title: Computer-aided high-throughput cloning of bacteria in liquid medium.
abstract: Bacterial cloning was first introduced over a century ago and has since become one of the most useful procedures in biological research, perhaps paralleled in its ubiquity only by PCR and DNA sequencing. However, unlike PCR and sequencing, cloning has generally remained a manual, labor-intensive, low-throughput procedure. Here we address this issue by developing an automated, computer-aided bacterial cloning method using liquid medium that is based on the principles of (i) limiting dilution of bacteria, (ii) inference of colony forming units (CFUs) based on optical density (OD) readings, and (iii) verification of monoclonality using a mixture of differently colored fluorescently labeled bacteria for transformation. We demonstrate the high-throughput utility of this method by employing it as a cloning platform for a DNA synthesis process.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Tuval
authors.initials: T
authors.lastname: Ben Yehezkel
authors.forename: Shiran
authors.initials: S
authors.lastname: Nagar
authors.forename: Danny
authors.initials: D
authors.lastname: Mackrants
authors.forename: Zipora
authors.initials: Z
authors.lastname: Marx
authors.forename: Gregory
authors.initials: G
authors.lastname: Linshiz
authors.forename: Uri
authors.initials: U
authors.lastname: Shabi
authors.forename: Ehud
authors.initials: E
authors.lastname: Shapiro
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D014170:Transformation, Genetic