Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker
Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we e...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Published: |
Oxford University Press (OUP)
2012
|
| Online Access: | https://eprints.nottingham.ac.uk/3062/ |
| _version_ | 1848790943879135232 |
|---|---|
| author | Heap, John T. Ehsaan, Muhammad Cooksley, Clare M. Ng, Yen-Kuan Cartman, Stephen T. Winzer, Klaus Minton, Nigel P. |
| author_facet | Heap, John T. Ehsaan, Muhammad Cooksley, Clare M. Ng, Yen-Kuan Cartman, Stephen T. Winzer, Klaus Minton, Nigel P. |
| author_sort | Heap, John T. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. |
| first_indexed | 2025-11-14T18:20:39Z |
| format | Article |
| id | nottingham-3062 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T18:20:39Z |
| publishDate | 2012 |
| publisher | Oxford University Press (OUP) |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-30622020-05-04T16:32:11Z https://eprints.nottingham.ac.uk/3062/ Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker Heap, John T. Ehsaan, Muhammad Cooksley, Clare M. Ng, Yen-Kuan Cartman, Stephen T. Winzer, Klaus Minton, Nigel P. Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. Oxford University Press (OUP) 2012-01-18 Article PeerReviewed Heap, John T., Ehsaan, Muhammad, Cooksley, Clare M., Ng, Yen-Kuan, Cartman, Stephen T., Winzer, Klaus and Minton, Nigel P. (2012) Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Research, 40 (8). 10/1-10/10. ISSN 0305-1048 http://nar.oxfordjournals.org/content/40/8/e59.full doi:10.1093/nar/gkr1321 doi:10.1093/nar/gkr1321 |
| spellingShingle | Heap, John T. Ehsaan, Muhammad Cooksley, Clare M. Ng, Yen-Kuan Cartman, Stephen T. Winzer, Klaus Minton, Nigel P. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker |
| title | Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker |
| title_full | Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker |
| title_fullStr | Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker |
| title_full_unstemmed | Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker |
| title_short | Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker |
| title_sort | integration of dna into bacterial chromosomes from plasmids without a counter-selection marker |
| url | https://eprints.nottingham.ac.uk/3062/ https://eprints.nottingham.ac.uk/3062/ https://eprints.nottingham.ac.uk/3062/ |