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US 6,673,567 B2Granted
Method of determination of gene function
AssigneeE. I. Du Pont de Nemours and Company
InventorsPamela L. Sharpe, Vasantha Nagarajan
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Abstract
The present method is useful for the identification of genes, ORF's and other nucleic acid molecules which are essential for the expression of a specific phenotype in microorganisms. The method employs In vitro transposition in conjunction with an chromosomal integration vector containing a specific gene or genetic element whose function is unknown. Subsequent transformation of a recombination proficient host with the vector and growth first under non-integrating conditions and then under integrating conditions, followed by a selection screen for either single or double crossover events results in transformants that may be subjected to phenotypic screens to determine gene function.
Claims34
A method for the identification of an essential gene responsible for the presence of a specific phenotype on a recombination proficient microorganism comprising:
- a) contacting In vitro:
- (i) a transposable element comprising at least one first genetic marker;
- (ii) a transposase for the insertion of the transposable element into the essential gene;
- and
- (iii) a target DNA containing the essential gene, said gene having a homolog in the genome of the recombination proficient microorganism;
- under suitable conditions whereby the transposable element inserts within the essential gene to form a transposon disrupted gene;
- b) cloning the transposon disrupted gene into a suitable vector to form a chromosomal integration vector, said vector comprising at least one second genetic marker;
- c) transforming a recombination proficient microorganism which is not naturally transformable with the chromosomal integration vector of step (b) to create at least one transformant;
- d) selecting the at least one transformant of step (c) under conditions whereby no chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant, by identifying transformants expressing the first genetic marker;
- e) culturing the identified transformants of step (d) under conditions whereby chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant;
- f) selecting transformants of step (e) which express either the first genetic marker or both the first and second genetic markers by which transformants having undergone either a single or double crossover event are identified;
- g) screening the transformants of step (f) which have undergone either a single or double crossover event, for the presence of a specific phenotype wherein the transformants which are positive for the specific phenotype contain a transposon disrupted gene;
- and
- h) isolating the transposon disrupted gene from the transformant of step (g) which is positive for the specific phenotype.
11 more elements
A method for the identification of an essential gene responsible for the cell growth under any condition in a recombination proficient microorganism comprising:
- a) contacting In vitro
- (i) a transposable element comprising at least one marker;
- (ii) a transposase for the insertion of the transposable element into the essential gene;
- and
- (iii) target DNA containing the essential gene, said gene having a homolog in the genome of the recombination proficient microorganism;
- under suitable conditions whereby the transposable element inserts within the essential gene to form a transposon disrupted gene;
- b) cloning the transposon disrupted gene into a temperature sensitive vector containing a second genetic marker to form a temperature sensitive chromosomal integration vector;
- c) transforming a recombination proficient microorganism, which is not naturally transformable, with the temperature sensitive chromosomal integration vector of step (b) to create at least one transformant;
- d) culturing the at least one transformant of step (c) at a permissive temperature whereby no chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant;
- e) identifying transformants of step (d) expressing the marker;
- f) culturing the identified transformants of step (e) at non-permissive temperatures whereby chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant;
- g) selecting transformants of step (e) which did not grow at step (f) after chromosomal integration between the chromosomal integration vector and the genome of the transformant and which contain a transposon disrupted gene;
- and
- h) isolating the transposon disrupted gene from the transformant of step (g) which is responsible for cell growth under any condition.
11 more elements
A method for the identification of an essential gene responsible for the presence of a specific phenotype in a recombination proficient microorganism comprising:
- a) contacting In vitro:
- (i) a transposable element comprising at least one first marker;
- (ii) a transposase for the insertion of the transposable element into the essential gene;
- and
- (iii) target DNA containing the essential gene, said gene having a homolog in the genome of the recombination proficient microorganism;
- under suitable conditions whereby the transposable element inserts within the essential gene to form a transposon disrupted gene;
- b) cloning the transposon disrupted gene into a suitable vector to form a chromosomal integration vector, said vector comprising at least one second marker and at least one third marker;
- c) transforming a recombination proficient microorganism which, is not naturally transformable, with the chromosomal integration vector of step (b) to create transformants;
- d) selecting the transformants of step (c) under conditions whereby no chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant, by identifying transformants expressing the first marker;
- e) culturing the identified transformants of step (d) under conditions whereby chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant;
- f) selecting transformants of step (e) which express either the first marker alone, the first and second markers alone, or the first, second and third markers, by which transformants having undergone either a single or double crossover event are identified;
- g) screening the transformants of step (f) which have undergone either a single or double crossover event, for the presence of a specific phenotype wherein the transformants which are positive for the specific phenotype contain a transposon disrupted gene;
- and
- h) isolating the transposon disrupted gene from the transformant of step (g) having the specific phenotype.
11 more elements
Dependencies are inferred from the claim text.