Question

Demo Problem You are planning a mapping experiment to map several genes in E. coli. You choose the following strains of E. coli:

lac+, gal+, trp+, his+, str^S          X        lac-, gal-, trp-, his- str^R

Trp refers to the amino acid tryptophan and his refers to the amino acid histidine; str refers to the antibiotic streptomycin with S indicating sensitivity and R indicating resistance. Lac and gal refer to the sugars lactose and galactose (glucose is the default carbon source). To assess phenotypes of the various “offspring”, you need to prepare plate media with various ingredients added or left out.

What additive(s) should you include or leave out of the medium to achieve the following purposes?

a. To select for lac+ and against str^s.

b. To select for trp+ and against str^s.

Answer 1

To select against any antibiotic sensitive gene would mean selecting the resistant cells, i.e. bacteria possessing the StrR gene. and therefore, you need to grow the bacteria in plates containing Streptomycin. All colonies that can grow on streptomycin containing plate would have the StrR gene, irrespective of their other metabolic capabilities.

Now, in the given scenario, there are two alternate carbon sources other than glucose that can be utilized by the cells, either lactose ( lac+) or galactose (gal+) or both (lac+, gal+) depending upon their genetic makeup. Also, the presence of a gene capable of producing an amino acid, say tryptophan by trp+ bacteria or histidine by his+ bacteria means that these bacteria can synthesize these amino acids if they are not present in the medium and therefore would be able to grow on plates lacking these amino acids.

a) to select for lac+ cells and against streptomycin sensitive cells, the bacteria would have to be grown on streptomycin containing plates having no glucose and no galactose but containing added lactose. Here, only lactose is available as sugar and so, only lac+ cells that have the ability to metabolize lactose can grow. However, as we are not screening for the ability to produce any of the amino acids, Histidine and tryptophan should be provided in the medium.

b) To select for trp+ and against strS, grow the bacterial cells on plates having minimal media with streptomycin, histidine, and glucose but no tryptophan. The trp+ cells would be able to synthesize the amino acid and therefore grow on the plate while trp- cells would not.