Induction of Mutation in Escherichia Coli by Ethyl Methane Sulfonate in Salmonella Typhimurium by Tn10-

In this experiment we will:

1. induce mutations in E. cli with the alkylating agent ethyl methane sulfonate (EMS)

2. Examine some of the properties of induced DNA changes in Salmonella typhimurium with transposable element, Tn10

a. We specifically select the EMS mutagenized E. coli on indicator media for cells that can no longer use lactose as a carbon source (Lac-)

b. We will obtain newly induced auxotrophs in S. typhimurium and try to identify the growth factor required by each mutant clone.

Procedure:

Make 10-5 and a 10-6 dilution of all cultures (E. coli and S. typhimurium).

For each dilution, plate 0.1 ml of diluted EMS on and 0.1 ml of each diluted E. coli control on one separate plate of MacConkey’s agar. Incubate plates overnight.

Look for white colonies on MacConkey’s plates and estimate their frequency.

Purify two by streaking out to single colonies on a new plate of MacConkey;s. Read them within 24 hours; look for white or (sometimes) pink colonies)

Once pure clones of lac- cells are picked from streaked plates, tape plates shut and show to TA

For the 10-5 and a 10-6 dilutions of S. typhimurium, plate 0.1 ml of mutagenized culture on nutrient agar and 0.1 ml of same dilutions of S. typhimurium control culture on nutrient agar. Incubate plates overnight.

Patch 100 colonies from mutagenized and non-mutagenized samples on minimal and then on nutrient agar. Incubate over night

Look for colonies on the nutrient agar plates that did not grow on minimal. Grow is thich if the colony is prototrophic. If a small amount of growth seen, do not score as a prototroph (they may be auxotrophs).

Spot presumptive auxotrophic colonies on a single plate of nutrient agar; include prototrophic colony as control. Incubate overnight.

Replica last plate to a minimal plate, then through the 9 pool plates and finish on a nutrient agar plate.

The plates are scored for colonies which appear on the supplemented minimal plate.

To replica:...