Question

You have a strain of E. coli which is ade- (adenine minus, meaning unable to make adenine on its own). In the biosynthesis of adenine, there are five steps as follows:            

In the biosynthetic pathway shown above, the ‘precursor’ is the starting material. Enzyme 1 converts this precursor to compound 1 (intermediate 1), enzyme 2 then acts on compound 1 to give rise to compound 2 (intermediate 2), so on and so forth, until enzyme 5 gives rise to the final product, which is adenine.

NOTE: i) Your strain of E. coli accumulates compound 3 inside its cytoplasm. This is because it never gets converted to compound 4 since enzyme 4 is not produced. Since compound 4 is not produced, adenine is not produced either. The significance of this is that these bacteria are unable to make/produce adenine on their own so will only be able to grow if adenine is supplied to them exogenously.   

ii) Also, this strain of E. coli happens to be resistant of ampicillin.

With this information, think of your experimental strategy to conduct genetic transformation of this strain of E. coli such that will be able to “glow in the dark” *** To answer the questions posed, the following information and hints will help:

1) Whatever plasmids you need, are available to you easily OR you can modify available plasmids and design them to suit your needs. Money is not a problem, for example, to buy restriction enzymes, ligase enzymes, etc.

2) You can give your newly designed plasmid a new ‘name’ (example PUC 21 or PUC 22 etc.)

3) Remember that Nutrient Agar (NA) is a type of ‘nutritionally complete’ medium, meaning it has ALL the ingredients needed for E. coli to grow

4) A ‘Synthetic Medium agar’ (called SMA) is NOT a nutritionally complete medium, you can design it the way you want. For example, you can add everything that E. coli needs to grow except –say a specific amino acid, or a particular nucleotide or a specific fatty acid, etc. (depending on the need of your experiment).

Questions:

State the types of agar media you will use in this exercise

What would be your ‘expected results’ be? Assume no mistakes are made. Be organized and clear in your answer! Take additional space if needed

Answer 1

The genetic transformation of E.coli such as to produce a luminescent population can be achieved by introducing a plasmid containing the LUX OPERON in the bacterial population. This operon is present in Vibrio fisheri , a luminescent bacteria.

STEPS FOR PREPARING GLOWING POPULATION OF E.COLI CAN BE SIMPLIFIED AS;

1. Prepare a desired plasmid which contains the gene of intest i.e, lux operon
2. Treating the bacterial population with CaCl₂ that increases the uptake of the plasmid by the bacteria.
3. incubate the transformed bacteria .
4. examine the transformed bacterial population in the dark.

As the strain of E.coli in our question is unable to produce adenine ,which is very much required for its growth . Also they are resistant to ampicillin. The agar medium which can be used to incubate and grow the transformed bateria can be a synthetic medium agar which consists of similar composition as that of a nutrient agar and aditionally it must contain adenine and ampicillin

Thus the agar plate contains ampicillin-adenine-nutrient agar for the growth of transformed population. As the strain is resistant to ampicillin ,it could easily grow in the ampicillin nutrient medium.

EXPECTED RESULT; After incubation for about 12-24hours , the population can be examined for luminescence . The E.coli population that takes up the plasmid would show luminescence under dark background where as the ones which did not take up the plasmid containing lux operon will not show any luminescence in that ampicillin-adinine-nutrient agar.