Construcción de una de las primeras genotecas en plásmidos

A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome.

Clarke and Carbon,  Cell, 9: 91-99 (1976)

Using the poly(dA-dT) “connector” method , a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle contained one molecule of poly(dT)-tailed CoI EI-DNA (L_RI) annealed to any one of a collection of poly(dA)-tailed linear DNA fragments, produced originally by shearing total E. coli DNA to an average size of 8.5 × 10⁶ daltons. This annealed DNA preparation (12 μg) was used to transform an F⁺recA E. coli strain (JA200), selecting transformants by their resistance to collcin EI. A collector or “bank” of over 2000 colicin EI-resistant clones was thereby obtained, 70% of which were shown to contain hybrid CoI EI-DNA (E. coli) plasmids. This colony bank is large enough to include hybrid plasmids representative of the entire E. coli genome. Individual plasmids have been readily identified by replica mating the collection onto plates seeded with cultures of various F⁻ auxotrophic recipients, selecting for complementation of the auxotrophic markers by F-mediated transfer of hybrid plasmids to the F⁻ recipients. In this manner, over 80 hybrid CoI EI-DNA (E. coli) plasmid-bearing clones have been identified in the colony bank, and about 40 known E. coli genes have been tentatively assigned to these various plasmids. The hybrid plasmids are transferred efficiently from F⁻ donors to appropriate F⁻ recipients. The use of this method to establish similar colony banks in E. coli containing hybrid plasmids representative of various simple eucaryotic genomes is discussed.

Acceso al trabajo