TY  - JOUR
A1  - Messerschmidt, Katrin
A1  - Hochrein, Lena
A1  - Dehm, Daniel
A1  - Schulz, Karina
A1  - Mueller-Roeber, Bernd
T1  - Characterizing seamless ligation cloning extract for synthetic biological applications
JF  - Analytical biochemistry : methods in the biological sciences
N2  - 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. (C) 2016 Elsevier Inc. All rights reserved.
KW  - SLiCE
KW  - Seamless ligation cloning
KW  - Homologous recombination
KW  - Synthetic biology
Y1  - 2016
U6  - https://doi.org/10.1016/j.ab.2016.05.029
SN  - 0003-2697
SN  - 1096-0309
VL  - 509
SP  - 24
EP  - 32
PB  - Elsevier
CY  - San Diego
ER  -