Linked Life Data

12853150

Source:http://linkedlifedata.com/resource/pubmed/id/12853150

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:dateCreated
2003-7-10
pubmed:abstractText
We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0378-1119
pubmed:author
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
311
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
153-63
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli.
pubmed:affiliation
Laboratory of Genetics, University of Wisconsin-Madison, 445 Henry Mall, Madison, WI 53706, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.