title: Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications.
abstract: Recombination-based DNA construction methods, such as Gibson assembly, have made it possible to easily and simultaneously assemble multiple DNA parts, and they hold promise for the development and optimization of metabolic pathways and functional genetic circuits. Over time, however, these pathways and circuits have become more complex, and the increasing need for standardization and insulation of genetic parts has resulted in sequence redundancies--for example, repeated terminator and insulator sequences--that complicate recombination-based assembly. We and others have recently developed DNA assembly methods, which we refer to collectively as unique nucleotide sequence (UNS)-guided assembly, in which individual DNA parts are flanked with UNSs to facilitate the ordered, recombination-based assembly of repetitive sequences. Here we present a detailed protocol for UNS-guided assembly that enables researchers to convert multiple DNA parts into sequenced, correctly assembled constructs, or into high-quality combinatorial libraries in only 2-3 d. If the DNA parts must be generated from scratch, an additional 2-5 d are necessary. This protocol requires no specialized equipment and can easily be implemented by a student with experience in basic cloning techniques.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Torella
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Florian
authors.initials: F
authors.lastname: Lienert
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Christian R
authors.initials: CR
authors.lastname: Boehm
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Jan-Hung
authors.initials: JH
authors.lastname: Chen
authors.affiliation: 1] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. [2] Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Way
authors.affiliation: 1] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. [2] Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Silver
mesh_terms: D001483:Base Sequence / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods*
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