Selection of restriction endonucleases using artificial cells.
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.
Zheng
Yu
Y
New England BioLabs, Inc., 240 County Road, Ipswich, MA 01938, USA. zhengy@neb.com
Roberts
Richard J
RJ
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
D004269:DNA, Bacterial; D004655:Emulsions; D004262:DNA Restriction Enzymes; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific