Recursive DNA Assembly Using Protected Oligonucleotide Duplex Assisted Cloning (PODAC).
A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication in vivo, unveiling a new cloning site in the process. We used this method to efficiently and accurately assemble a biosynthetic pathway and demonstrated its robustness toward sequence repeats by constructing artificial CRISPR arrays. As PODAC is readily amenable to standardization, it would make a useful addition to the synthetic biology toolkit.
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University , 9000 Ghent, Belgium.
Bob
B
Van Hove
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University , 9000 Ghent, Belgium.
Chiara
C
Guidi
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University , 9000 Ghent, Belgium.
Lien
L
De Wannemaeker
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University , 9000 Ghent, Belgium.
Jo
J
Maertens
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University , 9000 Ghent, Belgium.
Marjan
M
De Mey
D064113:CRISPR-Cas Systems / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation / Q000235:genetics; D005818:Genetic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*
D012313:RNA; D004247:DNA
CRISPR; DNA assembly; Golden Gate cloning; molecular cloning; pathway assembly; recursive cloning