Chromosome - Fapesp

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Gene Knock-out The ClosTron: A rapid, highly effective mutagenesis system for meatbolic engineering in Clostridia (http://clostron.com) • Carries a small segment of DNA encoding a mobile group II intron • The intron jumps from the plasmid into the host chromosome causing gene inactivation • Through DNA synthesis we control into which gene the intron inserts • Concomitant with insertion, host becomes resistant to erythromycin • This allows the rapid, selectable creation of stable mutants Kuehne SA, Heap JT, Cooksley CM, Cartman ST, Minton NP (2011) ClosTron-Mediated Engineering of Clostridium. Methods Mol Biol. 765: 389-407
Streamlining the System http://www.clostron.com • Free to use webbased algorithm removes reliance on Sigma Aldrich kit • Retargeted region synthesised and cloned into ClosTron vector by DNA2.0 • Retargeted plasmid delivered within 10- 14 days • Integrants (mutants) isolated within 5-7 days of receipt of plasmid
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