Characterizing seamless ligation cloning extract for synthetic biological applications.
Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular and synthetic biology projects.
University of Potsdam, Cell2Fab Research Unit, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany. Electronic address: messer@uni-potsdam.de.
Katrin
K
Messerschmidt
University of Potsdam, Cell2Fab Research Unit, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany.
Lena
L
Hochrein
University of Potsdam, Cell2Fab Research Unit, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany.
Daniel
D
Dehm
University of Potsdam, Cell2Fab Research Unit, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany.
Karina
K
Schulz
University of Potsdam, Department of Molecular Biology, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany; Center of Plant Systems Biology and Biotechnology (CPSBB), Ruski Blvd. 139, Plovdiv 4000, Bulgaria.
Bernd
B
Mueller-Roeber
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D011088:DNA Ligases / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000737:chemistry* / Q000235:genetics
D004247:DNA; D011088:DNA Ligases
Homologous recombination; SLiCE; Seamless ligation cloning; Synthetic biology