Journal
NUCLEIC ACIDS RESEARCH
Volume 40, Issue 8, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkr1321
Keywords
-
Categories
Funding
- Biotechnology and Biological Sciences Research Council [BB/F003390/1, BB/G016224/1, BB/E021271/1]
- Medical Research Council [G0601176]
- European Union [HEALTH-F3-2008-223585]
- TMO Renewables Ltd.
- University of Nottingham
- Biotechnology and Biological Sciences Research Council [BB/G016224/1, BB/E021271/1, BB/F003390/1] Funding Source: researchfish
- Medical Research Council [G0601176] Funding Source: researchfish
- BBSRC [BB/F003390/1, BB/G016224/1, BB/E021271/1] Funding Source: UKRI
- MRC [G0601176] Funding Source: UKRI
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available