期刊
NUCLEIC ACIDS RESEARCH
卷 42, 期 4, 页码 -出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkt1101
关键词
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资金
- UK Engineering and Physical Sciences Research Council [EP/H019154/1]
- UK Biotechnology and Biological Sciences Research Council [BB/K003356/1, BB/H005277/1, BB/J004561/1]
- National Science Foundation [0943392]
- John Innes Foundation
- University of Glasgow
- Biotechnology and Biological Sciences Research Council [BB/H005277/1, BBS/E/J/00000614, BB/L004321/1, BBS/E/J/000CA527, BB/E022200/1, BB/H001212/1, BBS/E/J/000CA397, BB/H005447/1, BB/K003356/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/K034359/1, EP/H019154/1] Funding Source: researchfish
- BBSRC [BB/K003356/1, BB/E022200/1, BBS/E/J/000CA397, BB/H005277/1, BBS/E/J/000CA527, BB/L004321/1, BB/H001212/1, BB/H005447/1] Funding Source: UKRI
- EPSRC [EP/K034359/1, EP/H019154/1] Funding Source: UKRI
Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by phi C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. phi C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.
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