a biobrick library for cloning custom eukaryotic plasmids一个倡导图书馆真核质粒克隆定制.pdfVIP

A Biobrick Library for Cloning Custom Eukaryotic Plasmids ¨ Marco Constante*, Raik Grunberg, Mark Isalan EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain Abstract Researchers often require customised variations of plasmids that are not commercially available. Here we demonstrate the applicability and versatility of standard synthetic biological parts (biobricks) to build custom plasmids. For this purpose we have built a collection of 52 parts that include multiple cloning sites (MCS) and common protein tags, protein reporters and selection markers, amongst others. Importantly, most of the parts are designed in a format to allow fusions that maintain the reading frame. We illustrate the collection by building several model contructs, including concatemers of protein binding-site motifs, and a variety of plasmids for eukaryotic stable cloning and chromosomal insertion. For example, in 3 biobrick iterations, we make a cerulean-reporter plasmid for cloning fluorescent protein fusions. Furthermore, we use the collection to implement a recombinase-mediated DNA insertion (RMDI), allowing chromosomal site-directed exchange of genes. By making one recipient stable cell line, many standardised cell lines can subsequently be generated, by fluorescent fusion-gene exchange. We propose that this biobrick collection may be distributed peer-to-peer as a stand-alone library, in addition to its distribution through the Registry of Standard Biological Parts (/). ¨ Citation: Constante M, Grunberg R, Isalan M (2011) A Biobrick Library for Cloning Custom Eukaryotic Plasmids. PLoS ONE 6(8): e23685. doi:10.1371/ journal.pone.0023685 ¨ Editor: Jorg D. Hoheisel, Deutsches Krebsforschungszentrum, Germany Received April