Prototrophy is often the phenotype selected to detect transformants. Prototrophi

Question

Prototrophy is often the phenotype selected to detect transformants. Prototrophic cells are used for donor DNA extraction; then this DNA is cloned and the clones are added to an auxotrophic recipient culture. Successful transformants are identified by plating the recipient cultural on minimal medium and looking for colonies. What experimental design would you use to make sure that a colony that you hope is a transformant is not in fact, a revertant (mutation back to prototrophy by a second mutation in the originally mutated gene) of the auxotrophic mutation?

Explanation / Answer

Auxotrophic and prototrophic organisms are opposites. Auxotrophic organisms are the ones incapable of producing a specific molecule needed for their growth, incapability caused by a mutation of a gene vital for the production of the molecule. Prototrophic organisms on the other side are the ones capable of producing all the molecules they need for their growth.

An adenine auxotrophic cell would be the one incapable to produce adenine, and therefore it would be unable to grow in a minimal medium unless adenine is added to it. A leucine auxotrophic cell would be incapable of synthesizing leucine, therefore it would grow only in a minimal medium that has leucine added to it. A prototrophic cell will be able to grow in both mediums, in a minimal medium, and in a minimal medium to which an extra nutrient was added, like adenine or leucine for the last 2 examples.

A revertant would be a cell that was auxotrophic, but that due to a mutation it will change to be prototrophic. In other words, the auxotrophic cell, that can't synthesize a molecule due to a mutation of a gene, suffers a mutation in the same gene that makes the gene functional again, turning the auxotrophic cell into a prototrophic cell.

In this problem, genes from prototrophic cells were cloned into plasmids, and then a laboratory procedure was performed to induce auxotrophic cells to introduce the plasmids in them. The cells that successfully introduce the plasmids (transformants) in them will now turn into prototrophic cells (able to grow in any media). To identify the transformants they were grown in minimal medium plates (if they are the will grow).

To be able to assure that the transformants are not revertants, you can start by cloning the prototrophic genes in plasmids that contain an antibiotic resistance gene (most plasmids do), so the possible transformants can be proven to be so by picking the colonies that grew in the minimal medium and transferring them to a medium that contains the antibiotic for which the truly transformants should be resistant if they successfully introduce the plasmids into them.

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