Directed evolution and identification of control regions of ColE1 plasmid replication origins using only nucleotide deletions.
J Mol Biol
; 351(4): 763-75, 2005 Aug 26.
Article
em En
| MEDLINE
| ID: mdl-16051272
ABSTRACT
Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions.
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Bases de dados:
MEDLINE
Assunto principal:
Plasmídeos
/
Origem de Replicação
/
Evolução Molecular Direcionada
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Revista:
J Mol Biol
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Estados Unidos