Method for isolating mutant cells
AssigneePresident and Fellows of Harvard College
InventorsEdmund Lin, Bryan L. Ray
Life of this patent
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Abstract
Disclosed is a method for isolating a mutant cell that secretes a desired compound at a level greater than that secreted by a starter cell from which the mutant cell is derived. The method includes contacting a plurality of auxotrophic feeder cells and auxotrophic starter cells together in the solid growth medium. The starter cells produce the desired compound required by the feeder cells and the feeder cells produce a metabolite required by the starter cells. These cells are cultured to produce a plurality of primary colonies, the largest of which include at least one of the mutant cells which overproduces the desired compound.
Claims15
A method for isolating a mutant cell that secretes a desired compound at a level greater than that secreted by a starter cell from which said mutant cell is derived, said method comprising the steps of:
- (a) providing a starter cell selected from the group consisting of bacterial, fungi, and animal cells, which:
- (i) is an auxotroph that has a requirement for a metabolite for survival or division of the starter cell in a solid growth medium, the metabolite being selected from the group consisting of nutrients, amino acids, growth factors, cofactors, interleukins, vitamins, and carbohydrates, the metabolite being other than the desired compound;
- (ii) mutating to lack the requirement at a frequency of less than 10.sup.-10 per cell division;
- (iii) has a rate of mutation at least 10.sup.-6 per nucleotide base per cell division, the rate resulting from the presence of a mutator gene or mutation resulting from mutagenesis in the DNA of the starter cell;
- and
- (iv) secretes the desired compound into the solid growth medium;
- (b) providing a feeder cell selected from the group consisting of bacterial, fungi, and animal cells, which:
- (i) is an auxotroph that requires the desired compound for its survival or division in the solid growth medium, the desired compound being selected from the group consisting of nutrients, amino acids, growth factors, interleukins, cofactors, vitamins, and carbohydrates, the desired compound being other than the metabolite;
- and
- (ii) secretes the metabolite into solid growth medium;
- (c) contacting a plurality of the feeder cells and the starter cells together in or on the surface of the solid growth medium to allow the starter cells to produce the desired compound and the feeder cells to produce the metabolite;
- and
- (d) culturing the feeder and starter cells to grow, divide, produce a plurality of primary colonies,
- wherein the primary colonies which are the largest include at least one of the mutant cells.
Dependencies are inferred from the claim text.