title: Selection of restriction endonucleases using artificial cells.
abstract: We describe in this article an in vitro system for the selection of restriction endonucleases using artificial cells. The artificial cells are generated in the form of a water-in-oil emulsion by in vitro compartmentalization. Each aqueous compartment contains a reconstituted transcription/translation mix along with the dispersed DNA templates. In the compartments containing endonuclease genes, an endonuclease expressed in vitro cleaves its own DNA template adjacent to the gene, leaving a sticky end. The pooled DNA templates are then ligated to an adaptor with a compatible end. The endonuclease genes are then enriched by adaptor-specific PCR on the ligation mix. We demonstrate that the system can achieve at least 100-fold enrichment in a single round of selection. It is sensitive enough to enrich an active endonuclease gene from a 1:10(5) model library in 2-3 rounds of selection. Finally, we describe experiments where we selected endonuclease genes directly from a bacterial genomic DNA source in three rounds of selections: the known PstI gene from Providencia stuartii and the new TspMI gene from Thermus sp. manalii. This method provides a unique tool for cloning restriction endonuclease genes and has many other potential applications.
authors.affiliation: New England BioLabs, Inc., 240 County Road, Ipswich, MA 01938, USA. zhengy@neb.com
authors.forename: Yu
authors.initials: Y
authors.lastname: Zheng
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Roberts
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D004655:Emulsions; D015723:Gene Library; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D023281:Genomics; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D004655:Emulsions; D004262:DNA Restriction Enzymes; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific