title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost.
abstract: Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides.
authors.affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland.
authors.forename: Roger M
authors.initials: RM
authors.lastname: Benoit
authors.affiliation: Novartis Pharma AG, Basel, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Ostermeier
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Geiser
authors.affiliation: The Kellog School of Science and Technology, The Scripps Research Institute, La Jolla, United States of America.
authors.forename: Julia Su Zhou
authors.initials: JS
authors.lastname: Li
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Hans
authors.initials: H
authors.lastname: Widmer
authors.affiliation: University of Edinburgh, School of Biological Sciences (CSE) and School of Biomedical Sciences (CMVM), Edinburgh, United Kingdom.
authors.forename: Manfred
authors.initials: M
authors.lastname: Auer
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant / Q000191:economics / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000191:economics / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000191:economics / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods
chemical_list: D004274:DNA, Recombinant; D004259:DNA-Directed DNA Polymerase