategy | ||
| 520 | |a CONCLUSION: This recombinational cloning strategy provides us a new method to modify large DNA fragment of bacteria | ||
| 650 | 4 | |a English Abstract | |
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a DNA, Bacterial |2 NLM | |
| 650 | 7 | |a DNA, Recombinant |2 NLM | |
| 700 | 1 | |a Chen, Zeliang |e verfasserin |4 aut | |
| 700 | 1 | |a Qiao, Feng |e verfasserin |4 aut | |
| 700 | 1 | |a Du, Xinying |e verfasserin |4 aut | |
| 700 | 1 | |a Jia, Leili |e verfasserin |4 aut | |
| 700 | 1 | |a Yuan, Jing |e verfasserin |4 aut | |
| 700 | 1 | |a Huang, Liuyu |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Wei sheng wu xue bao = Acta microbiologica Sinica |d 1985 |g 48(2008), 4 vom: 16. Apr., Seite 532-8 |w (DE-627)NLM012631655 |x 0001-6209 |7 nnns |
| 773 | 1 | 8 | |g volume:48 |g year:2008 |g number:4 |g day:16 |g month:04 |g pages:532-8 |
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