title: Model-guided ligation strategy for optimal assembly of DNA libraries.
abstract: DNA ligation is essential to many molecular biology manipulations, but this reaction is often carried out by following generic guidelines or by trial and error. Maximizing the desired ligation product is especially important in DNA library construction for directed evolution experiments since library diversity is directly affected by ligation efficiency. Here, we suggest that display vectors that rely on Type IIP restriction sites for cloning should be redesigned to utilize Type IIS restriction sites instead because ligation yield is significantly improved: we observed up to 15- and 2.6-fold increases in desired products for circular and linear ligation reactions, respectively. To guide ligation optimization more rationally, we developed an easily parameterized thermodynamic model that predicts product distributions based on input DNA concentrations and free energies of the ligation events. We applied this model to study ligation reactions using a ribosome display vector redesigned with Type IIS restriction sites (pRDV2). We computationally predicted and experimentally validated the relative abundance of various products in three-piece linear ligations as well as the extent of transformation from vector-insert circular ligations. Based on our results, we provide general insights into ligation and we outline guidelines for optimizing this reaction for both in vivo and in vitro display methodologies.
authors.affiliation: Department of Bioengineering,  University of Pennsylvania, Philadelphia, PA 19104,  USA.
authors.forename: Daphne T W
authors.initials: DT
authors.lastname: Ng
authors.forename: Casim A
authors.initials: CA
authors.lastname: Sarkar
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019020:Directed Molecular Evolution; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical; D013816:Thermodynamics
chemical_list: D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases